Untitled - Repositório Aberto da Universidade do Porto

This work was performed at the Microbiology Group of the Faculty of Pharmacy, University of Porto (FFUP, Porto, Portugal). The author received financial support through an individual pre-doctoral fellowship funded by the Foundation for Science and Technology (FCT) of Portugal with the reference SFRH/BD/84341/2012. Additionaly, a Federation of European Microbiologies Societies (FEMS) Research Grant (FEMS-RG-2014-0089) was attributed to the author to realize a three months internship in Hospital Universitario Ramón y Cajal (Instituto Ramón y Cajal de Investigación Sanitaria, IRYCIS), Madrid, Spain. The experimental research performed in this work was supported by funds from the European Union (FEDER funds) through Programa Operacional Fatores de Competitividade – COMPETE; through project POCI/01/0145/FEDER/007728), and national funds [FCT and Quadro de Referência Estratégica Nacional (QREN)] through projects numbers Pest-C/EQB/LA0006/2013, UID/Multi/04546/2013, NORTE-07-0124-FEDER-000066 and under the Partnership Agreement PT2020 UID/MULTI/04378/2013.

Carla Alexandra Parada Rodrigues

In depth characterization of contemporary Escherichia coli and

Klebsiella pneumoniae resistant to extended-spectrum β-lactams: from key molecular drivers to clonal delineation

Tese do 3.º Ciclo de Estudos Conducente ao Grau de Doutoramento em Ciências

Farmacêuticas na Especialidade de Microbiologia

Trabalho realizado sob a orientação de:

Professora Doutora Luísa Peixe (Professora Associada com agregação da Faculdade

de Farmácia, Universidade do Porto, Portugal; chefe de grupo BacT_Drugs Lab do

UCIBIO/REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal)

Doutora Ângela Novais (Investigadora UCIBIO/REQUIMTE, Faculdade de Farmácia,

Universidade do Porto, Portugal)

Professora Doutora Elisabete Machado (Professora Associada da Faculdade de

Ciências de Saúde, Universidade Fernando Pessoa, Porto, Portugal; Investigadora

UCIBIO/REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal).

May 2017

É AUTORIZADA A REPRODUÇÃO INTEGRAL DESTA TESE APENAS PARA EFEITOS

DE INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO INTERESSADO, QUE A

TAL SE COMPROMETE;

Carla Alexandra Parada Rodrigues

Ao Luís, aos meus pais e ao meu irmão

Por serem o melhor de mim!

v

AGRADECIMENTOS (ACKNOWLEDGMENTS)

Ao concluir esta Tese de Doutoramento quero expressar a minha gratidão e

reconhecimento a instituições, mas acima de tudo, a todas as pessoas que direta ou

indiretamente me acompanharam pelo maravilhoso mundo da Ciência e por todo o auxílio

que me dedicaram e que, de forma crucial, contribuíram para que a realização desta tese

se tornasse uma realidade. MUITO OBRIGADA!

“Coming together is a beginning; keeping together is progress; working together

is success”

Henry Ford

Em primeiro lugar à minha orientadora Prof. Doutora Luísa Peixe, e

coorientadoras Doutora Ângela Novais e Prof. Doutora Elisabete Machado quero deixar

expresso o meu profundo agradecimento por terem aceite esta tarefa tão árdua que é a

orientação de um aluno de Doutoramento. Agradeço todo o apoio durante estes quatro

anos e a partilha de conhecimentos constante.

À Prof. Doutora Luísa Peixe um muito obrigada por me ter permitido realizar o

meu trabalho de doutoramento na sua equipa, por me fornecer os meios para a realização

do mesmo e por estimular todos os dias o meu pensamento criativo mas também crítico

para alcançar o sucesso! Obrigada ainda pelo carinho, amizade e por acreditar em mim.

À Doutora Ângela Novais, não quero deixar passar esta oportunidade para

expressar um agradecimento muito especial. Sem dúvida que a conclusão desta tese não

teria sido possível sem o seu esforço e contributo inigualáveis. Muito obrigada do fundo do

coração pela paciência, por me inspirar a querer fazer mais e melhor, pelos conhecimentos

transmitidos, e pelas longas horas de discussões tão importantes na minha formação, no

desenvolvimento do meu espírito crítico e criatividade! Sinto esta Tese tão minha quanto

sua! Mais do que tudo, obrigada pela constante disponibilidade! Não posso deixar de

manifestar a minha gratidão, admiração, carinho, e acima de tudo amizade.

À Prof. Doutora Elisabete Machado pelas palavras de carinho, incentivo, força e

motivação proferidas sempre nas alturas certas. Obrigada pela amizade, pelo apoio, pela

compreensão e pela transmissão de conhecimentos.

vi

Em segundo lugar quero agradecer todo o apoio financeiro concedido ao longo

destes quatro anos: Fundação para a Ciência e a Tecnologia (FCT), Ministério da

Educação e da Ciência (MEC) (Bolsa SFRH/BD/84341/2012, QREN – POPH – Tipologia

4.1 – Formação Avançada, comparticipado pelo Fundo Social Europeu e por fundos

sociais do MEC). Agradeço também à Federation of European Microbiologies Societies

(FEMS) pela bolsa de investigação (FEMS Research Grant - FEMS-RG-2014-0089) que

me foi concedida para poder realizar um estágio essencial na minha formação no

Laboratório de Microbiologia do Hospital Universitario Ramón y Cajal em Madrid,

Espanha. Adicionalmente, gostaria de agradecer à Unidade de Investigação/FCT –

UCIBIO/REQUIMTE (através do projeto PEst-C/EQB/LA0006/2011 e

UID/MULTI/04378/2013, fundos FEDER POCI/01/0145/FEDER/007728, e FCT/MEC

ao abrigo do programa PT2020 UID/MULTI/04378/2013).

À Prof. Doutora São José Nascimento agradeço ter-me dado a oportunidade de

realizar este trabalho no Laboratório de Microbiologia da Faculdade de Farmácia da

Universidade do Porto. A todos os Prof. Doutores do Laboratório de Microbiologia quero

agradecer todas as palavras de carinho, disponibilidade e a simpatia com que me

receberam.

À Joana Campos que mais que uma companheira de trabalho se transformou num

pilar durante estes anos, não tenho palavras para dizer o quanto te devo, e o quão

orgulhosa estou de tudo o que alcançaste! Nunca te esqueças que estarei sempre aqui

como tens estado para mim. Acho que no fundo somos irmãs de mães diferentes!! À

minha companheira de bancada e confidente ao longo destes anos, Teresa Ribeiro,

obrigada pela experiência partilhada e por toda a amizade, e mais que tudo obrigada por

me ouvires sempre e pelo apoio prestado. Aos amigos e companheiros assíduos Liliana

Silva, Joana Mourão, João Pires, João Botelho, Michael Brilhante, Eduarda Silveira e

Raquel Branquinho quero agradecer todos os momentos de companheirismo, amizade,

risadas e confidências (bem sei que ás vezes as minhas eram um pouco exageradas),

partilha e auxílio nos bons e nos maus momentos. Sem dúvida que tornaram este caminho

muito especial e mais fácil de superar. Obrigado por fazerem com que todos os dias de

trabalho valessem a pena.

Às “seniores”, Prof. Doutora Carla Novais, Prof. Doutora Patrícia Antunes, Doutora

Ana Freitas e Doutora Filipa Grosso quero agradecer todo o apoio, boa disposição,

incentivo, disponibilidade e partilha de conhecimentos. Muito obrigada!

vii

Aos membros e companheiros mais recentes, mas também importantes, Joana

Rocha, Magda Księżareke e Svetlana Perovic o meu muito obrigada pelo carinho e

simpatia.

Não posso deixar de agradecer a duas pessoas muito especiais, Doutora Teresa

Coque e Doutor Val Lanza, que além da determinante contribuição na minha formação no

mundo da genómica, me receberam e acolheram durante três meses incríveis no

Laboratório de Microbiologia do Hospital Universitario Ramón y Cajal em Madrid

(Espanha) com todo o carinho e simpatia! A toda a equipa deste laboratório, em especial

Ana Sofia, Marta Hernández, Irene Merino, Elsa Freitas e Ricardo León por me fazerem

sentir em casa! Obrigada!

Gostaria também de agradecer à Doutora Clara Sousa, Professor Doutor João

Lopes e ao Doutor Hugo Osório, pelo conhecimento partilhado e disponibilidade

demonstrada.

À Cristina Pinto da Costa (a minha “Tininha”) um agradecimento muito especial,

por toda a ajuda prestada, pelos desabafos, pela companhia e acima de tudo pela amizade

que guardarei para sempre no meu coração (acredita que “a peste” não se vai esquecer

nunca de ti). Muito obrigada por tudo. Ao Nuno Oliveira agradeço todo o apoio ao longo

destes anos.

A todos os alunos de projeto e de iniciação à investigação da Faculdade de

Farmácia da Universidade do Porto (Sofia Fernandes, Carolina Montenegro, Carolina

Pereira) muito obrigada pelo carinho, simpatia e por toda a ajuda prestada. Foi sem

dúvida um prazer conhecer-vos.

A todos os alunos ERASMUS com quem tive oportunidade de trabalhar (Irina

Gheorghe, Ilda Czobor e Valérie De Scheerder, Filip Sima, Jan Bavlovič,) o meu muito

obrigada pela partilha de experiências quer laborais, quer culturais. Foi um prazer

conhecer-vos.

Às amigas de uma vida, Maria João, Bruna, Mafalda, Cris, Xana e Tanita, obrigado

pelo apoio incondicional, por perdoarem a minha ausência e por torcerem por mim em

todos os momentos. Obrigado por estarem sempre lá e por comprovarem que a verdadeira

amizade pode fortalecer-se todos os dias, fazendo jus à frase “longe da vista, mas perto do

coração”.

viii

Ao Luís, meu futuro marido, companheiro de longos anos e já parte da minha

família. Muito obrigada pelo amor, apoio, compreensão e por seres diariamente o meu

pilar, que me faz acreditar que sou capaz de tudo. Às vezes tenho a sensação que me

conheces melhor do que eu a mim mesma! Sei que nem sempre foi fácil lidar com tudo

que a vida de um investigador implica, mas mesmo assim estiveste SEMPRE lá. Nunca

terei palavras suficientes para te agradecer o apoio incondicional, mas tenho a certeza que

terei amor para te dar SEMPRE!

Aos meus pais e ao meu irmão, sem dúvida as pessoas mais importantes da minha

vida juntamente com o Luís. Obrigado por acreditarem em mim, por apoiarem os meus

sonhos e nunca me deixarem desistir, e sobretudo por contribuírem para ser quem sou

hoje. Sem vocês esta tese não seria possível. Mãe obrigada por me ouvires todos os dias e

por sofreres mais do que eu por mim...um dia saberei o que isso é! OBRIGADA!

Também gostaria de agradecer à Isabel, ao senhor Filipe e à “Anginha” pelo apoio

que me deram sempre! No fundo são já a minha família! Obrigada!

Por fim e por estranho que possa parecer a quem está neste momento a ler estes

agradecimentos há um ser muito especial a quem tenho que agradecer! A minha cadela

Kira!! Foste a minha companheira fiel nestes meses de escrita e foste também a minha

“sanidade mental”. Acho que nunca poderás ter noção do significado que tiveste nesta

fase. Parece que ainda estou a ver-te olhar para mim do sofá enquanto eu trabalhava!

Obrigada fiel companheira!

“As coisas vulgares que há na vida

Não deixam saudade

Só as lembranças que doem

Ou fazem sorrir

Há gente que fica na história

Da história da gente

E outras de quem nem o nome

Lembramos ouvir”

Jorge Fernando “Chuva”

ix

ABSTRACT

The rise on antibiotic resistance (ABR) rates among main bacterial pathogens is a growing

global Public Health threat with significant social and economic impact. Of particular concern is

the ever-increasing detection of Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) strains

resistant to multiple clinically important antibiotics for human medicine, including relevant and

even last-resource therapeutic choices [extended-spectrum cephalosporins (ESC), carbapenems,

fluoroquinolones or colistin] seriously compromising the eradication of common infections.

Besides the endemicity of extended-spectrum β-lactamase (ESBL)-producing Ec since long time in

different settings, the situation is nowadays particularly worrying for Kp due to the explosive

dispersion of strains producing ESBLs and especially carbapenemases among different healthcare

settings [hospitals, ambulatory, nursing homes (NHs), long-term care facilities (LTCFs)]. However,

bacterial dispersion drivers (clones and mobile genetic elements) behind this scenario are still

poorly understood and explored for Kp and in recent national collections of Ec. The availability of

molecular tools for accurate and detailed characterization at strain [e.g. whole genome sequencing

(WGS)] or plasmid levels provides an excellent opportunity for a more precise definition of

evolutionary processes of a bacterial pathogen as well as the recognition of key dispersion drivers of

ABR genes, in order to understand possible transmission pathways. Moreover, epidemiological

surveillance of the multidrug resistant (MDR) Ec and Kp clones is essential for clinical laboratories

to guide infection control measures and therapeutic decisions, but accurate, quick and low cost

methods are lacking.

The main goals of this work were to determine the current epidemiological scenario

involving acquired resistance to extended-spectrum β-lactams in recent collections of Ec and Kp

from different clinical and non-clinical human settings in Portugal; and to explore different high-

throughput omics approaches for accurate and quick typing of MDR Kp strains, identifying key

molecular features behind their discriminatory potential. To accomplish these purposes, a

comprehensive multilevel characterization of recent Ec and Kp isolates non-susceptible to

extended-spectrum β-lactams from different clinical (hospital, ambulatory, LTCFs, NHs) and non-

clinical (healthy volunteers) human settings mainly from the North region of Portugal (2006-2016)

was performed. Additionally, a representative international collection of main MDR Kp clones and

genomes included in public databases were used to assess the potential of different high-

throughput methodologies [Fourier transform infrared (FTIR) spectroscopy, matrix assisted laser

desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and WGS] for typing and

characterization of Kp.

The results of this Thesis revealed high resistance rates to extended-spectrum β-lactams

among clinical Ec and Kp and significant epidemiological changes in the period analysed (2006-

2016), though fosfomycin and colistin seem to be valuable alternatives. Currently, CTX-M-15-

producing Ec B2-ST131 is still dominating ESBL-producing Ec population, though different

subclones (fimH30, fimH22) presumably associated with variable pathogenic/persistence abilities

were identified. On the other hand, a remarkable increase in the proportion of ESBL-producing Kp

(a ca. five-fold increase in 3 years) was noted (especially CTX-M-15) linked mostly to worldwide

x

spread lineages (mainly ST15, ST147) known to have an increased ability to spread and persist, and

plasmids apparently well adapted to this species (IncR, IncFIIK). Moreover, carbapenemase-

producing Kp (mostly KPC-3 and ST147/ST15) were detected since 2011, and rapidly disseminated

among hospitalized patients and also residents of LTCFs and NHs, a situation of high concern that

requires reinforced surveillance and a concerted infection control action. It is also of note the

identification of specific capsular types among lineages belonging to ST15 (K19, K24, KL110, KL112)

or ST147 (K64) infecting or colonizing humans and with ability to carry variable MDR plasmids,

reflecting their open genome. Lineages belonging to the major clonal groups (CGs) 15 and 14 were

precisely defined by comparative genomics analysis showing circulation in different geographic

regions for several years and a correspondence with specific capsular types. Moreover, a turbulent

flux of plasmids of disparate families containing a wide number of adaptive traits (including ABR

genes) was observed, with IncF and IncR plasmids being pervasive and greatly contributing to the

CG15/14 pangenome. Besides supporting the intraclonal genomic variability inferred by genotypic

methods, FTIR spectroscopy proved to be able to accurately differentiate Kp capsular types,

highlighting the potential of this methodology as a fast and accurate alternative K-typing tool, and

eventually its potential for Kp typing in routine laboratory conditions, as demonstrated by the

application of this approach in a real-time context. Conversely, MALDI-TOF MS showed a limited

potential to discriminate MDR Kp clones, apparently explained by the high degree of conservation

of Kp ribosomal proteins.

This Thesis provides a comprehensive overview of the molecular basis for ESC and

carbapenem resistance among Ec and Kp, unveiling particular lineages and species-specific

plasmids at the origin of the dispersion of genes encoding ESBL or carbapenemases. Moreover,

alternative K-typing tools (FTIR spectroscopy) with potential routine application are proposed, and

ultimately the high congruence observed between genotypic, genomics and metabolomics

approaches for Kp typing points to a great relevance of surface bacterial components (and

particularly the capsule) on the evolution, host adaptation and/or virulence features in this species

that deserves to be further explored.

Keywords: extended-spectrum acquired β-lactamases, drivers, typing, high-throughput methods

xi

RESUMO

O aumento da frequência de resistência a antibióticos (RA) em bactérias patogénicas

relevantes representa uma ameaça global para a saúde pública com significativos impactos sociais

e económico. De particular preocupação é a crescente deteção de Escherichia coli (Ec) e Klebsiella

pneumoniae (Kp) resistentes a múltiplos antibióticos importantes para a medicina humana,

incluindo antibióticos de primeira e última linha [cefalosporinas de largo espectro (CLE),

carbapenemos, fluoroquinolonas, colistina], comprometendo seriamente a erradicação de infeções

frequentes. Adicionalmente à já antiga endemicidade de Ec produtoras de β-lactamases de

espectro alargado (BLEA) em diferentes nichos, o aparecimento e disseminação de estirpes de Kp

produtoras de BLEAs e carbapenemases em diferentes instituições de saúde [hospitais,

ambulatório, lares, unidades de cuidados continuados (UCCs)] agravou significativamente a

problemática da resistência.

No entanto, os veículos de dispersão (clones e elementos genéticos móveis) subjacentes a

este cenário encontram-se ainda insuficientemente explorados em Kp e em coleções recentes

nacionais de Ec. Com a recente disponibilização de ferramentas moleculares com maior resolução

para a discriminação bacteriana ao nível da infraespécie [e.g. sequenciação do genoma total

(WGS)] e para a tipagem de plasmídeos abriu-se uma excelente oportunidade para uma definição

mais precisa dos processos evolutivos de bactérias patogénicas assim como para o reconhecimento

dos veículos chave de dispersão de genes de RA essenciais para compreender possíveis vias de

transmissão. Além disso, a vigilância epidemiológica de clones multirresistentes (MR) de Ec e Kp é

fundamental na rotina clinica, para a adoção de medidas de controlo de infeção efetivas e para

guiar decisões terapêuticas, sendo, no entanto, necessários métodos de tipagem precisos, rápidos e

de baixo custo.

Os principais objetivos deste trabalho compreenderam a caracterização epidemiológica

da resistência adquirida a β-lactâmicos de espectro alargado em Ec e Kp provenientes de diferentes

nichos clínicos e não clínicos humanos em Portugal; e a exploração de diferentes abordagens

ómicas de alto rendimento para a tipagem rápida de Kp MR, identificando características

moleculares chave subjacentes ao seu poder discriminatório. Assim, procedeu-se através de uma

aproximação multinível à caracterização de isolados recentes de Ec e Kp não suscetíveis a β-

lactâmicos de espectro alargado provenientes de diferentes nichos clínicos (hospital, ambulatório,

UCCS, lares) e não-clínicos (voluntários saudáveis) e maioritariamente da região Norte de Portugal

(2006-2016). Além disso, uma coleção internacional de Kp MR representativa dos principais

clones foi utilizada para avaliar o potencial de diferentes metodologias de alto rendimento, como a

espectroscopia de infravermelho com transformada de Fourier (FTIR) e a espectrometria de massa

com analisador por tempo de voo e fonte de libertação/ionização por laser e matriz (MALDI-TOF

MS) para a tipagem de Kp, e utilizando-se como referência a análise resultante da sequenciação de

todo o genoma (WGS) (coleção representativa de clones internacionais e genomas disponíveis em

bases de dados públicas).

xii

Os resultados desta tese revelaram uma elevada frequência de resistência a β-lactâmicos

de espectro alargado em Ec e a Kp de origem clínico e importantes alterações epidemiológicas no

período analisado (2006-2016), embora a fosfomicina e a colistina pareçam ser ainda alternativas

valiosas. Atualmente, Ec B2-ST131 produtora de CTX-M-15 continua a dominar a população de Ec

produtoras de BLEAs, embora tenham sido detetados diferentes subclones (fimH30, fimH22),

presumivelmente associados a um potencial patogénico e/ou de persistência variável. Por outro

lado, observou-se um aumento notável na proporção de Kp produtoras de BLEAs (cerca de 5 vezes

em 3 anos), especialmente CTX-M-15, associada a clones globalmente disseminados (ST15, ST147),

conhecidos pela sua capacidade de propagação e persistência, e plasmídeos aparentemente bem

adaptados a esta espécie (IncR, IncFIIK). Além disso, detetou-se a ocorrência de Kp produtoras de

carbapenemases (principalmente KPC-3 e ST147/ST15) desde 2011, que se disseminaram

rapidamente entre pacientes hospitalizados e também residentes de UCCs ou lares, situação

preocupante e que requer uma ação concertada de vigilância e controlo de infeção. É também de

salientar a identificação de tipos capsulares específicos entre linhagens pertencentes a ST15 (K19,

K24, KL110, KL112) ou ST147 (K64), associados a infeção ou colonização em humanos e com

variáveis plasmídeos de multirresistência, refletindo o seu “open genome”. Linhagens pertencentes

a grupos clonais (GCs) maioritários como GC15 e GC14 foram definidas com precisão por genómica

comparativa, revelando a sua circulação em diferentes regiões geográficas ao longo de vários anos

e uma correspondência com determinados tipos capsulares. Além disso, observou-se um fluxo

turbulento de plasmídeos de diferentes famílias contendo um elevado número de características

adaptativas, incluindo genes de RA, sendo os de tipo IncF e IncR os que mais contribuem para o

pangenoma de GC15/GC14. Para além de suportar a variabilidade genómica intraclonal inferida

por métodos genotípicos, a espectroscopia de FTIR provou ser capaz de diferenciar com precisão

os tipos capsulares de Kp, destacando o potencial desta metodologia como uma ferramenta

alternativa rápida para tipagem capsular, eventualmente adaptável à rotina de microbiologia

clínica, conforme demonstrado pela aplicação desta abordagem num contexto em “tempo real”.

Inversamente, o MALDI-TOF MS mostrou um limitado potencial de discriminação, aparentemente

explicado pelo elevado grau de conservação das proteínas ribossomais em Kp.

Esta tese fornece uma visão abrangente da base molecular da resistência a CLE e

carbapenemos em Ec e Kp, revelando o papel de linhagens particulares e plasmídeos específicos de

espécie como veículos de dispersão de genes que codificam para BLEAs ou carbapenemases. Além

disso, são propostas ferramentas de tipagem de Kp alternativas (espectroscopia de FTIR) com

potencial aplicação na rotina. Por último, a elevada congruência observada entre abordagens

genotípicas, genómicas e metabolómicas na tipagem de Kp aponta para a relevância dos

componentes bacterianos de superfície (particularmente a cápsula) na evolução, adaptação ao

hospedeiro e/ou características de virulência nesta espécie que merecem ser alvo de um estudo

aprofundado.

Palavras-chave: β-lactamases adquiridas de espectro alargado, veículos de dispersão, tipagem,

metodologias de alto rendimento

xiii

CONTENTS CHAPTER 1 – INTRODUCTION..........................................................................1 1.1. Fundaments for understanding the epidemiology of Escherichia coli and Klebsiella

pneumoniae resistant to extended-spectrum β-lactam antibiotics in the 21st century ......... 3

1.1.1. The growing burden of multidrug resistant (MDR) Escherichia coli and

Klebsiella pneumoniae ....................................................................................................... 3

1.1.2. Acquired resistance mechanisms to extended-spectrum β-lactams in

Escherichia coli and Klebsiella pneumoniae: a current perspective ............................... 13

1.1.3. Update on clinical relevance, virulence and population structure of

Escherichia coli and Klebsiella pneumoniae isolates resistant to extended-spectrum β-

lactams from clinical and non-clinical human settings ................................................... 29

1.1.3.1. Escherichia coli ......................................................................................... 30

1.1.3.2. Klebsiella pneumoniae ............................................................................. 38

1.1.4. Mobile genetic elements involved in the spread of bla genes among

Escherichia coli and Klebsiella pneumoniae ................................................................... 44

1.2. Overview of typing methods for Escherichia coli and Klebsiella pneumoniae:

exploring the potential of omics approaches ........................................................................ 50

1.2.1. Phenotyping methods ....................................................................................... 53

1.2.2. Genotyping methods ......................................................................................... 57

1.2.3. Proteomics ........................................................................................................ 63

1.2.4. Metabolomics .................................................................................................... 66

REFERENCES ............................................................................................ 69

CHAPTER 2 – OBJECTIVES AND OUTLINE OF THE STUDY.................113 2.1. Statement of objectives ............................................................................................ 115

2.2. Outline of the thesis ................................................................................................. 119

CHAPTER 3 – RESULTS AND DISCUSSION………………………….................123 3.1. Multi-niche dispersal of contemporary antibiotic resistance genetic backgrounds

associated with Escherichia coli from human origin ......................................................... 125

3.1.1. Increase of widespread A, B1 and D Escherichia coli clones producing a high-

diversity of CTX-M-types in a Portuguese hospital ....................................................... 127

xiv

3.1.2. Occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli

in Portuguese nursing homes: the identification of different B2-ST131 subclones

(fimH30, fimH22).....…..…………………………………………………………………………………… 139

3.1.3. An update on faecal carriage of ESBL-producing Enterobacteriaceae by

Portuguese healthy humans: detection of the H30 subclone of B2-ST131 Escherichia

coli producing CTX-M-27 ................................................................................................ 145

3.1.4. Importation of fosfomycin resistance fosA3 gene to Europe ............................... 151

3.2. Elucidating the drivers for expansion of Klebsiella pneumoniae resistant to

extended-spectrum β-lactams in different Portuguese clinical settings ............................ 155

3.2.1. Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized patients: a

successful story of international clones (ST15, ST147, ST336) and epidemic plasmids

(IncR, IncFIIK) ................................................................................................................. 157

3.2.2. KPC-3-Producing Klebsiella pneumoniae in Portugal linked to previously

circulating non-CG258 lineages and uncommon genetic platforms (Tn4401d-IncFIA

and Tn4401d-IncN) ......................................................................................................... 167

3.2.3. Detection of VIM-34, a novel VIM-1 variant identified in the intercontinental

ST15 Klebsiella pneumoniae clone ................................................................................. 177

3.2.4. High rates of long-term care facilities (LTCFs) residents colonized with multidrug

resistant Klebsiella pneumoniae lineages frequently causing infections in Portuguese

clinical institutions .......................................................................................................... 181

3.3. Detailing multidrug resistant (MDR) Klebsiella pneumoniae by omic approaches:

from classical to high-throughput modern typing .............................................................. 189

3.3.1. Congruence between capsular genotypic and phenotypic features of multidrug-

resistant (MDR) Klebsiella pneumoniae clones: a step-forward on K-typing by Fourier

Transform Infrared (FTIR) Spectroscopy ....................................................................... 191

3.3.2. Fourier Transform Infrared (FTIR) spectroscopy based-typing for “real-time”

analysis of an outbreak by carbapenem-resistant Klebsiella pneumoniae isolates ...... 219

3.3.3. Elucidating constraints for differentiation of major human Klebsiella

pneumoniae clones by MALDI-TOF MS ....................................................................... 227

3.3.4. High-resolution genomic analysis of the globally disseminated multidrug

resistance Klebsiella pneumoniae Clonal Groups 14 and 15 ......................................... 239

CHAPTER 4 - CONCLUSIONS.........................................................................291 APPENDIX…………………………………………………………………………………...............297

xv

LIST OF FIGURES Chapter 1 – Introduction FIGURE 1. Ceftazidime MIC distribution and ECOFF values for Escherichia coli isolates..

......................................................................................................................................... 4

FIGURE 2. Schematic representation of the dissemination routes of antibiotic resistant

bacteria ............................................................................................................................ 5

FIGURE 3. Targets of the main antibiotics used in Enterobacteriaceae infections ........... 6

FIGURE 4. Trends in antibiotic resistance (third-generation cephalosporins,

carbapenems, fluoroquinolones and aminoglycosides) among E. coli clinical isolates

by world region, with Portugal detailed for comparison ............................................... 7

FIGURE 5. Trends in antibiotic resistance (third-generation cephalosporins,

carbapenems, fluoroquinolones and aminoglycosides) among K. pneumoniae clinical

isolates by world region, with Portugal detailed for comparison .................................. 8

FIGURE 6. Structure of different β-lactam antibiotic families ......................................... 13

FIGURE 7. Timeline representation of the introduction of β-lactams into clinical practice

and further sequential emergence of β-lactamases among Enterobacteriaceae ........ 16

FIGURE 8. Distribution of different carbapenemases types among Enterobacteriaceae

isolates by world region (2008-14) ............................................................................... 24

FIGURE 9. Worldwide geographic distribution of KPC-producing K. pneumoniae in

clinical and non-clinical human settings ...................................................................... 25

FIGURE 10. Worldwide geographic distribution of OXA-48-like-producing K.

pneumoniae in clinical and non-clinical human settings. ........................................... 26

FIGURE 11. Worldwide geographic distribution of OXA-48-like-producing E. coli in


FIGURE 12. Worldwide geographic distribution of NDM-producing K. pneumoniae in


FIGURE 13. Mechanisms of dissemination of antibiotic resistance genes ....................... 30

FIGURE 14. Proportion of ExPEC isolates identified among all bacterial pathogens

isolated in 80.089 hospital admissions in 19 US hospitals (2007 and 2010) ............. 31

FIGURE 15. Global dissemination of E. coli B2-ST131 clone among clinical and non-

clinical human hosts (until 2013) ................................................................................. 34

FIGURE 16. Evolution of E. coli ST131 lineages driven by selection pressure due to

fluoroquinolones and ESC use ...................................................................................... 35

FIGURE 17. Taxonomic restructuration of K. pneumoniae .............................................. 38

xvi

FIGURE 18. Well-characterized virulence factors among K. pneumoniae strains and

differences among MDR and hypervirulent strains .................................................... 39

FIGURE 19. The modular and hierarchical composition of different mobile and

mobilizable genetic elements ....................................................................................... 44

FIGURE 20. Genetic organization of a conjugative and a mobilizable plasmid .............. 46

FIGURE 21. Applications of bacterial typing. ................................................................... 50

FIGURE 22. Pan-genome schematic representation.. ...................................................... 52

FIGURE 23. Scheme of the methodology applied in phage typing ................................... 55

FIGURE 24. Number of bacterial and archaeal genomes sequenced each year (between

1995 and 2014) and submitted to NCBI ...................................................................... 62

FIGURE 25. Schematic representation showing the technical fundament of MALDI-TOF

MS ................................................................................................................................. 64

FIGURE 26. FTIR bacterial characteristic spectrum. ....................................................... 66

xvii

LIST OF TABLES

Chapter 1 – Introduction

TABLE 1. Studies reporting E. coli and K. pneumoniae isolates from clinical and non-

clinical human origin harbouring plasmid-mediated fosfomycin resistance genes ... 12

TABLE 2. Functional and molecular classification of β-lactamases .................................. 15

TABLE 3. Studies characterizing the ESBL-types harboured by E. coli and K. pneumoniae

isolates colonizing LTCFs or NHs residents (2006-16) ............................................... 20

TABLE 4. Studies characterizing the ESBL-types harboured by E. coli and K.

pneumoniae isolates colonizing healthy volunteers (2006-16) ................................... 21

TABLE 5. Hydrolytic and inhibition profiles of the most prevalent carbapenemases

identified among Enterobacteriaceae .......................................................................... 23

TABLE 6. Virulence factors associated with ExPEC by functional category and

associations between some of them and specific clinical infections ............................ 32

TABLE 7. MGE associated with the most widespread bla genes conferring resistance to

extended-spectrum β-lactams among E. coli and K. pneumoniae from human sources

....................................................................................................................................... 47

TABLE 8. Characteristics and applications of the main genotyping methods used in

analysis of E. coli and K. pneumoniae populations ..................................................... 58

TABLE 9. Proteotyping studies performed in E. coli and K. pneumoniae using MALDI-

TOF MS ......................................................................................................................... 64

TABLE 10. Overview, advantages and disadvantages of the main bacterial strain typing

methods currently used for E. coli and K. pneumoniae populations .......................... 68

xviii

LIST OF BOXES Chapter 1 – Introduction BOX 1. Antibiotic Resistance Concepts ................................................................................ 4

BOX 2. Fosfomycin and Polymyxins – Mechanism of action, spectrum of activity and

acquired resistance mechanisms .................................................................................. 10

BOX 3. Mechanism of action, classification and spectrum of activity of β-lactam

antibiotics ...................................................................................................................... 13

BOX 4. Population structure concepts and methodologies applied in the typing and

subtyping of E. coli ....................................................................................................... 33

BOX 5. Methodologies applied in the typing and subtyping of K. pneumoniae ................ 41

BOX 6. Concepts and definitions related to mobile and mobilizable genetic elements .... 45

BOX 7. Glossary of concepts commonly used in bacterial typing. ...................................... 51

xix

LIST OF ABBREVIATIONS

Description ABR Antibiotic resistance AFLP Amplified Fragment Length Polymorphism AIEC Adherent-invasive Escherichia coli AP-PCR Arbitrarily Primed PCR ATR Attenuated total reflectance BIGSdb Bacterial Isolate Genome Sequence Database BSAC British Society for Antimicrobial Chemotherapy CA Clavulanic acid CAP Community-acquired Pneumonia CARA Canadian Antimicrobial Resistance Alliance CDC Centers for Disease Control and Prevention CG(s) Clonal Group(s) CTn(s) Conjugative transposon(s) cgMLST Core genome multilocus sequence typing CLSI Clinical and Laboratory Standards Institute CPS Capsular polysaccharide DAEC Diffusely Adherent Escherichia coli DNA Deoxyribonucleic acid EAEC Enteroaggregative Escherichia coli EARS-Net European Antimicrobial Resistance Surveillance Network ECDC European Center for Disease Prevention and Control ECOFF(s) Epidemiological Cut-Off(s) EHEC Enterohaemorrhagic Escherichia coli EIEC Enteroinvasive Escherichia coli ETEC Enterotoxigenic Escherichia coli EPEC Enteropathogenic Escherichia coli EUCAST European Committee on Antimicrobial Susceptibility Testing EDTA Ethylenediaminetetraacetic acid EMA European Medicines Agency ESC Extended-spectrum cephalosporins ESBL(s) Extended-Spectrum β-Lactamase(s) EUCAST European Committee on Antimicrobial Susceptibility Testing ExPEC Extraintestinal Escherichia coli FTIR Fourier transform infrared GI(s) Genomic island(s) HAI Healthcare-associated infections HAP Hospital-acquired pneumonia HGT Horizontal gene transfer IS Insertion Sequences JSP Japanese Society for Chemotherapy K-typing Capsular typing K-type Capsular types LPS Lipopolysaccharide LTCF(s) Long-Term Care Facility(ies) MALDI-TOF MS Matrix assisted laser desorption ionization-time of flight mass spectrometry MBL(s) Metallo-β-lactamase(s) MDA Multivariate data analysis

xx

MDR Multidrug resistant MIC Minimum Inhibitory Concentration MGE Mobile Genetic Elements MNEC Meningitis-associated Escherichia coli MLEE Multilocus Enzyme Electrophoresis MLST Multilocus sequence typing MLVA Multiple-locus variable-number tandem repeat analysis MOB Plasmid mobility genes MPF Mating pair formation MRSA Methicillin resistant Staphylococcus aureus NCBI National Center for Biotechnology Information NH(s) Nursing Home(s) NHSN National Healthcare Safety Network PAIs Pathogenicity-associated islands PBPs Penicillin Binding Proteins PBRT PCR-based replicon typing PCR Polymerase Chain Reaction PFGE Pulsed-Field Gel Electrophoresis PLACNET PLAsmid Constellation NETwork RAPD Random Amplification of Polymorphic DNA ReLAVRA Red Latinoamericana de Vigilancia de la Resistencia a los Antimicrobianos rep-PCR Repetitive element palindromic PCR RFLP Restriction Fragment Length Polymorphism RNA Ribonucleic Acid rRNA Ribosomal ribonucleic acid SNP(s) Single nucleotide polymorphism(s) SMART Study for Monitoring Antimicrobial Resistance Trends SERS Surface-enhanced Raman spectroscopy ST Sequence Type STEAEC Shiga-toxin producing enteroaggregative Escherichia coli T4CP Type IV coupling proteins T4SS Type 4 secretion system TB Tuberculosis Tn Transposon TZB Tazobactam UPEC Uropathogenic Escherichia coli USA United States of America UTI(s) Urinary Tract Infection(s) VRE Vancomycin resistant Enterococcus spp. VRSA Vancomycin resistant Staphylococcus aureus WGS Whole-genome sequencing WHO World Health Organization XDR Extensively drug-resistant

“Devote yourself to an idea, go make it happen, struggle on it, overcome your fears,

smile, don't you forget this is your dream.”

Anonymous

Introduction

Chapter 1

Chapter 1 - Introduction

3

1.1. Fundaments for understanding the epidemiology of Escherichia coli and Klebsiella pneumoniae resistant to extended-spectrum β-lactam antibiotics in the 21st century

1.1.1. The growing burden of multidrug resistant (MDR) Escherichia

coli and Klebsiella pneumoniae

“The greatest possibility of evil in self-medication is the use of too small doses so that

instead of clearing up infection the microbes are educated to resist penicillin and a host of

penicillin-fast organisms is bred out which can be passed to other individuals and from

them to others until they reach someone who gets a septicemia or pneumonia which

penicillin cannot save.”

Alexander Fleming (The New York Times in 1945)

The accidental discovery of penicillin by Alexander Fleming, described in a

publication in the late 1920s and commercialized later in the 1940s was revolutionary (1).

However, in Nobel Prize award speech in 1945 (partially transcribed above), he envisioned

the capacity of microbes to develop antimicrobial resistance under selective pressure, as if

he already anticipated the current global crisis involving antimicrobial resistant bacteria.

Antibiotic resistance (ABR) [see Box 1 and Figure 1 to review concepts regarding this

topic] has been considered one of the major threats to human health by several world health

entities, such as the World Health Organization (WHO), the Centers for Disease Control

(CDC) or the European Centre for Disease Prevention and Control (ECDC) (2–4), and a

priority research topic in European Horizon 2020 programs

(https://ec.europa.eu/research/health/index.cfm?pg=area&areaname=amdr) (5). The

problem resides on the exponential increase at a global level of infections caused by

multidrug resistant (MDR) bacterial pathogens, defined as bacteria resistant to three or

more antimicrobial classes to which they do not show intrinsic resistance. Consequently,

the depletion of therapeutic options increases the number of untreatable infections and

envisions a panorama similar to that of the pre-antibiotic era (6,7). Mortality rates and

associated costs are high either in Europe (ca. 25,000 deaths, €1.6 billion annually, data

from 2007) or in the United States of America (USA) (23,000 deaths, $20 billion, data from

2013) (4,8). The ABR crisis has been attributed to the overuse and misuse of antibiotics in

both humans and food-producing animals (Figure 2), the globalization of tourism and food


4

industry, together with an alarming decline in the discovery and development of new

classes of antibiotics (5).

BOX 1. Antibiotic Resistance Concepts

ABR can be defined from the clinical and epidemiological point of views (9,10).

Clinical resistance is defined using clinical breakpoints. A clinical breakpoint is calculated taking into

account different parameters: minimum inhibitory concentration (MIC) distributions (MIC is the lowest concentration of an antimicrobial agent that inhibits the growth of bacterial cells), clinical outcome data,

accepted dosing and pharmacokinetic/pharmacodynamic data. The clinical breakpoint is a MIC value that is

used to predict in vitro, the in vivo antibiotic efficacy in a given isolate (9,10). Based on these clinical

breakpoints, the isolate can be defined as: susceptible (level of antimicrobial activity associated with a high

likelihood of therapeutic success), intermediate (level of antimicrobial activity associated with uncertain

therapeutic effect) or resistant (level of antimicrobial activity associated with a high likelihood of

therapeutic failure), according to annual published guidelines and recommendations such as those published

by the Clinical and Laboratory Standards Institute (CLSI - http://clsi.org/), the European Committee on

Antimicrobial Susceptibility Testing (EUCAST - http://mic.eucast.org/Eucast2/) or other national organisms

[e.g. British Society for Antimicrobial Chemotherapy (BSAC); Japanese Society for Chemotherapy (JSC)].

Epidemiological resistance is defined using epidemiological cut-offs (ECOFFs) values. ECOFFs

are defined as the MIC value that corresponds to the upper-limit of the wild-type population (considered as being deprived of any acquired resistance mechanisms to a particular antibiotic) of a particular bacterial

species. ECOFFs can be used to distinguish within a particular species the strains without (wild-type) or with

(non wild-type) phenotypically expressed resistance mechanisms (Figure 1)

(https://mic.eucast.org/Eucast2/SearchController/search.jsp?action=init) (9).

FIGURE 1. Ceftazidime MIC distribution and ECOFF values for Escherichia coli isolates. (https://mic.eucast.org/Eucast2/).Clinical susceptible (S) and resistant (R) breakpoints defined by EUCAST are indicated.

S breakpoint EUCAST

R breakpoint EUCAST

9

ECOFF


5

FIGURE 2. Schematic representation of the dissemination routes of antibiotic resistant bacteria.

(adapted from http://www.spiegel.de/international/world/bild-811560-309342.html).

The main pathogens associated with the ABR crisis are Mycobacterium tuberculosis

[MDR-tuberculosis (TB) and extensively drug-resistant (XDR-TB)], fluoroquinolone

resistant Clostridium difficile, methicillin and vancomycin resistant Staphylococcus aureus

(MRSA and VRSA, respectively), vancomycin resistant Enterococcus spp. (VRE), macrolide

and penicillin resistant Streptococcus pneumoniae, third-generation cephalosporins

resistant Neisseria gonorrhoeae, MDR Acinetobacter spp., MDR Pseudomonas aeruginosa

and MDR Enterobacteriaceae (in this specific group generally the combined resistance to

extended-spectrum β-lactams [extended-spectrum cephalosporins (ESC) and

carbapenems], fluoroquinolones and aminoglycosides) (2,4).

One of the most worrying epidemiological problems is the increasing number of

infections involving Enterobacteriaceae resistant to “critically important antimicrobials for

human medicine” such as ESC, carbapenems, fluoroquinolones and aminoglycosides,

frequently in combination as referred above (Figure 3) (2–4). In particular, the recent

widespread of Enterobacteriaceae resistant to carbapenems or other last resource

antibiotics, such as fosfomycin or polymyxins, raised the alarm and received increased

attention by different organizations worldwide due to the lack of therapeutic options

(Figure 3) (11,12).

Air, Dust

Humans travel Trade of animals and food


6

FIGURE 3. Targets of the main antibiotics used in Enterobacteriaceae infections [adapted from

(13)]. Antibiotics considered “critically important antimicrobials for human medicine” are highlighted by red line boxes.

E. coli and Klebsiella pneumoniae are the Enterobacteriaceae species more

frequently implicated in human infections and increasingly linked to multidrug resistance

phenotypes, constituting the main species analysed in this thesis for these reasons. An

overview of the evolution in resistance rates to extended-spectrum β-lactams (third-

generation cephalosporins and carbapenems), fluoroquinolones, aminoglycosides,

fosfomycin and polymyxins among clinical E. coli and K. pneumoniae from the last decade

(2006-2015) is presented below.

In some geographic areas, ABR trends are monitored by large surveillance networks,

such as the European Antimicrobial Resistance Surveillance Network (EARS-Net;

http://ecdc.europa.eu/en/Pages/home.aspx) including only invasive isolates (blood and

cerebrospinal fluid), the National Healthcare Safety Network (NHSN) Antibiotic Resistance

Data network from CDC (https://gis.cdc.gov/grasp/psa/MapView.html) with isolates

restricted to healthcare-associated infections (HAI) in the USA, the Canadian Antimicrobial

Resistance Alliance (CARA, http://www.can-r.com/index.php) or the Red Latinoamericana

de Vigilancia de la Resistencia a los Antimicrobianos (ReLAVRA,

http://antimicrobianos.com.ar/category/resistencia/relavra/), these latter considering only

hospital isolates. Data collected from these surveillance networks or published in

independent studies are very useful but limited to particular periods of time, sources,

geographic areas and/or specific pathogens. In order to gather global ABR trends, data

regarding E. coli and K. pneumoniae exhibiting resistance to different antibiotic classes

were compiled from the different networks and articles available. Results from this

compilation are represented for different geographic regions in Figure 4 and Figure 5,

respectively.

RifampicinQuinolones

TrimethoprimSulphonamides

Beta-lactamsFosfomycin

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RifampicinQuinolones

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DNA-directed RNA polymerase

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DNA gyrase Cell-wall synthesis

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30S 50S

Aminoglycosides Tigecycicline Tetracyclines

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9

Regarding E. coli, the main current concern is the high rates of resistance observed

for third-generation cephalosporins and fluoroquinolones and the increasing resistance to

carbapenems (Figure 4). The rates of resistance to third-generation cephalosporins vary

according to the region analysed, from 47% for Asia-Pacific region to 12%-21% for the other

geographic regions in 2014. Resistance rates to fluoroquinolones vary from 22%-32%, being

also higher in Asia-Pacific region (43%). Carbapenem resistance among E. coli increased

since 2006, but the rates are still low (<3% in all world regions) (Figure 4). In Portugal,

resistance to third-generation cephalosporins among clinical invasive E. coli isolates

experienced an approximately 1.5-fold increase between 2006 and 2015 (10% to 16%), with

values above the European average. Resistance to fluoroquinolones is above 25% since

2006, with Portugal being among the ten European countries with higher resistance rates to

these antibiotics in 2015 (Figure 4)

(http://atlas.ecdc.europa.eu/public/index.aspx?Instance=GeneralAtlas).

Regarding K. pneumoniae, the scenario is even more worrying, with an increasing

expansion of resistance to third-generation cephalosporins and carbapenems (Figure 5).

Resistance to third-generation cephalosporins is much higher in this species in most

regions analysed, with the exception of Asia-Pacific region. Increasing trends have been

observed in Europe (from 19% in 2007 to 30% in 2015), contrasting with the situation in

North America (29% in 2006-07 to 19% in 2014). Regarding carbapenem resistance, rates

observed in Europe between 2006 and 2010 (5-8%) are almost exclusively influenced by the

situation in Greece, Italy and Cyprus, whereas after 2012 a massive increase was reported in

almost all European countries

(http://atlas.ecdc.europa.eu/public/index.aspx?Instance=GeneralAtlas). In North America,

a two-fold increase (5% to 11%) is observed between 2006 and 2010, being nowadays

around 8-9%, a situation similar to that of Asia-Pacific (10%) and Latin America (14%). In

Portugal, resistance to carbapenems emerged in 2007 (0,7%) and remained stable till 2013,

from which an approximately 2-fold increase (1,8% in 2013 to 3,4% in 2015) (Figure 5)

occurred, turning it the sixth country with the highest resistance rates to carbapenems in

Europe (although quite far from the rates reported for Greece-62%, Italy-34% and

Romania-25%) (3).

Regarding multidrug resistance phenotypes (combined resistance to third-

generation cephalosporins, fluoroquinolones and aminoglycosides), rates ranged from 2%

and 5% for E. coli, and 10% and 19% for K. pneumoniae, between 2006 and 2015 in Europe

(http://atlas.ecdc.europa.eu/public/index.aspx?Instance=GeneralAtlas), a similar scenario

also reported in the USA (ranging between 2% and 8% for E. coli and 13% to 16% for K.

pneumoniae, 2009-2014) (https://gis.cdc.gov/grasp/psa/MapView.html) (17).


10

1 Resistance mechanisms to antibiotics can be intrinsic (innate ability of a bacterial species to resist to the activity of a particular antimicrobial agent through its inherent structural or functional characteristics) or acquired [via mutations in chromosomal genes and/or via exogenous acquisition by horizontal gene transfer (HGT), mainly mediated by plasmids] (474).

BOX 2. Fosfomycin and Polymyxins – Mechanism of action, spectrum of activity and acquired resistance mechanisms1

1. Fosfomycin

• Mechanism of action: bactericidal antibiotic that links to the active site of MurA enzyme, preventing

the formation of N-acetylmuramic acid, an essential precursor for the synthesis of the bacterial wall (Figure 3) (18).

• Spectrum of activity: broad-spectrum activity, including Gram-positive and Gram-negative bacteria.

Commonly used in the treatment of low urinary tract infections (UTIs) (18).

• Acquired resistance mechanisms: decrease in permeability by chromosomal mutations affecting the transporters (GlpT, UhpT), enzymatic inactivation by plasmid-encoded glutathione S-transferases (mainly FosA3) or alterations in the target site MurA (the first two mechanisms

are common in Enterobacteriaceae) (18).

2. Polymyxins

• Mechanism of action: colistin (also known as polymyxin E) and other polymyxins (e.g. polymyxin B) are bactericidal antibiotics that attach to the outer membrane of Gram-negative bacteria leading to

disruption of membrane, and thereby promoting cell death (Figure 3) (12).

• Spectrum of activity: narrow-spectrum, as they are mainly active against common Gram-negative bacteria, such as E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., Salmonella spp.,

Shigella spp., Acinetobacter baumanni and P. aeruginosa. (12). Their use was abandoned in 1970

due to the high incidence of nephrotoxicity (12), but have been recently re-introduced as last resort

drug in the treatment of infections caused by MDR Gram-negative bacteria (12).

• Acquired resistance mechanisms: ü chromosomal mutations in genes encoding proteins involved in the PmrAB-PhoPQ two

component systems (e.g. mutations in pmrA, pmrB, phoQ and phoP genes), and the

corresponding regulators [e.g. mgrB gene, which regulates negatively the PhoPQ system and has

relevance in K. pneumoniae; crrAB involved in the regulation of the PmrAB system], which lead to

changes in the lipopolysaccharide (LPS) charges, making difficult the binding of polymyxins;

upregulation of capsular polysaccharide (CPS) production (described in K. pneumoniae);

ü plasmid-mediated colistin resistance genes (mcr-1, mcr-1.2, mcr-1.3 and mcr-2) encoding a phosphoethanolamine transferase that confers low to moderate polymyxin resistance [firstly

described in late 2015 and increasingly reported worldwide among Enterobacteriaceae strains

(mainly in E. coli) from human and animal origin] (12).


11

Resistance rates to fosfomycin [see Box 2 for details] are still low but increasing

lately among strains resistant to extended-spectrum β-lactams and especially in K.

pneumoniae. Regarding clinical isolates resistant to extended-spectrum β-lactams,

fosfomycin resistance rates vary between 0% and 19% for E. coli, with the lowest rates

observed for North (0-5%; 2009-2013) and Latin (2-3%; 2011-2013) America. For K.

pneumoniae, resistance rates diverge between 0% and 57% [Europe (9-54%, 2006-2013),

North America (14-38%, 2006-2010), Latin America (0-6%, 2010-2013) and Asia (5-57%,

2008-2013)] (11,19). The increasing identification of plasmid-mediated fosfomycin

resistance genes, mainly among E. coli from human clinical and non-clinical origins (Table

1) and also among animals, predominantly in Asian countries, is of remark, being more

likely to spread than chromosomal resistance mechanisms (11). Meanwhile, fosfomycin is

one of the antibiotics retaining good activity in other geographical regions, including in

isolates resistant to extended-spectrum β-lactams.

The increased use of polymyxins [see Box 2 for details], mainly colistin, to treat

infections caused by MDR E. coli and K. pneumoniae led to the emergence of polymyxin

resistance in several countries worldwide, particularly among carbapenemase-producing K.

pneumoniae. Since 2013, EARS-Net reports include data regarding resistance to

polymyxins for E. coli and K. pneumoniae, although not all countries report numbers. For

E. coli, resistance rates between 2013 and 2015 increased, although remain still low (0,5%

to 1%), whereas for K. pneumoniae resistance trends were superior and stable in the last

three years (8-9%), with higher rates (29-32%) being reported within carbapenem-resistant

K. pneumoniae in specific countries (Greece, Italy, Romania and Hungary) (3,20,21). The

scenario observed in other world regions is similar to the European one, with low rates of

colistin resistance among clinical E. coli and K. pneumoniae isolates without resistance to

extended-spectrum β-lactams [e.g. rates below 3% for E. coli and K. pneumoniae in North

America, Asia-Pacific and Latin American regions (2006-2010)] (22–25). Higher colistin

resistance rates are also observed among carbapenemase-producing K. pneumoniae in

particular countries of these world regions (e.g. 27,5% in Brazil in 2015; 17% in Taiwan in

2010 and 12% in 2012; 13% in the USA between 2011 and 2014) (26–28). Plasmid-mediated

colistin resistance [see Box 2 for details] has been described mainly in E. coli but also

among K. pneumoniae from clinical and non-clinical human origin, although its prevalence

still seems relatively low in these niches (12). However, the high-risk of further spread

mainly among carbapenemase producers is of extreme concern, since it will compromise

the clinical utility of colistin. For this reason, guidelines for the use of these antibiotics in

clinical setting and also in animals were revised by world health organizations (29,30).


12

TAB

LE 1

. St

udie

s re

port

ing

E. c

oli a

nd K

. pne

umon

iae

isol

ates

from

clin

ical

and

non

-clin

ical

hum

an o

rigi

n ha

rbou

ring

pla

smid

-med

iate

d fo

sfom

ycin

re

sist

ance

gen

es.

NR

, not

repo

rted

; 1 The

six

E. c

oli i

sola

tes w

ere

reco

vere

d fr

om th

e sa

me

patie

nt b

etw

een

2007

and

201

0.

Is

olat

ion

da

te

Cou

ntr

y So

urc

e Sp

ecie

s (n

o.)

Res

ista

nce

to

fosf

omyc

in

[%, (

no.)]

Pla

smid

-med

iate

d fo

sfom

ycin

re

sist

ance

gen

e (n

o.)

Ref

eren

ce

1996

-200

8 Ch

ina

Clin

ical

E.

col

i (18

78)

1,0

(18)

fo

sA2

(6)

fosA

5 (2

) (3

1)

2002

-200

7 Ja

pan

Clin

ical

E.

col

i (19

2)

3,6

(7)

fosA

3 (2

) fo

sC2

(1)

(32)

2007

-201

0 U

SA

Clin

ical

E.

col

i (6)

1 -

fosA

3 (5

) (3

3)

2008

Ch

ina

Clin

ical

E.

col

i (1)

-

fosA

5 (1

) (3

4)

2009

K

orea

Cl

inic

al

E. c

oli (

165)

K

. pne

umon

iae

(182

) 4,

8 (8

) 7,

1 (1

3)

fosA

3 (5

) fo

sA3

(2)

(35)

2009

-201

0 Ch

ina

Clin

ical

E.

col

i (11

09)

7,8

(86)

fo

sA3

(69)

(3

6)

2010

Ja

pan

Hea

lthy

volu

ntee

rs

E. c

oli (

138)

5,8

(8)

fosA

3 (5

) (3

7)

2006

-201

1 Ch

ina

Clin

ical

E.

col

i (1)

-

fosA

3 (1

) (3

8)

2010

-201

3 Ch

ina

Clin

ical

K

. pne

umon

iae

(278

) K

. pne

umon

iae

(80)

61

(169

) 13

(10)

fo

sA3

(94)

fo

sA3

(7)

(39)

2010

-201

4 Bo

livia

Cl

inic

al

E. c

oli (

170)

K

. pne

umon

iae

(19)

1,

8 (3

) 5,

3 (1

) fo

sA3

(3)

fosA

3 (1

) (4

0)

2010

-201

4 Ch

ina

Clin

ical

E.

col

i (46

5)

12,3

(57)

fo

sA3

(50)

fo

sA1

(1)

(41)

2011

-201

2 Ch

ina

Clin

ical

E.

col

i (4)

-

fosA

3 (2

) (4

2)

2012

Ch

ina

Clin

ical

E.

col

i (1)

-

fosA

3 (1

) (4

3)

2013

Ta

iwan

Cl

inic

al

E. c

oli (

123)

6,

5 (8

) fo

sA3

(1)

(44)

N

R

USA

Cl

inic

al

E. c

oli (

1)

- fo

sA6

(1)

(45)


13

1.1.2. Acquired resistance mechanisms to extended-spectrum β-lactams in Escherichia coli and Klebsiella pneumoniae: a current perspective

The β-lactam antibiotics [see Box 3 for details] were introduced in the clinical

practice in 1940, being one of the oldest and widely used classes of antimicrobial agents

(46). They are main therapeutic choices (mainly ESC and carbapenems) for the treatment of

nosocomial and community-acquired infections caused by Enterobacteriaceae isolates,

given their therapeutic efficacy, large spectrum of activity, low cost and low toxicity (46).

BOX 3. Mechanism of action, classification and spectrum of activity of β-lactam antibiotics

Mechanism of action: β-lactam antibiotics are characterized by the presence of a four-membered, nitrogen-

containing, β-lactam ring at the core of their structure. Their targets are the bacterial transpeptidases (PBPs-

penicillin binding proteins) that participate in the synthesis of peptidoglycan, the main component of the

bacterial cell wall. The β-lactam ring binds to these different PBPs, rendering them unable to perform their

role and leading to bacterial death by osmotic instability or autolysis (46). For these reasons, β-lactams are

considered bactericidal antibiotics.

According to their chemical structure, β-lactams can be divided into four different families: penicillins,

cephalosporins, carbapenems and monobactams (Figure 6).

FIGURE 6. Structure of different β-lactam antibiotic families [adapted from reference (47)].

Spectrum of activity:

• Narrow-spectrum – penicillins (e.g. benzylpenicillin, amoxicillin, ampicillin, ticarcillin, piperacillin, mecillinam), 1st-, 2nd-generation cephalosporins [e.g. cefazoline, cephalothin,

cefuroxime and cephamycins (e.g. cefoxitin)], monobactams (e.g. aztreonam, only active in Gram-

negative bacteria) – active against Gram-positive bacteria, moderate activity against Gram-

negative ;

• Extended-spectrum – 3rd-, 4th- and 5th-generation cephalosporins (e.g. ceftazidime, cefotaxime, cefepime, ceftaroline, ceftobiprole) and carbapenems (imipenem, ertapenem, meropenem,

doripenem)– acting on both Gram-negative and Gram-positive bacteria (46,47).


14

Acquired resistance (footnote page 10) to β-lactams might be mediated by different

mechanisms: alteration of the antibiotic target (common among Gram-positive bacteria),

increased activity of efflux pumps, changes in membrane permeability (mediated by

loss/alteration of porins) and antibiotic degradation by the production of β-lactamases (46).

The latter is, by far, the most frequent mechanism of resistance to β-lactams among Gram-

negative bacteria. β-lactamases are enzymes that are able to hydrolyse the β-lactam ring

leading to its inactivation (46). They can be classified based either on functional (Bush-

Jacoby-Medeiros classification, based on substrate and inhibitor profiles) or molecular

(Ambler classification scheme, based on amino acid sequence homology) properties, which

have been recently updated (Table 2) (46,48).

The first plasmid-mediated β-lactamases described in Enterobacteriaceae (TEM-1

in 1962 and SHV-1 in 1970s, both in E. coli) have the ability to hydrolyse narrow-spectrum

β-lactams [see Box 3 for details] and rapidly spread among members of Enterobacteriaceae

(particularly among E. coli) and to other bacterial hosts (e.g. N. gonorrhoeae, P.

aeruginosa) (46,49). In response to the increased prevalence of these narrow-spectrum β-

lactamases, new β-lactams, the ESC, were developed, but soon after, extended-spectrum β-

lactamases (ESBLs), able to hydrolyse these new compounds, emerged. Subsequently,

carbapenems, β-lactams with higher stability towards ESBLs were introduced in

therapeutics, being also observed soon after the emergence of carbapenemases (Figure 7).

The contemporary epidemiology (2006-2016) of resistance to extended-spectrum β-

lactams among E. coli and K. pneumoniae isolates causing human infections is dominated

by the predominance of ESBLs, and more recently by the emergence and rapid spread of

carbapenemases, especially among K. pneumoniae isolates. The resistance to extended-

spectrum β-lactams is not confined to the human setting, especially for E. coli where this

problem in non-human sources, mainly in food-producing animals, has been extended.

However, in this thesis the epidemiology review was limited to the human setting, since the

worrisome situation observed for K. pneumoniae is mainly related with humans, in

particular with the hospital setting. This section will provide a current perspective on the

occurrence and diversity of the different extended-spectrum acquired β-lactamases (ESBLs,

carbapenemases and plasmid-mediated AmpCs) circulating among E. coli and K.

pneumoniae from humans, covering epidemiologically related clinical settings [hospital,

long-term care facilities (LTCFs) and nursing homes (NHs)] and healthy volunteers.


15

TABLE 2. Functional and molecular classification of β-lactamases [adapted from (48)].

Functional Class1

Molecular Class2

Distinctive substrate(s)

Inhibition profile Representative examples

Classic β-lactamase inhibitors (e.g. CA or TZB)

EDTA

1 C Cephalosporins No No CMY-2, DHA-1, FOX-7, ACT-1

1e C Cephalosporins (increase activity against ESC)

No No CMY-37, GC1

2a A Penicillins Yes No PC1 2b A Penicillins, 1st- and

2nd-generation cephalosporins

Yes No TEM-1, TEM-2, SHV-1

2be A ESC, monobactams Yes No TEM-24, SHV-12, CTX-M-15, PER-1, VEB-1, GES-1

2br A Penicillins No No TEM-30 (IRT-2), SHV-10

2ber A ESC, monobactams No No TEM-50 (CMT-1)

2c A Carbenicillin Yes No PSE-1, CARB-3 2ce A Carbenicillin,

cefepime Yes No RTG-4

2d D Cloxacillin Variable No OXA-1, OXA-10 2de D ESC Variable No OXA-11, OXA-17

2df D Carbapenems Variable No OXA-48, OXA-181, OXA-232

2e A ESC Yes No CepA

2f A Carbapenems Variable No KPC-2, KPC-3 GES (some variants)

3a B All β-lactams except monobactams

No Yes VIM-1, NDM-1, IMP-1, SPM-1

3b B Carbapenems No Yes CphA, Sfh-1

CA, clavulanic acid; TZB, tazobactam; EDTA, ethylenediamine tetraacetic acid; ESC, Extended-spectrum

cephalosporins. The grey shading represents the groups of extended-spectrum acquired β-lactamases most

disseminated among Enterobacteriaceae and which will be focused on this section. 1According to Bush-Jacoby-Medeiros classification; 2According to Ambler classification - β-lactamases from

class A, C and D are serine β-lactamases (serine residue in the active site), whereas class B are metallo-β-lactamases (MBLs) (zinc atom in the active site).


16

FIGURE 7. Timeline representation of the introduction of β-lactams into clinical practice and

further sequential emergence of β-lactamases among Enterobacteriaceae. Note: The year in which each antibiotic was firstly introduced in the clinical practice, independent of the

country, is presented. aAvibactam is a novel non-β-lactam β-lactamase inhibitor able to retain activity in the presence of OXA-48 and

KPC-producing bacteria, which has been used in combination with ceftazidime in the treatment of

carbapenemase-producing Enterobacteriaceae infections (mainly complicated UTIs and intra-abdominal

infections). Until date low resistance levels have been reported (0-1%) (50).

A) Extended-spectrum β-lactamases (ESBLs)

ESBLs are a plasmid-mediated group of enzymes with ability to hydrolyse ESC and

monobactams but not cephamycins (cefoxitin) and carbapenems, and they are inhibited by

β-lactamase classical inhibitors (Table 2) (46). According to the classification schemes

above referred, ESBLs are distributed in classes A/2be and D/2de (Table 2). Different

families of ESBLs have been assigned according to their amino acid sequence, with those

belonging to TEM, SHV and CTX-M types (class A/2be) being worldwide predominant

among Enterobacteriaceae (46,51,52), whereas other families (PER, GES and VEB from

class A/2be; OXA from class D/2de) seem to be more confined to other Gram-negative

species (e.g. P. aeruginosa, A. baumannii) and/or specific geographic areas (53).

The first report of a plasmid-encoded ESBL occurred in 1983 in Germany in a

Klebsiella ozaenae isolate producing SHV-2 (differing from SHV-1 by one amino acid

mutation) (Figure 7) (54), followed by the emergence in 1985 of TEM-3 in a K. pneumoniae

(at that time named CTX-1, differing from the narrow-spectrum TEM-2 by two amino acid

1940

1950

1960

1970

1980

1990

2000

2010

Intr

oduc

tion

of t

he d

iffe

rent

β-l

acta

ms

Em

erge

nce

of r

esis

tanc

e

Peni

cilli

n (1

942)

Plasmid-mediated β-lactamases (1962) TEM-1

Sem

i-sy

nthe

tic

peni

cilli

ns

Ampi

cilli

n (1

962)

1st

gen

erat

ion

ceph

alos

pori

ns

(cep

halo

tin a

nd c

epha

lori

dine

– m

id 19

60s)

2nd g

ener

atio

n ce

phal

ospo

rins

(c

efom

ando

le-1

973,

cef

urox

ime-

1984

)

3rd g

ener

atio

n ce

phal

ospo

rins

(c

efot

axim

e -1

980,

cce

fatz

idim

e-19

84)

Extended-spectrum β-lactamases (ESBLs) (1983)

4th g

ener

atio

n ce

phal

ospo

rins

(c

efep

ime-

1994

)

Mon

obac

tam

s (a

ztre

onam

-198

6)

Inhi

bito

rs o

f β-l

acta

mas

es

(cla

vula

nic

acid

-198

4)

Car

bape

nem

s (I

mip

enem

-198

5)

Car

bape

nem

s

(Ert

apen

em-2

001)

Car

bape

nem

s

Mer

open

em -1

996)

5th g

ener

atio

n ce

phal

ospo

rins

(c

efbi

prol

e-20

08,

cefta

rolin

e - 2

010)

Plasmid-mediated AmpCs β-lactamases (CMY-1 - 1989)

Plasmid-encoded carbapenemases (IMP-1-metallo-β-lactamase- 1991 KPC-2-serine-β-lactamase - 1996)

ceft

azid

ime-

avib

acta

ma

( 201

5/20

16)


17

mutations) (55,56). The first CTX-M enzyme emerged in 1986 in a E. coli in Japan (57), and

different variants of this ESBL family rapidly spread worldwide. While TEM and SHV-

ESBLs were prevalent until the end of 1990s mainly among nosocomial K. pneumoniae

isolates, a shift was observed in the beginning of 2000 with the emergence and expansion of

CTX-M enzymes, mainly among E. coli isolates from nosocomial and community-acquired

infections. However, in the end of 2000s a new trend was observed with the worldwide

expansion of CTX-M and in a lesser extent SHV-ESBLs enzymes in K. pneumoniae of

clinical origin (51,52). Current distribution of TEM, SHV and CTX-M enzymes in isolates

from the hospital setting is detailed below (58,59).

Among the more than 220 variants of TEM enzymes described, at least 91 have been

defined as TEM-ESBLs (http://www.lahey.org/Studies/temtable.asp). From these, only a

few variants [e.g. TEM-3-like in France, Spain, Portugal, Bulgaria; TEM-4 in Spain; TEM-

10 in Portugal, United Kingdom, Argentina and the USA; TEM-24 in France, Spain,

Portugal, Belgium and Italy; TEM-26 in the USA; TEM-52 in different European countries

and Korea] were frequently identified amongst nosocomial K. pneumoniae (often

associated with clonal outbreaks) or E. coli isolates between the end of the 1980s and the

middle of 2000s (52,60–62). Nowadays, these enzymes seem to be residual in clinical

epidemiological surveillance studies (63–65), with the exception of TEM-52, which is still

being reported among E. coli from healthy humans (Table 4), and also from food-producing

animals (66).

From the more than 190 SHV variants identified, at least 46 are ESBLs

(http://www.lahey.org/Studies/). However, only few of them have successfully spread

worldwide (SHV-2, SHV-2a, SHV-5 and SHV-12), whereas others seem to be limited to

specific geographic areas (e.g. SHV-4 in France and the United Kingdom; SHV-7 in the

USA; SHV-55/-106 in Portugal) (52,60–62,67,68). Contrarily to the high prevalence of

TEM-ESBLs enzymes in European countries, SHV-ESBLs predominated among K.

pneumoniae and E. coli clinical isolates in Asia, Latin and North American countries,

between 1990 and middle 2000s (60–62). Currently, SHV-ESBLs (mainly SHV-2 and SHV-

12) are still frequently identified in many countries in isolates from different clinical

settings (Table 3) and from healthy volunteers (Table 4), mainly in K. pneumoniae,

sometimes concomitantly with the production of carbapenemases (mainly VIM-1 and

NDM-1) (67).

CTX-M enzymes were designated cefotaximases due to the preferential hydrolysis

over cefotaxime than ceftazidime observed in particular variants (51). Nevertheless, other

variants have been described with variable ability to hydrolyse cephalosporins and other β-

lactams (51,69,70). Currently, 170 CTX-M allelic variants have been recognized and

clustered into five main groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25)


18

(http://www.lahey.org/Studies/other.asp#table1) (51). Different CTX-M enzymes emerged

in distinct continents in the late 1980s till mid-1990s [CTX-M-3 in Asia in 1986, CTX-M-1

in Europe in 1989, CTX-M-2 in South America in 1990] (51). The selective pressure exerted

by the extensive use of ESC and other β-lactams, favoured CTX-M enzymes diversification

(69,70) and subsequent dispersion of specific variants in certain countries (e.g. CTX-M-1 -

Italy, Russia, The Netherlands; CTX-M-2 - Israel, Japan, South American countries; CTX-

M-3 - Bulgaria, China, Poland, Taiwan; CTX-M-9 - Japan, Spain, Taiwan, United Kingdom;

CTX-M-14 - Asian countries, Canada, Portugal, Spain, United Kingdom; and CTX-M-15 -

India, Italy, Portugal, Scandinavia, Spain, Turkey, United Kingdom, USA) (51,52). Finally,

since 2000 we assisted to an explosion in multiple settings of CTX-M β-lactamases

(especially CTX-M-15 and CTX-M-14 variants) among E. coli and more recently K.

pneumoniae, including in Portugal (51,52,62,68,71,72). Nevertheless, the distribution of

particular CTX-M enzymes among E. coli and K. pneumoniae is quite distinct. In E. coli

isolates, CTX-M-15 and CTX-M-14 are predominant and while CTX-M-15 is widely

distributed in humans but poorly in animals, CTX-M-14 is commonly reported in isolates

from humans, animals and the environment (51,72,73). Other CTX-M variants are still

being reported among E. coli but limited to specific regions (e.g. CTX-M-2 and CTX-M-8 in

South America; CTX-M-27 in Europe and Asia) (51,74–76). In K. pneumoniae, the

expansion of CTX-M (and specifically CTX-M-15) has been prominent mainly since 2005.

Other CTX-M variants have been only sporadically identified (with the exception of CTX-M-

14 frequently reported in Asian countries and Canada) (58,59,76,77). Other relevant

question is the interface between community and hospitals, with the number of studies

reporting ESBL-producing E. coli (mainly CTX-M) involved in community-acquired

infections being superior. However, increases in the detection of ESBL-producing K.

pneumoniae (mainly CTX-M-15 and SHV-12) involved in community-acquired infections

has also been recently observed in different regions (58,76).

LTCFs and NHs play a major role in contemporary societies due to the ageing of the

population. Residents are mainly elderly people living in community, often with multiple

comorbidities, which result in frequent hospitalizations, recurrent antibiotic consumption

and use of medical invasive devices. For these reasons, they are a highly susceptible

population for acquisition of infections by MDR bacteria and furthermore represent a high

risk for further spread within and outside these institutions (78). Colonization by ESBL-

producing Enterobacteriaceae in LTCFs or NHs residents varies according to the country

analyzed (rates between 9,2% and 64%, with most of the studies performed in European

countries) and species (most frequently in E. coli) (Table 3). Besides, CTX-M enzymes (and

mainly CTX-M-15 and CTX-M-14) are the predominant ESBL types detected in E. coli,

although some SHV-ESBLs (SHV-2, SHV-5 and SHV-12) are also detected in E. coli and K.


19

pneumoniae, reflecting current epidemiology in hospitals. It is of note to highlight that the

genotypic characterization of ESBLs among K. pneumoniae isolates is frequently not

performed (Table 3).

Different works have recognized the role of human intestinal flora as a reservoir of

Enterobacteriaceae producing ESBL (79). Most studies surveying the faecal flora of healthy

volunteers report variable rates of ESBL-producing E. coli, partly because some studies only

sought for E. coli, and also cause E. coli is more abundant than K. pneumoniae in the

intestinal flora of humans and, the composition of agar mediums used major favours the

growth of this species. Globally, the faecal carriage rates of ESBL-producing

Enterobacteriaceae greatly vary according to the world regions, with African and Asian

countries presenting the highest rates (Europe-0,6-6,7%; America-7,3-27%; Africa-5,7-59%

and Asia-6,4-50,6%). Although the dominance of ESBL-producing E. coli among healthy

volunteers, some studies report the presence of ESBL-producing K. pneumoniae in high

numbers, mainly in Asian and African countries (Table 4). Besides the predominance of

CTX-M enzymes (mainly CTX-M-groups 1 and 9) in both species (followed by SHV-ESBLs

and TEM-52), it is of note the high variability of CTX-M allelic variants observed for E. coli

isolates in healthy volunteers, which is not detected in K. pneumoniae (Table 4).


20

TAB

LE 3

. Stu

dies

cha

ract

eriz

ing

the

ESBL

-typ

es h

arbo

ured

by

E. c

oli a

nd K

. pne

umon

iae

isol

ates

col

oniz

ing

LTCF

s or N

Hs r

esid

ents

(200

6-16

).

ESBL

-E, E

SBL-

prod

ucin

g En

tero

bact

eria

ceae

; NI,

not i

dent

ified

. Ec,

E. c

oli,

Kp,

K. p

neum

onia

e; *

Oth

er E

nter

obac

teri

acea

e sp

ecie

s de

tect

ed. G

rey

shad

ing

repr

esen

t

the

stud

ies w

here

onl

y ES

BL-p

rodu

cing

E. c

oli w

ere

anal

ysed

. 1 Onl

y ES

BL-p

rodu

cing

E. c

oli a

nd E

SBL-

prod

ucin

g K

. pne

umon

iae

wer

e di

scri

min

ated

in th

is ta

ble.

TABL

E 1.

Stud

ies ch

arac

teriz

ing t

he E

SBL-

type

s har

bour

ed by

E. c

oli an

d K. p

neum

onia

e iso

lates

colon

izing

LTC

Fs or

NHs

resid

ents

(200

6-16

).

Cont

inen

t Co

untr

y/Ye

ar o

f is

olat

ion

Type

of

heal

thca

re

faci

lity (

no.)

No. o

f re

side

nts

stud

ied

Spec

imen

ty

pe

Occu

rren

ce o

f ES

BL-E

%

(no.

of sa

mpl

es)

Spec

ies

(no.)

1 ES

BL-ty

pe(s

) (no

.) Re

fere

nce

Euro

pe

Italy/

2006

LT

CF (2

3)

211

Cath

eter

urin

e 54

,0 (1

14)

Ec (4

3)

Kp (7

) *

CTX-

M (4

1), SH

V-5 (

1), SH

V-2 (

1) CT

X-M

(6),

TEM

-151 (

1) (8

0)

Ita

ly/20

08

LTCF

(1)

111

Recta

l swa

b an

d urin

e 64

,0 (7

1) Ec

(46)

Kp

(13)

*

CTX-

M-g

roup

1 (3

4), S

HV-5

(6),

CTX-

M-4

(1),

NI (5

) NI

(13)

(81)

Ita

ly/20

12

LTCF

(2)

106

Cath

eter

urin

e and

re

ctal s

wab

49,0

(52)

Ec

(56)

Kp

(10)

*

CTXM

-15 (4

5), C

TX-M

-9 (4

), SH

V-5/

12 (6

), TE

M-E

SBL

(1),

SHV-

12 (9

), CT

X-M

-15 (1

)

(82)

Ge

rman

y/20

06-0

7 NH

(8)

178

Recta

l swa

b 11

,2 (2

0)

Ec

CTX-

M-15

(8

3)

Ge

rman

y/20

10-11

NH

(11)

240

Recta

l swa

b 9,

2 (22

) Ec

(24)

Kp

(1)

CTX-

M-15

(15)

, CTX

-M-14

(5),

CTX-

M-1

(4)

CTX-

M-15

(8

4)

Ge

rman

y/20

13-14

NH

(26)

15

6 Re

ctal s

wab

14,7

(23)

Ec

(23)

CT

X-M

-15 (1

5), C

TX-M

-27 (

5), C

TX-M

-1 (2

), CT

X-M

-14 (1

)

(85)

Po

rtuga

l/200

8-12

NH

(7)

LTCF

(3)

322

Recta

l swa

b 24

,5 (7

9)

E. co

li (65

) Kp

(14)

* CT

X-M

-15 (3

1), C

TX-M

-32 (

1), O

ther

ESB

L (3

3)

CTX-

M gr

oup 1

(11),

Oth

er E

SBL

(3)

(86)

Ne

ther

lands

/201

2

NH (1

) 16

0 Re

ctal s

wab

20,6

(33)

Ec

(32)

Kp

(1)

CTX-

M-15

(22)

, CTX

-M-1

(5),

CTX-

M-3

(3),

CTX-

M- g

roup

9 (2

) NI

(87)

Amer

ica

USA/

2006

-07

LTCF

(1)

214

Recta

l swa

b 13

,0 (2

7)

Kp (2

3)

E. co

li (7)

SH

V-12

(17)

, SHV

-5 (5

) SH

V-31

(1),

SHV-

5 (6)

, CTX

-M-15

(1)

(88)

Asia

Ch

ina/

2014

NH

(7)

390

Recta

l swa

b 46

,5 (18

0)

Ec (1

67)

Kp (8

) *

CTX-

M-15

(73)

, CTX

-M-14

(68)

, CT

X-M

-27 (

8), C

TX-M

-24 (

7), C

TX-M

-3 (6

), CT

X-M

-69 (

5), C

TX-M

-3 (2

), CT

X-M

-132 (

2),

CTX-

M-6

4 (1),

CTX

-M-8

2 (1),

CTX

-M-12

3 (1),

CT

X-M

-65 (

1)

CTX-

M-3

(2),

CTX-

M-14

(2),

CTX-

M-6

5 (1),

CT

X-M

-15 (1

), CT

X-M

-22 (

1), SH

V-2 (

1)

(89)


21

TAB

LE 4

. Stu

dies

cha

ract

eriz

ing

the

ESBL

-typ

es h

arbo

ured

by

E. c

oli a

nd K

. pne

umon

iae

isol

ates

col

oniz

ing

heal

thy

volu

ntee

rs (2

006-

16).

Co

ntin

ent

Coun

try/

Year

of

isol

atio

n N

o. o

f pa

rtic

ipan

ts

incl

uded

ESBL

-E

(%, n

o. o

f sam

ples

) Sp

ecie

s (n

o.)1

ESBL

-typ

e(s)

(no.

) R

efer

ence

Euro

pe

Port

ugal

/200

1-04

11

3 1,8

(2)

Ec (2

) CT

X-M

-14

(1),

TEM

-153

(1)

(90)

Port

ugal

/200

7-08

11

2 2,

7 (3)

Ec

(3)

CTX-

M-1

(1),

TEM

-52

(1),

SHV-

12 (1

) (9

1)

Fr

ance

/200

6 33

2 0,

6 (2

) Ec

(2)

TEM

-52

(1),

SHV-

12 (1

) (9

2)

Fr

ance

/200

8 50

0 6,

6 (3

3)

Ec (2

6)

Kp (5

) *

CTX-

M-1

(7),

CTX-

M-1

5 (5

), CT

X-M

-14

(6),

CTX-

M-2

7 (1)

, CTX

-M-6

5 (2

), TE

M-5

2 (3

), TE

M-1

5 (1

), SH

V-12

(1)

CTX-

M-1

5 (4

), SH

V-2

(1)

(93)

Fr

ance

/201

1 34

5 6

(21)

Ec

(22)

CT

X-M

-15

(7),

CTX-

M-1

(7),

CTX-

M-1

4 (4

), CT

X-M

-2 (1

), SH

V-12

(3)

(94)

Sp

ain/

2007

10

5 6,

7 (7)

Ec

(7)

CTX-

M-1

4 (2

), CT

X-M

-1 (2

), CT

X-M

-32

(1),

CTX-

M-8

(1),

TEM

-52

(1)

(95)

D

enm

ark/

2008

84

3,

6 (3

) Ec

(3)

CTX-

M-1

4 (3

) (9

6)

H

unga

ry/2

009-

10

1102

2,

0 (2

2)

Ec (2

2)

CTX-

M-1

(6),

CTX-

M-1

5 (5

), CT

X-M

-8 (3

), CT

X-M

-2-g

roup

(2),

CTX-

M-9

-gro

up (1

), CT

X-M

-32

(1),

SHV-

12 (4

)

(97)

H

unga

ry/2

013-

14

779

2,3

(18)

Ec

(18)

CT

X-M

-15

(14)

, CTX

-M-1

(4)

(98)

Germ

any/

2009

-13

3344

6,

3 (2

11)

Ec (2

12)

CTX-

M-1

5 (9

7), C

TX-M

-1 (5

1), C

TX-M

-14

(31)

, CT

X-M

-3 (1

1), C

TX-M

-27 (

8), C

TX-M

-2 (2

) CT

X-M

-32

(1),

CTX-

M-5

5 (1

), SH

V-12

(5)

TEM

-52

(5)

(99)

Sw

itzer

land

/201

0 58

6 5,

8 (3

4)

Ec (3

4)

CTX-

M-1

5 (1

4), C

TX-M

-1 (1

0), C

TX-M

-14

(7),

CTX-

M-2

(2),

SHV-

12 (1

) (1

00)

N

ethe

rland

s/20

11

1033

4,

7 (49

) Ec

(51)

Kp

(2)

CTX-

M-1

(17)

, CTX

-M-1

5 (1

3), C

TX-M

-14

(9),

CTX-

M-2

(3),

CTX-

M-3

(2),

CTX-

M-2

4 (2

), CT

X-M

-27 (

1), C

TX-M

-32

(1),

SHV-

12 (2

), TE

M-5

2 (1

) CT

X-M

-15

(1),

SHV-

65 (1

)

(101

)

N

orwa

y/20

14-1

6 28

4 4,

9 (1

4)

Ec (1

4)

Kp (1

)

CTX-

M-1

5 (6

), CT

X-M

-1 (1

), CT

X-M

-55

(1),

CTX-

M-3

(2),

CTX-

M-2

7 (2)

, CTX

-M-1

4 (1

) CT

X-M

-24

(1)

SHV-

12 (1

)

(102

)

Asia

Ch

ina/

2009

10

9 50

,6 (5

5)

Ec (5

5)

CT

X-M

-14

(39)

, CTX

-M-1

5 (8

), CT

X-M

-55

(3),

CT

X-M

-79

(3),

CTX-

M-3

(2),

CTX-

M-2

4 (1

), CT

X-M

-27 (

1)

(103

)

2


22

ESBL

-E, E

SBL-

prod

ucin

g En

tero

bact

eria

ceae

; N

I, no

t id

entif

ied.

Ec,

E. c

oli,

Kp,

K. p

neum

onia

e; *

Oth

er E

SBL-

prod

ucin

g En

tero

bact

eria

ceae

spe

cies

wer

e al

so

iden

tifie

d. G

rey

shad

ing

repr

esen

t the

stu

dies

whe

re o

nly

ESBL

-pro

duci

ng E

. col

i wer

e an

alys

ed.1 O

nly

ESBL

-pro

duci

ng E

. col

i and

ESB

L-pr

oduc

ing

K. p

neum

onia

e

wer

e di

scri

min

ated

in th

is ta

ble.

2 Dat

a fr

om P

ortu

gal (

2001

-04)

wer

e in

clud

ed fo

r com

pari

son.

TA

BLE

4. S

tudi

es c

hara

cter

izin

g th

e ES

BL-t

ypes

har

bour

ed b

y E.

col

i and

K. p

neum

onia

e is

olat

es c

olon

izin

g he

alth

y vo

lunt

eers

(200

6-16

) (co

nt.).

Con

tine

nt

Cou

ntry

/Yea

r of

is

olat

ion

No.

of

part

icip

ants

in

clud

ed

ESB

L-E

(%

, no.

of s

ampl

es)

Spec

ies

(no.

)1

ESB

L-ty

pe(s

) (n

o.)

Ref

eren

ce

Asi

a (c

ont.)

Ch

ina/

2014

17

32

30,5

(528

) Ec

(528

) CT

X-M

-14

(258

), CT

X-M

-15

(155

), CT

X-M

-27

(50)

, CT

X-M

-65

(21)

, CTX

-M-3

(16)

, CTX

-M-6

4 (8

), CT

X-M

-55

(7),

CTX

-M-9

8 (5

), CT

X-M

-123

(5),

CTX

-M-1

05 (1

), CT

X-M

-132

(1),

CTX

-M-1

37 (1

)

(104

)

Ja

pan/

2009

-10

218

6,4

(14)

Ec

(12)

K

p (2

)

CTX

-M-1

4 (4

), CT

X-M

-2 (4

), CT

X-M

-8 (2

),

CTX

-M-1

5 (1

), SH

V-12

(1)

CTX

-M-3

(1),

CTX

-M-1

4 (1

)

(105

)

La

os/2

011

397

23,2

(92)

Ec

(78)

K

p (1

8)

CTX

-M-1

4 (3

6), C

TX-M

-55

(13)

, CTX

-M-1

5 (1

0),

CTX

-M-2

7 (9

), CT

X-M

-64

(5),

CTX

-M-2

4 (3

) CT

X-M

-101

(1)

SHV-

2a (1

3), C

TX-M

-14

(5)

(106

)

Le

bano

n/20

13

125

24,8

(31)

Ec

(25)

K

p (3

) *

CTX

-M-9

+CTX

-M-1

5 (1

0), C

TX-M

-9 (8

),

CTX

-M-1

5+CT

X-M

-2+C

TX-M

-9 (6

) O

ther

CTX

-M (1

) CT

X-M

-15

(1),

CTX

-M-9

(1),

CT

X-M

-15+

CTX

-M-2

+CTX

-M-9

(1)

(107

)

Am

eric

a Fr

ench

Gui

ana/

2006

16

3 7,

3 (1

2)

Ec (1

4)

CTX

-M-2

(11)

, CTX

-M-8

(1),

SHV-

2 (2

) (1

08)

Ch

ile/2

010

49

12,2

(6)

Ec (5

) CT

X-M

-1 (2

), CT

X-M

-15

(2),

CTX

-M-3

0 (1

) (1

09)

Bo

livia

and

Per

u/20

11

482

12,4

(60)

Ec

(6o)

CT

X-M

-15

(23)

, CTX

-M-3

(1),

CTX

-M-1

4 (4

) CT

X-M

-65

(20)

, CTX

-M-8

(7),

CTX

-M-2

(2)

CTX

-M-1

4+CT

X-M

-15

(1),

Oth

er E

SBL

(2)

(110

)

N

icar

agua

/201

2 10

0 27

(27)

Ec

(23)

K

p (5

) CT

X-M

-15

(20)

, CTX

-M-3

2 (1

), CT

X-M

-22

(1)

CTX

-M-1

5 (5

) (1

11)

Afr

ica

Liby

a/20

07

243

8,2

(49)

Ec

(4)

CTX

-M-1

5 (4

) (1

12)

Tu

nisi

a/20

09-1

0 15

0 7,

3 (1

1)

Ec (1

1)

CTX

-M-1

(10)

, TEM

-52

(1)

(113

)

Tuni

sia/

2012

-13

105

5,7

(6)

Ec (6

) CT

X-M

-1 (4

), CT

X-M

-15

(2)

(114

)

Mad

agas

car/

2009

48

4 10

,1 (4

9)

Ec (3

1)

Kp

(14)

*

CTX

-M-1

5 (2

8), C

TX-M

-3 (2

), CT

X-M

-1 (1

) CT

X-M

-15

(13)

, SH

V-2a

(1)

(115

)

Ca

mer

oon/

2009

15

0 6,

7 (1

0)

Ec (9

) K

p (1

) CT

X-M

-15

(10)

(1

16)

Ce

ntra

l Af

rica

n R

epub

lic/2

011-

13

134

59,0

(79)

Ec

(51)

K

p (2

0) *

CT

X-M

-15

(4),

CTX

-M-2

7 (1

), CT

X-M

-127

(1)

CTX

-M-1

5 (2

0)

(117

)

An

gola

/201

3 18

22

,4 (4

) Ec

(5)*

CT

X-M

-15

(5)

(118

)


23

B) Carbapenemases

Plasmid-mediated carbapenemases are a group of enzymes with ability to hydrolyse

almost all β-lactams, including carbapenems. They represent the most versatile family of β-

lactamases with a variable hydrolysis spectrum (mainly with respect ESC, monobactams

and β-lactamase inhibitors), which sometimes difficult their identification by phenotypic

methods (Table 5). According to the classification schemes for β-lactamases,

carbapenemases can be classified in three classes: A/2f, B/3a and D/2df (Table 2).

The inadequate and uncontrolled use of carbapenems to treat infections caused by

ESBL-producing Enterobacteriaceae seems to have favoured the recent emergence and

abrupt expansion of carbapenemases (Figure 7). The first carbapenemase identified in an

Enterobacteriaceae isolate was described in Japan in 1991 (IMP-1-producing Serratia

marcescens) (Figure 7) (119), and since then different carbapenemases have been identified

in different Enterobacteriaceae species, but especially in K. pneumoniae clinical isolates.

The most prevalent carbapenemases among Enterobacteriaceae are KPC (class A/2f),

MBLs (mainly plasmid-encoded NDM, VIM, IMP types; class B/3a) and OXA-48-like (class

D/2df) enzymes (Table 5).

TABLE 5. Hydrolytic and inhibition profiles of the most prevalent carbapenemases identified among Enterobacteriaceae.

aOXA-48-like enzymes usually show high-level of resistance to temocillin. bCephamycins are poorly hydrolysed

by most of class A enzymes. cVariable resistance to 3rd and 4th generation cephalosporins.

In a recent SMART (Study for Monitoring Antimicrobial Resistance Trends) study,

the distribution of carbapenemase genes among Enterobacteriaceae isolates recovered

from intra-abdominal infections and UTIs by country (2008-2014) was evaluated (120).

The study revealed a high prevalence of KPC producers in American and European

countries, NDM and IMP in Asia and the South Pacific region, OXA-48-like in African,

Middle East and European countries, and VIM mainly in European countries (Figure 8)

(120). In Portugal, KPC-3 producers were firstly detected in 2009 in a central hospital

Carbapenemase

Hydrolytic Profile Inhibitory profile

Penicillins Cephalosporins Monobactams Carbapenems classical β-lactamase inhibitors

in-vitro

1st and 2nd

3rd and 4th

KPC (A/2f)

+++ +++ b +++ ++ +++ ± Boronic acid

MBLs (B/3a)

+++ +++ +++ - ++ - EDTA

OXA-48-like (D/2df)

+++ a +++ ± c + + - NaCl


24

among K. pneumoniae isolates (121), and are currently widespread in different Portuguese

hospitals, mainly associated with K. pneumoniae isolates (19,122,123). Other

carbapenemases have been sporadically detected (KPC-2 in an environmental E. coli in

2010; VIM-1-like in environmental E. coli isolates in 2015; GES-5 in clinical K. pneumoniae

isolates between 2011 and 2012; OXA-48 in clinical K. pneumoniae and E. coli isolates

between 2013-2015) (122,124–126) (Peixe L, unpublished results). The occurrence and

distribution of the predominant carbapenemase families (KPC, OXA-48-like, and MBLs)

among K. pneumoniae and E. coli isolates is reviewed in detail below.

FIGURE 8. Distribution of different carbapenemases types among Enterobacteriaceae isolates by

world region (2008-14) [reprinted with permission from (120)]. a data from India and China were not included.

KPCs (Klebsiella pneumoniae carbapenemases) were firstly identified in a clinical K.

pneumoniae isolate in 1996 in North Carolina (USA) (127), and soon after were reported

worldwide, predominantly among K. pneumoniae isolates (Figure 9) (128). Twenty-three

KPC variants have been described, with KPC-2 and KPC-3 being the most commonly

reported (differing in just one amino acid), although with variable epidemiology

(http://www.lahey.org/Studies/other.asp#table1). In some countries, both variants are

endemic (USA, Colombia, Italy and Israel) among K. pneumoniae, whereas in others KPC-2

is almost exclusively found (Argentina, Brazil, Greece, Poland and China) (Figure 9)

(128,129).

KPC-producing bacteria have mainly been linked to the nosocomial setting but also

increasingly among LTCFs and NHs, either involved in infection or colonization of the

patients/residents, and rarely among healthy volunteers (130–133). Its detection in gowns


25

and gloves of healthcare workers, as well as in sink drains in the nosocomial setting,

suggests a major role of the hospital environment in the spread of KPC producers in

outbreak situations (134,135).

FIGURE 9. Worldwide geographic distribution of KPC-producing K. pneumoniae in clinical and non-clinical human settings [adapted from (129)].

OXA-48 carbapenemase (the name derives from oxacillinases due to the increased

ability to hydrolyse isoxazolylpenicillins faster than benzylpenicillin) was firstly identified in

a K. pneumoniae isolate in Istanbul, Turkey, in 2001 (136). Since its first description,

several variants of OXA-48 (differing by a few amino acid substitutions) have been

reported, such as OXA-162, OXA-163, OXA-181, OXA-204, OXA-232, OXA-244, OXA-245,

OXA-247 and OXA-370. However, the less efficient and slow hydrolysis observed for

carbapenems among these enzymes occasionally hampers their laboratory identification,

which possibly explains that their prevalence might be underestimated (129).

OXA-48-like enzymes have been reported worldwide among nosocomial K.

pneumoniae isolates and also frequently among E. coli, most of the times associated with

the co-production of CTX-M enzymes (mainly CTX-M-15). OXA-48-like-producing K.

pneumoniae are endemic in the Middle East (Turkey, Egypt), North African countries

(Tunisia, Morocco, and Libya) and India (mainly OXA-181) (Figure 10). The recent

emergence and subsequent spread of OXA-48-like-producing K. pneumoniae in other

countries (mainly European) has been mostly associated with population exchanges with

endemic areas. It is of note the high prevalence of OXA-48 among carbapenemase-

producing K. pneumoniae in Spain and France (>70%) (Figure 10) (129,137,138),

contrasting with the very low frequency of OXA-48-like-producing K. pneumoniae enzymes

among North American countries (120,129). OXA-48-like-producing E. coli reports are also

Endemic spread of KPC producers

Sporadic spread of KPC producers

KPC producers reported


26

increasing in endemic areas and some European countries (France, Spain and Germany)

(Figure 11) (19), and are more frequently observed among healthy volunteers (99,139,140).

FIGURE 10. Worldwide geographic distribution of OXA-48-like-producing K. pneumoniae in clinical and non-clinical human settings [adapted from (129)].

FIGURE 11. Worldwide geographic distribution of OXA-48-like-producing E. coli in clinical and non-clinical human settings (19,99,126,139,141–209).

Regarding plasmid-encoded MBLs, three different families are frequently

encountered among Enterobacteriaceae: VIM (Verona integron-encoded MBL), IMP (from

its hydrolytic activity on imipenem) and NDM (New Delhi MBL) (19,120,129).

Forty-six VIM enzymes have been described (most of them in P. aeruginosa)

(http://www.lahey.org/Studies/other.asp#table1), being VIM-1 and in lesser extent VIM-2

the most disseminated variants (mainly among K. pneumoniae and Enterobacter cloacae).

Endemic spread of OXA-48-like producers

Sporadic spread of OXA-48-like producers

OXA-48-like producers reported

1 2-10

11-50

51-150

>150

No. of isolates reported


27

In fact, VIM-1-producing K. pneumoniae are endemic in Greece and multiple outbreaks

have been reported in other European countries (Spain, Italy, France, Hungary, Belgium)

(19). Other variants are confined to specific geographic areas (e.g. VIM-4-producing K.

pneumoniae in clinical isolates from Hungary and Tunisia) (210,211).

IMP carbapenemases have been mostly reported among P. aeruginosa and

Acinetobacter spp. isolates worldwide, whereas reports among K. pneumoniae and E. coli

isolates are mainly confined to the Asia-Pacific region (120,212). From the 53 IMP enzymes

described, IMP-1, IMP-4, IMP-6, IMP-8 and IMP-26 are the ones more frequently detected

among K. pneumoniae and E. coli clinical isolates (212,213).

NDM enzymes were firstly described in 2008 simultaneously in a K. pneumoniae

and E. coli isolate from a Swedish patient previously hospitalized in New Delhi, India. Since

then, 16 NDM variants have been described (most often in Asian countries) but

undoubtedly NDM-1 is the most disseminated variant, followed by NDM-5

(http://www.lahey.org/Studies/other.asp#table1) (129). The Indian subcontinent (India,

Pakistan, Sri Lanka) seems to be primary reservoir of NDM producers, whereas other

possible significant reservoirs have also been discussed, such as the Balkan states and the

Arabian Peninsula (129). Additionally, a high prevalence has also been reported in different

European (United Kingdom, Poland, Denmark, France, Belgium) and Asian (China,

Singapore, Nepal) countries (Figure 12) (129,138,214). The international spread of NDM

producers has been strongly associated with travel to or medical tourism in endemic areas,

although cases of NDM producers in individuals with no history of travel to high risk areas

have also been reported (214,215). Although K. pneumoniae and E. coli are the main

sources of NDM-1 in hospital and community-acquired infections, respectively, multiple

other Enterobacteriaceae or non-fermenting Gram-negative species have been reported as

NDM-producers (214). NDM-producing K. pneumoniae and E. coli have been also reported

as colonizers of travellers and healthy humans, especially from endemic areas (140,216–

218). It is of note the extensive dissemination of this carbapenemase in non-clinical human

settings such as in animals (72) and in the environment, particularly in areas of poor

sanitation (219,220).


28

FIGURE 12. Worldwide geographic distribution of NDM-producing K. pneumoniae in clinical and non-clinical human settings [adapted from (129)].

C) Plasmid-mediated AmpC β-lactamases

Plasmid-mediated AmpC β-lactamases can hydrolyse penicillins, first-, second- and

third-generation cephalosporins, cephamycins (e.g. cefoxitin), and more rarely fourth

generation cephalosporins, while they are generally resistant to classical β-lactamase

inhibitors (221). AmpC enzymes are classified as C/1 or C/1e (Table 2) and belong to nine

different families (ACC, ACT, CFE, CMY, DHA, FOX, LAT, MIR, MOX)

(http://www.lahey.org/Studies/other.asp#table1), many of them encoded by the

chromosome of diverse Enterobacteriaceae species (e.g. E. coli, Shigella spp., Citrobacter

spp., Serratia spp., Hafnia alvei, Morganella spp., Enterobacter spp.). The first plasmid-

mediated AmpC β-lactamase (CMY-1) was isolated in 1989 in a K. pneumoniae strain from

Korea, and nowadays different variants have already been described in diverse

Enterobacteriaceae, including in species naturally lacking the chromosomal blaAmpC genes,

such as Klebsiella spp., Proteus mirabilis or Salmonella spp. (221).

More than 240 allelic variants of AmpC β-lactamases have been described

worldwide, but only a few are plasmid-mediated. CMY-2 is the most worldwide spread

plasmid-acquired AmpC, reported mostly in E. coli isolates (but also noticeable among

Salmonella spp.) from different clinical and non-clinical human settings and non-human

hosts (mainly food-producing animals) (66,71,72,221). DHA-1 is the second most frequent

plasmid-acquired AmpC and has been predominantly detected among nosocomial K.

pneumoniae isolates causing outbreaks worldwide (76,221), including in Portugal (222)

(Ribeiro T et al., unpublished results).

Endemic spread of NDM producers

Sporadic spread of NDM producers

NDM producers reported


29

1.1.3. Update on clinical relevance, virulence and population structure of Escherichia coli and Klebsiella pneumoniae isolates resistant to extended-spectrum β-lactams from clinical and non-clinical human settings

Enterobacteriaceae constitutes a large family of Gram-negative bacteria including

53 genera and more than 170 species. Members of Enterobacteriaceae family are facultative

anaerobic roads or coccobacilli, typically glucose fermenters, oxidase negative and non-

spore forming with variable mobility (223). Enterobacteriaceae species have an ubiquitous

distribution, being found amongst the intestinal flora of humans and animals and also in

different ecological niches such as plants, soil and water (223). Nevertheless, some species

from the Enterobacteriaceae family are commonly recognized as important human

pathogens, either associated with enteric diseases (E. coli, Salmonella enterica, Shigella

spp., Yersinia enterocolitica) or acting as opportunistic pathogens, involved mainly in

extraintestinal infections, especially UTIs, pneumonia or septicaemia (E. coli, K.

pneumoniae, Enterobacter spp., P. mirabilis and S. marcescens) (46,223).

As explained in section 1.1.1, the central problematic addressed in this thesis (the

expansion of MDR Enterobacteriaceae) is particularly relevant for E. coli and K.

pneumoniae. Established the differences in the incidence and impact of human infections

caused by MDR strains belonging to these species (see section 1.1.3) and in the

epidemiology of main plasmid-encoded ABR genes (see section 1.1.4), it is critical to deepen

aspects that are on the basis of clinical manifestations and of selection, emergence and

dissemination of ABR strains. Transmission of bla genes across bacterial populations is the

result of interplay between clonal expansion and/or HGT mediated more frequently by

plasmids, transposons or integrons (Figure 13). Details regarding the clinical relevance,

virulence and population structure will be reviewed in detail for E. coli (subsection 1.1.3.1)

and K. pneumoniae (subsection 1.1.3.2) separately. The impact of particular plasmid

families and specific mobile genetic elements (MGE) associations in the dissemination of

bla genes will be reviewed in section 1.1.4.


30

Clone A

Acquired resistance gene

Vertical gene transfer Horizontal gene transfer

Clone A

Clone A

Clone A Clone B

Conjugation, Transformation

Transduction

FIGURE 13. Mechanisms of dissemination of antibiotic resistance genes (adapted from http://evolution.berkeley.edu/evolibrary/news/161206_blackwidow).

1.1.3.1. Escherichia coli

Clinical Relevance

E. coli is the most prevalent commensal inhabitant of the gastrointestinal tract of

humans and animals among Enterobacteriaceae family, establishing a symbiotic

relationship with the host (224). The acquisition of certain MGE carrying virulence or ABR

genes capacitated E. coli to cause a broad spectrum of diseases, from gastroenteritis to

extraintestinal infections (223,224).

Pathogenic E. coli strains comprise two main subsets, diarrhoeagenic and

extraintestinal E. coli (ExPEC) strains. Diarrhoeagenic strains cause diarrheal syndromes

that vary in clinical manifestations and pathogenesis depending on distinctive virulence

traits, on the basis for discrimination of eight distinct pathovars - enteropathogenic E. coli

(EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive

E. coli (EIEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC), and

the recently proposed adherent-invasive E. coli (AIEC) and Shiga-toxin producing

enteroaggregative E. coli (STEAEC) (223,224). Regarding ExPEC, two pathovars have been

traditionally designated - uropathogenic E. coli (UPEC) and meningitis-associated E. coli

(MNEC). However, these categories are deceptively narrow since these strains have the

ability to cause infections in multiple extraintestinal anatomical sites, and it is more


31

acceptable the more embracing ExPEC term (225). ExPEC strains are linked to a variety of

diseases involving high morbidity and mortality and are strongly associated with ABR.

ExPEC are the most common cause of UTIs (responsible for approximately 70-95%

of community-acquired UTIs and 50% of nosocomial UTIs) and bacteraemia (involved in

47,4% of all bacteraemia reported in Europe in 2008) (226), but also neonates meningitis,

skin structure infections, myositis, osteomyelitis and respiratory tract infections (Figure 14)

in the hospital or community settings (226).

FIGURE 14. Proportion of ExPEC isolates identified among all bacterial pathogens isolated in

80.089 hospital admissions in 19 US hospitals (2007 and 2010) [reprinted with permission from

(226)].

Virulence factors of ExPEC strains

The ability of E. coli to cause disease in extraintestinal sites has been associated with

the presence of a diversified combination of virulence factors positively linked to enhanced

adaptability, competitiveness and ability to colonize the human body, and considered by

many authors as “fitness factors” (225,227). Many different combinations of virulence

factors can be found among ExPEC isolates as well as multiple representatives of a

particular virulence functional category (e.g. adhesins, siderophores), supporting diverse

pathways of extraintestinal virulence in E. coli (227). Variations in virulence profiles are

common even within the same lineage, evidencing the role of HGT [plasmids, phage-

mediated transduction, or recombination involving pathogenicity-associated islands (PAIs)]

or deletions, and the genome plasticity among ExPEC strains (225). Virulence factors

described among ExPEC strains, and, associations between some of them and site-specific

diseases are listed in Table 6 (227,228).


32

TABLE 6. Virulence factors associated with ExPEC by functional category and associations between some of them and specific clinical infections.

*Highly prevalent amongst patients with pyelonephritis; NM, neonatal meningitis. References used to construct Table 6 (224,225,228).

Functional Category

Gene(s) or operon

Description Clinical infection(s) commonly associated

Adhesins

fimH Type 1 fimbriae, D-mannose-specific adhesin UTI, NM papACEFG P fimbriae operon (pyelonephritis associated) UTI ecpA E. coli common pilus sfa S (sialic-acid specific) UTI foc F1C fimbriae UTI afa/dra Dr antigen-specific binding adhesins UTI afaE-8 Afimbrial adhesin VII Iha Adhesion siderophore bmaE Blood group M-specific adhesin gafD N-acetyl D-glucosamine-specific fimbriae adhesin clpG CS31A adhesin (K88 related) nfa Non-fimbrial adhesin csgA Curli fimbriae UTI hra Heat-resistant haemagglutinin

Toxins

hly α-haemolysin UTI* cnf1 Cytotoxic necrotizing factor 1 NM cdtB Cytolethal distending toxin sat Secreted autotransporter toxin UTI pic Serine protease autotransporter UTI vat Vacuolating toxin UTI astA Enteroaggregative E. coli heat-stable toxin

Siderophores

iuc, iutA Aerobactin siderophore (synthesis and receptor) UTI*, bacteraemia irp, fyuA Yersiniabactin siderophore (synthesis and receptor) UTI, bacteraemia iroN Salmochelin siderophore (receptor) UTI, bacteraemia ireA Siderophore receptor sitA Periplasmic-binding protein bacteraemia

Surface polysaccharides

kpsMT II Group II capsular polysaccharide synthesis (K1, K5, K12)

kpsMT III Group III capsular polysaccharide synthesis (K3, K10, K54)

K1 K1 group II capsule variants NM K2 K2 group II capsule variants K5 K5 group II capsule variants UTI, NM rfc O4 LPS synthesis UTI

Protectins and invasins

ompT Outer membrane protein T (protease) ompA Outer membrane protein A (cellular invasion) NM cvaC Colicin V (bacteriocin) clb Colibactin synthesis (bacteriocin) iss Increased serum survival UTI, bacteraemia traT Conjugal transfers surface exclusion protein UTI*, bacteraemia ibeABC Invasion of brain endothelium NM aslA Cellular invasion NM traJ Cellular invasion

Miscellaneous fliC H7 Flagellin variant UTI usp Uropathogenic-specific protein (bacteriocin) UTI malX Pathogenicity island marker


33

Population structure of ExPEC strains

A relationship between phylogeny [to review typing methods of E. coli isolates see

Box 4] and pathogenicity has traditionally been established for E. coli, in which virulent

BOX 4. Population structure concepts and methodologies applied in the typing and subtyping of E. coli

Clone: an isolate or a group of isolates descending from a common ancestor and exhibiting identical or closely similar phenotypic or genotypic traits. They can be detected and characterized by strain-typing

methods as belonging to the same group (229).

High-risk clones: clones with a global distribution and showing enhanced ability to colonize, spread, and

persist in a variety of niches (230). High-risk clones play a major role in the spread of different genetic

platforms, virulence and ABR genes at global scale (230,231).

Genotyping methods most commonly used to define the population structure of E. coli at different levels

are:

• Phylo-typing - Polymerase chain reaction (PCR)-based method that allows the classification of E. coli into seven main phylogenetic groups - A, B1, B2, C, D, E and F (232).

• Pulsed-field gel electrophoresis (PFGE) - based on the analysis of restriction fragments after digestion of genomic DNA with a macrorrestriction enzyme, usually XbaI in E. coli (and also K.

pneumoniae) (233,234).

• Multilocus sequence typing (MLST) - based on allelic variation in seven housekeeping genes to define a sequence type (ST) with a numerical designation, according to the most widely used and

standardized schemes - Achtman (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) and Pasteur

(http://bigsdb.web.pasteur.fr/ecoli/).

• Whole-genome sequencing (WGS) - determines the complete DNA sequence of a bacterial genome at a single time, providing a highest resolution and discriminatory power in phylogenetic

analysis (228,234).

Subtyping of E. coli B2-ST131 clone:

• Molecular serogrouping - PCR targeting the O25b and O16 rfb region (gene from LPS locus) (235).

• CH typing- sequence-based typing of fumC gene based on Achtman MLST scheme and of the internal fragment (489 bp) of fimH gene (236).

• Clade Rx identification - PCR detection of a specific single-nucleotide polymorphism (SNP) within the allantoin-encoding gene (ybbW) (237).

• Virulence factors and virotypes - PCR-based classification into five virotypes (i.e., A, B, C, D

and E) that is based on the distribution of eleven distinctive virulence factors (afa/draBC, iroN,

ibeE, sat, papGII, cnf1, hlyA papGIII, cdtB and neuC-K1) (238,239).

• WGS - phylogenetic analysis of the main lineages associated with ST131 and in silico fimH

determination (FimTyper, https://cge.cbs.dtu.dk/services/FimTyper/).


34

ExPEC strains were frequently linked to phylogroup B2 and to a lesser extent phylogroup D,

while commensal strains were more frequently associated with A and B1 phylogroups

(232,240). Phylogroups C, E and F were only recently discriminated by molecular tools and

their distribution is still underexplored (232). However, studies focusing on the

characterization of MDR E. coli populations have shown that these barriers are no longer

useful due to the increasing identification of A and B1 E. coli causing human infections

enriched with ExPEC virulence factors, unveiling the role of non-human reservoirs in the

landscape of ExPEC infections (240–242)

The pandemics caused by ESC resistant E. coli has mostly been attributed to the

global expansion of certain MDR high-risk clones, with the most representative example

being the E. coli B2-ST131 clone. This clone emerged during the early 2000s associated with

production of CTX-M-15, initially identified in the United Kingdom and Canada and

designated as clone A and 15A/15AR (by PFGE), respectively (243,244). But it was only in

2008 that international studies including isolates from several countries (Canada, India,

Kuwait, Lebanon, Korea, France, Switzerland, Portugal and Spain) recognized the

worldwide spread of a single E. coli clone producing CTX-M-15, identified by MLST as

ST131 (245,246). Since then, E. coli B2-ST131 was extensively reported throughout the

world (Figure 15) associated mainly with CTX-M-15 (but also with other ESBL,

carbapenemases and/or plasmid-acquired AmpC) and fluoroquinolone resistance in

isolates from multiple origins (73).

FIGURE 15. Global dissemination of E. coli B2-ST131 clone among clinical and non-clinical human

hosts (until 2013) [adapted from (73)]. Red stars indicate isolates producing ESBL enzymes, and blue stars indicate fluoroquinolone resistant, non-

ESBL-producing isolates.


35

Recent molecular epidemiology studies using WGS and deep phylogenetic analysis

[see Box 4 to review subtyping methods of B2-ST131 isolates] depicted several different

ST131 subclonal lineages linked to variable ABR patterns, and a retrospective phylogenetic

analysis allowed establishing their evolutionary routes. These clades had characteristic

fimH alleles and virulence gene profiles that are used for large-scale subtyping of ST131

isolates (247). It has been proposed that ST131 clone was rare among E. coli isolates from

the 1990s and early 2000s, and the main lineages identified belonged to O25b:H4 serotype

carried fimH22 and ibeA (virotype D) and fimH35 and were fluoroquinolone susceptible.

During the mid- to late 2000s, the explosion of O25b:H4 ST131 linked to fluoroquinolone

resistance was driven by the expansion of the fluoroquinolone resistant ST131-fimH30

clade carrying specific mutations in GyrA (S83L and D87N) and ParC (S80I and E84V)

harbouring (H30-Rx) or not (H30-R) CTX-M-15. ST131-fimH30 sublineages are generally

associated with virotypes A (afa/draBC presence), B (iroN presence) and C (sat presence),

being the latter the most common. The strong selective pressure exerted by the extensive

use of fluoroquinolones and furthermore of ESC might have contributed to the expansion of

these lineages (Figure 16) (73,248,249). It is of note to highlight the recent expansion of E.

coli B2-ST131 H30-R-producing CTX-M-27 in different countries, among clinical (Thailand,

Australia, Canada, Korea, Czech Republic, France and USA) (73,74) and non-clinical human

(China, France) sources (75,250). Other ST131 lineages, such as O16:H5 fimH41 represent

only 1-5% of ST131 isolates, are susceptible to fluoroquinolones but resistant to

trimethoprim-sulfamethoxazole and gentamicin and have been associated mainly with the

production of CTX-M-14 (247,251–253).

FIGURE 16. Evolution of E. coli ST131 lineages driven by selection pressure due to

fluoroquinolones and ESC use [adapted from (249)]. FQ-R, fluoroquinolone resistant.


36

Due to the extraordinary global success of ST131, carbapenemase production has

been monitored and fortunately only a limited number of isolates have been detected.

However, a recent study from the USA documents twenty KPC-producing E. coli, 60% of

them belonging to the ST131 H30 lineage, whereas another global surveillance report from

SMART and Astra Zeneca program reports 116 carbapenemase-producing E. coli, 35% of

them producing KPC enzymes and belonging to the ST131-H30 lineage and virotype C

(254,255).

E. coli B2-ST131 clone is highly prevalent among residents in LTCFs/NHs, with

these settings regarded as reservoirs of this clone [colonization rates ranging between 4,6%

and 55% in different studies (2006-2014)] (82,84,86,87,256–259). E. coli B2-ST131 has

also been increasingly detected among healthy humans [colonization rates ranging between

0,5% and 29% (2006-2014)] (75,94,101,103,117). Despite it has occasionally been detected

in non-human sources (companion animals and food-producing animals, environment)

(73), its higher prevalence among human infections and human colonizing isolates,

probably reflects its primarily adaptation to the human host.

Other important ExPEC lineages have been detected worldwide in clinical isolates,

also linked to the expansion of CTX-M enzymes and/or carbapenemases. Other B2-E. coli

lineages which are frequently MDR but only occasionally linked to the production of β-

lactamases, are also highly represented worldwide among clinical ExPEC isolates, such as

ST73 and ST95 (228,242,260,261). From phylogroup D, the most representative clones

among clinical isolates are ST38 (mainly associated with production of CTX-M-9, -14, and

OXA-48) (162,184,262–267), ST69 (usually presenting high rates of resistance to

trimethoprim-sulfamethoxazole and frequently linked to the production of CTX-M-14)

(242,262,264,268,269), ST393 (mainly associated with CTX-M-14) (242,268) and ST405

(linked to the worldwide expansion of CTX-M-15 and also associated with NDM and OXA-

48) (137,263–267,270,271). From B1 phylogenetic group, ST101 (mostly linked to NDM-1

outbreaks) is widely distributed (272,273), whereas in phylogroup A, CC10 (including ST10,

ST167 or ST617) commonly producing different CTX-Ms (65,263,270,274) and ST410

(associated with CTX-M-15, and less frequently with CTX-M-14, KPC and NDM) have been

frequently reported (274–277). E. coli F-ST648, formerly classified as phylogroup D, is

mainly associated with production of CTX-M-15 and different NDM variants among clinical

isolates worldwide (241,263,267,278).

It is important to highlight the frequent detection of D-ST38, A-ST410, A-CC10 and

F-ST648 clones among non-human hosts (domestic, companion and food-producing

animals, food-products and environment), and the high identity observed between these

clones in these different settings, suggesting the role of these settings as reservoirs of


37

ExPEC lineages, especially trough the food chain. This aspect is out of the scope of this

thesis but it is very nicely reviewed in several papers (72,240,241).


38

1.1.3.2. Klebsiella pneumoniae

Clinical Relevance

K. pneumoniae is an encapsulated and non-flagellated bacterium frequently

encountered colonizing diverse animals and sites of the human body (intestinal and

respiratory tract, nasopharynx, oropharynx and skin), water, soil and plants (279,280).

Recently, K. pneumoniae sensu latu underwent an important taxonomic

restructuration. The populations previously assigned to phylogenetic groups KpI, KpII and

KpIII were recognized as belonging to three distinct species, namely K. pneumoniae, K.

quasipneumoniae and K. variicola, respectively. In turn, K. pneumoniae comprises three

subspecies: K. pneumoniae subsp. pneumoniae (K. pneumoniae sensu stricto), K.

pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis (Figure 17).

Whereas the latter are rarer and associated with specific diseases (rhinoscleroma and

ozena, respectively), K. pneumoniae subsp. pneumoniae is by far the subspecies with the

highest clinical relevance, being responsible for a wide range of infections worldwide and

will be the focus of this subsection (281–283).

FIGURE 17. Taxonomic restructuration of K. pneumoniae.

In the last years, K. pneumoniae gained notoriety due to the alarming rates of

isolates involved in hospital- (mainly pneumonia, UTIs, intra-abdominal and wound

infections or bacteraemia) and community-acquired (pyogenic liver abscess, meningitis,

pneumonia and UTIs) infections, increasingly associated with multidrug resistance

phenotypes (see sections 1.1.1 and 1.1.2) (2,280). K. pneumoniae population is linked to

different clinical syndromes and is asymmetrically distributed in human populations. While

MDR strains are most frequently linked to hospital and community-acquired infections

such as UTIs, pneumonia and bacteraemia and are worldwide distributed, the hypervirulent

Klebsiella pneumoniae sensu lato

KpI

Klebsiella pneumoniae (chromosomal blaSHV)

K. pneumoniae subsp. pneumoniae

(Klebsiella pneumoniae sensu stricto)

K. pneumoniae subsp.

rhinoscleromatisK. pneumoniae subsp. ozaenae

KpII

Klebsiella quasipneumoniae (chromosomal blaOKP)

K. quasipneumoniae

subsp. similipneumoniae

K. quasipneumoniae

subsp. quasipneumoniae

KpIII

Klebsiella variicola (chromosomal blaLEN)


39

strains are associated with community-acquired invasive infections such as pyogenic liver

abscess and are more prevalent in Asian countries and involving mainly young and healthy

individuals) causing diseases (280,284,285).

K. pneumoniae is the second or third (depending on the country) most frequent

agent of UTI, accounting for 2-7% of nosocomial and community-acquired UTIs. A large

proportion of these infections is caused by MDR strains, and often involve clinical

complications, such as bacteraemia, due to therapeutic failures (286). K. pneumoniae

strains (and particularly MDR) are also frequent agents of hospital-acquired pneumonia

(HAP, 10-12%) (287,288). Community-acquired pneumonia (CAP) is infrequently

associated with K. pneumoniae strains at least in North American and European countries

(approximately 3-5%), whereas in the Asia-Pacific and African countries the rates are

higher (around 15%) mainly due to a higher prevalence of hypervirulent strains in these

regions (289,290). It is of interest to highlight the description of a few hypervirulent strains

harbouring blaCTX-M and other genes conferring resistance to aminoglycosides and

fluoroquinolones, glimpsing a darker scenario with the emergence of strains combining

virulence and multidrug resistance features (284,291).

Virulence factors among K. pneumoniae strains

In comparison to ExPEC strains, a fewer number of virulence factors are recognized

in K. pneumoniae strains and a significant part of them are associated with the

hypervirulent population (Figure 18). K. pneumoniae presents a variety of virulence factors

associated with the ability to colonize the human body and to evade from host immune

system during infection, namely capsule polysaccharide, LPS, adhesins (type 1 and 3

fimbriae), iron acquisition structures (siderophores and iron uptake systems) and nitrogen

source utilization.

FIGURE 18. Well-characterized virulence factors among K. pneumoniae strains and differences among MDR and hypervirulent strains [adapted from (286)].

MDR K. pneumoniae

Hypervirulent K. pneumoniae


40

The capsule is an extracellular acidic polysaccharide matrix that envelops the

bacteria and has been recognized as a major virulence factor, with 77 capsular types

phenotypically recognized until date (279). Capsule protects bacteria from phagocytosis and

opsonophagocytosis by immune cells, confers resistance to the bactericidal action of

antimicrobial peptides (e.g. human defensins 1 to 3 and lactoferrin), blocks the action of the

complement and leads to the suppression of early inflammatory responses (286,292,293).

Serotyping was crucial to allow the identification of specific capsular types associated with

hypervirulent K. pneumoniae strains (mainly K1 and K2, and to a lesser extent K5, K20,

K54). These capsular types are linked to the expression of two plasmid-born transcriptional

regulators - rmpA and rmpA2 (regulator of mucoid phenotype) enhancing capsule

production, which seem to play a major role in the ability of hypervirulent K. pneumoniae

to cause invasive infections (283–285,294). Regarding MDR K. pneumoniae isolates, the

recent availability of molecular tools for CPS characterization were crucial to unveil a

greater diversity of capsular types than that previously recognized (284,285).

The LPS is composed by three parts - the highly conserved and hydrophobic lipid A

attached to the outer membrane; the variable O-antigen (9 antigens recognized until date)

as the outermost component of LPS; and the core polysaccharide connecting lipid A and O-

antigen (286,295). LPS plays a double role in the K. pneumoniae infection, it protects

against humoral defenses and is a strong activator of the immune system (mainly lipid A).

To avoid the immune system activation, some strains express capsular types that mask their

own LPS, whereas others modify the LPS (mainly the lipid A structure) to a form that is no

longer recognized by the immune system receptors, increasing the virulence of K.

pneumoniae. Moreover, strains with full-length (“smooth LPS”) O-antigen are resistant to

complement-mediated killing, whereas strains presenting a truncated or absent O-antigen

(“rough LPS”) are more susceptible to the action of the complement (286,293).

Fimbriae are also well-recognized virulence factors among K. pneumoniae strains

due to its major role in K. pneumoniae adhesion. Type 1 and 3 fimbriae are the major

adhesive structures that have been characterized as pathogenicity factors in K. pneumoniae

strains. Type 1 fimbriae (fim cluster) projects beyond the capsule mediating adhesion to

mannose-containing structures on host cells or to abiotic surfaces (but not the attachment

to intestine or the lung) and are important in the adhesion to uroepithelial cells and initial

establishment of UTI (286,293). Type 3 fimbriae (mrk cluster) have the ability to link to the

extracellular matrix in tissues and indwelling devices and, as type 1 fimbriae, both promote

biofilm formation. Type 1 and 3 fimbriae are highly prevalent among MDR isolates, but not

in hypervirulent K. pneumoniae (296).

Iron is essential for bacterial growth, and different iron-uptake systems have been

described among K. pneumoniae strains. Siderophores (enterobactin, yersiniabactin,


41

aerobactin and salmochelin) and ABC transporters (Kfu system) are the most commonly

implicated in K. pneumoniae virulence. The number of different siderophores produced

seems to be related with an increased ability of K. pneumoniae to colonize and spread to

different sites in the host, with niche specific roles for each siderophore (286,292,293). In

fact, there is a strong association between hypervirulent K. pneumoniae strains and the

expression of yersiniabactin and aerobactin (both expressed during lung infection),

salmochelin (enhances nasopharyngeal colonization) and Kfu system. However, some

studies have recently pointed out a high presence of yersiniabactin and Kfu among MDR K.

pneumoniae strains, which is worrisome, as mentioned above (283–285,294).

Other virulence factor mostly associated with hypervirulent K. pneumoniae K1

strains is the allantoinase cluster, which is involved in the ability to compete for allantoin as

a nitrogen and carbon source in mammalian hosts (286,292,293). Production of urease

(enzyme responsible for hydrolyzing urea to ammonia and carbamate) has been suggested

as a virulence factor. Urea hydrolysis leads to a localized increase in pH, resulting in the

precipitation of inorganic salts. This precipitation leads to encrustation, particularly on

indwelling urinary catheters, which may influence the development of infection. However

more studies are needed to clarify this aspect (292).

Population structure of MDR K. pneumoniae strains

Recent studies suggested an evolutionary radiation among K. pneumoniae and the

identification of distinct clonal groups (CGs; a clonal group includes one central genotype,

its single-locus variants (first circle SLVs) and the SLVs of the first-circle SLVs) among

hypervirulent and MDR K. pneumoniae strains [see Box 5 to review typing methods of K.

BOX 5. Methodologies applied in the typing and subtyping of K. pneumoniae

Genotyping methods most commonly used to define the population structure of K. pneumoniae are:

• PFGE [see Box 4 for details regarding the methodology].

• MLST [see Box 4 for details regarding the methodology] - only Pasteur scheme is available for K.

pneumoniae strains (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html).

• WGS [see Box 4 for details regarding the methodology] - two core genome MLST schemes

(cgMLST) are available for K. pneumoniae isolates (284,297)

(http://bigsdb.pasteur.fr/perl/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef_public&page=sequ

enceQuery), although phylogenetic analysis using SNPs have also been used (302,317).

• Molecular capsular typing - PCR and sequencing of wzi and wzc genes located in cps locus, or of

wzy for specific capsular types (298,299).

• Molecular determination of LPS O-antigen - PCR-based method covering the different O-types recognized (295).


42

pneumoniae strains] (284). Similarity to what happened in E. coli, the increase on MDR K.

pneumoniae strains occurred at the expense of the expansion of strains belonging to a

reduced number of high-risk CGs, such as CG14, CG15, CG17, CG101, CG147 and CG258.

They are reported worldwide and linked to the spread of ESBL and carbapenemases, with

specific associations being reported (e.g. CG15 and CTX-M-15; CG258 and KPC; CG101 and

OXA-48) (284,300–302).

K. pneumoniae CG258 is one of the most well-studied CGs among this species. This

CG includes different STs, being ST258, ST11, ST340 and ST512 the more frequent ones

(284). ST258 has been the main responsible for the worldwide expansion of KPC enzymes,

being endemic in different world regions (USA, Israel, Italy, Greece, Poland, Colombia,

Argentina and Brazil) (128). Characterization of large collections of CG258 isolates (due to

the problematic of KPC production) by WGS studies allowed insights into the evolution and

intraclonal diversity of this CG. These studies suggest that ST258 is a hybrid strain that

results from a first large recombination event between ST11 and ST442, with acquisition of

the cps locus of ST442 giving rise to ST258-clade II (wzi154), and a second event involving

the horizontal transfer of the cps locus from ST42 to ST258 resulting in ST259-clade I

(wzi29/K41). These two well-defined ST258 clades have been associated with different KPC

variants, clade I to KPC-2 and clade II to KPC-3 (301,303). Though pioneers in the

delineation of particular MDR K. pneumoniae lineages within certain groups defined

previously by MLST, available studies are biased towards CG258 and other relevant CGs

have been underexplored (301,304). Other STs from CG258 have also spread worldwide

linked to the production of different β-lactamases. ST11 has been associated with different

carbapenemases (KPC, NDM, OXA-48-like, VIM, IMP), ESBLs (mainly CTX-M-15 and

CTX-M-3) and plasmid-acquired AmpCs (mainly DHA-1) (77,128,211,305–310) (Ribeiro T

et al., unpublished results). The other members of CG258 (ST512, ST340) have mainly been

associated with KPC production in different countries (128), although some reports of

NDM- and VIM-producing K. pneumoniae ST340 have also been described (305,306).

K. pneumoniae CG14 and CG15 (central genotype ST14 and ST15, respectively) have

been described worldwide as involved in nosocomial and community-acquired infections

and multiple outbreaks. K. pneumoniae CG14 has been linked mainly to the expansion of

different carbapenemases (NDM-1, KPC, OXA-48-like), sometimes concomitantly with

CTX-M-15 production (162,311–313), or only with ESBL (310,314). On the other hand, K.

pneumoniae CG15 has been associated mostly with the worldwide expansion of CTX-M-15

(77,315–318), but also with different carbapenemases (NDM, VIM, OXA-48-like, KPC) with

or without production of CTX-M-15 or SHV-ESBLs (162,210,306,319,320).

K. pneumoniae CG147 isolates (central genotype ST147) have been linked to the

expansion of different carbapenemases (VIM, KPC, NDM, OXA-48-like) mainly in


43

European countries, most of the times simultaneously with different ESBLs, and less

frequently only with ESBL production (64,213,311,315,321). Regarding K. pneumoniae

CG101 (central genotype ST1o1), multiple outbreaks mainly linked to the production of

CTX-M-15, OXA-48-like plus CTX-M-15 and KPC have been reported (162,322–325). K.

pneumoniae CG20 (central genotype is ST20, includes three STs widely spread - ST16, ST17

and ST20) have been involved in the expansion of CTX-M-15, NDM, OXA-48-like and

DHA-1 (326–329).

The faecal carriage of carbapenemase-producing K. pneumoniae among hospitalized

or LTCFs patients by some of these CGs (CG15, CG20, CG147 CG258) is worrisome, due to

the risk of further development of an infection (330,331). Moreover, the recent

demonstration that K. pneumoniae strains causing hospital infections often corresponds to

the patient own colonizing isolates stresses the need to survey MDR bacteria in different

clinical settings, such as hospitals and LTCFs, mainly in endemic areas. This surveillance is

important to avoid the evolution for infection and also to adopt effective control measures

to prevent the spread of these high-risk clones (332).


44

1.1.4. Mobile genetic elements involved in the spread of bla genes among Escherichia coli and Klebsiella pneumoniae

In this section, an overview of main plasmid families and other MGE [transposons,

insertion sequences (IS)] [see Box 6 for fundamental concepts regarding mobile and

mobilizable genetic elements] linked to carriage of bla genes and multidrug resistance

phenotypes in E. coli and K. pneumoniae (Table 7), and plasmid classification methods will

be presented.

MGE are fundamental in the adaptation of bacterial cells to environmental

conditions, but they are also crucial in the dissemination and persistence of ABR genes

among bacterial populations (333,334). MGE can be generally divided in two types. The

first type includes elements that can move from one genetic location to another in the same

cell (e.g. from the chromosome to a plasmid or between plasmids), such as transposons and

IS. The second type comprise elements such as plasmids and transposons with ability to

transfer from one bacteria to another via conjugation (transfers of genetic material from

donor to recipients cells through bacterial pilus), transformation (uptake of free DNA) or

transduction (via bacteriophages) (Figure 19) (334).

FIGURE 19. The modular and hierarchical composition of different mobile and mobilizable genetic elements [reprinted with permission from (335)].

Level 1

Level 2

Level 3

Level 4

genecassette

integron

transposon

conjugativeplasmid

insertionsequence


45

BOX 6. Concepts and definitions related to mobile and mobilizable genetic elements

Gene cassettes - small mobilizable elements comprising one gene (frequently encoding ABR) and a

recombination site (attC) (336).

Integrons - are site-specific recombination systems able to capture and express gene cassettes. Several gene

cassettes can be integrated originating multiple gene cassette arrays. Integrons comprise three key elements:

a gene encoding an integrase (intI), a primary recombination site (attI) and a promoter (Pc) that directs the

transcription of the captured genes. According to the integrase sequence (int) they can be classified into five

different classes (336,337). Class 1 integrons (the most widespread in Enterobacteriaceae) are defective for self-transposition and their dissemination occurs through association with transposons (mostly Tn3/Tn21

family) and/or plasmids (336).

Transposable elements - comprise insertion sequences (IS) and transposons.

Insertion sequences (IS) - correspond to the simplest transposable element, consisting on a gene

codifying for a transposase, usually flanked by inverted repeats (IR) sequences (single stranded sequence of

nucleotides followed downstream by its reverse complement). IS are able to move from one location to

another in the genome, and they can exist as autonomous or as part of class I transposons (338,339).

Transposons - according with the mechanism of transposition, transposons in Enterobacteriaceae can be

assigned to four different classes, being class I (composite transposons), II and conjugative transposons the most frequently found.

• Class I or composite transposons comprise two copies of the same IS (or two closely related ISs)

flanking a variable number of genes (generally ABR genes) (334).

• Class II transposons include genes coding for a transposase (tnpA, enzyme involved in excision and integration) and resolvase (tnpR, a site-specific recombinase for co-integrate resolution within the

res site), and one variable DNA fragment flanked by two IR sequences. According with the

orientation of tnpA and tnpR, two subtypes can be defined: Tn21 (tnpA and tnpR are transcribed in

opposite directions) and Tn3 subfamily (tnpA and tnpR are transcribed in same direction). The transposition mechanism is replicative (after transposition both donor and recipient have a copy of

the genetic unit) (333).

• Tn5053/Tn402 family includes a transposition module formed by four different genes - transposase genes [tniA, tniB and tniQ (or tniD)] separated from a resolvase gene [tniR (or tniC)] by a res site.

The mechanism of transposition is replicative and it’s a two-step process (cointegration formation

and resolution in res site) (338).

• Conjugative transposons (CTns) are very heterogeneous self-transmissible elements, also know as CONSTIN (conjugative, self-transmissible, integrating element) or ICEs (integrative and

conjugative elements), that move from the chromosome of a donor bacteria to the chromosome (or

less frequently to plasmids) of a recipient bacteria (340).

Genomic islands (GIs) - are large chromosomal regions that can be mobilized by plasmids, CTns or

phages. GIs are flanked by direct perfect repeats and usually carry mobility genes coding for integrases or

transposases that are needed for chromosomal integration and excision, and also other additional genes

codifying for adaptive features. GIs may derive from ICEs that for different causes are defective of self-transfer (341).


46

BOX 6 (cont.). Concepts and definitions related to mobile and mobilizable genetic elements

Plasmids - are extra chromosomal genetic units able to replicate autonomously in a given host bacterial cell,

and which can be transmitted to other cells by conjugation, transformation or transduction. Plasmids

comprise genes coding for essential (replication, maintenance and transfer) and adaptive (e.g. ABR genes,

metal tolerance, virulence) functions (335). They can be classified according to the number of copies, host range, replication (incompatibility group) and mobility. According to the mobility, plasmids can be classified

as conjugative or mobilizable. The mobility machinery includes a set of plasmid mobility (MOB) genes [oriT,

relaxase and type IV coupling proteins (T4CP)] necessary for the conjugative process, and a mating pair

formation (MPF) complex which is a type 4 secretion system (T4SS) (Figure 20) (342). Conjugative plasmids (e.g. IncF, IncN, IncA/C) encode the functions needed for their own transfer (MOB and MPF),

replication and stability, and are generally large (>30 kb) and in low copy numbers. They can be classified

according to the plasmid incompatibility group (Inc) [PCR-based replicon typing (PBRT)] and also according

to their relaxases (Degenerate Primer MOB Typing) (342–344). By definition two plasmids that belong to the

same Inc group cannot stably coexist in the same cell. Mobilizable plasmids (e.g. ColE1; IncQ) use the T4SS

of another genetic element present in the cell to be transferred, are usually smaller and present in high copy

numbers (Figure 20) (342).

Transfer

Conjugative Plasmid

Mobilizable Plasmid

FIGURE 20. Genetic organization of a conjugative and a mobilizable plasmid [adapted from (342)].


47

TAB

LE 7

. M

GE

asso

ciat

ed w

ith t

he m

ost

wid

espr

ead

bla

gene

s co

nfer

ring

res

ista

nce

to e

xten

ded-

spec

trum

β-la

ctam

s am

ong

E. c

oli

and

K.

pneu

mon

iae

from

hum

an so

urce

s.

NR

, not

repo

rted

; Tn,

tran

spos

on. P

aren

thes

is in

dica

ted

vari

able

pre

senc

e or

abs

ence

of I

ncFI

A an

d In

cFIB

repl

icon

s. T

he p

lasm

id in

bol

d re

pres

ent t

he m

ost w

ides

prea

d on

es.

a Tn4

401

is a

cla

ss I

I Tn

fro

m T

n3 s

ubfa

mily

, with

the

fol

low

ing

stru

ctur

e: t

npR

-tnp

A-IS

Kpn

7-bl

a KPC

-ISK

pn6.

Tn4

401

has

five

isof

orm

s w

hich

diff

er b

y de

letio

ns (

68–2

55 b

p) b

etw

een

ISK

pn7

and

the

bla K

PC g

ene

[(a)

del

etio

n of

99

bp; (

b) n

o de

letio

n; (c

) del

etio

n of

215

bp;

(d) d

elet

ion

of 6

8 bp

; (e)

del

etio

n of

255

bp)

]. N

TEK

PC-I

a re

fers

to n

on-T

n440

1 m

obile

ele

men

ts;

b Tn1

999

is a

cla

ss I

Tn

with

the

follo

win

g st

ruct

ure:

IS1

999-

bla O

XA

-48-

lysR

-IS1

999.

One

var

iant

of T

n199

9 is

als

o re

port

ed (T

n199

9.1)

diff

erin

g by

the

inse

rtio

n of

ISR

1 up

stre

am b

laO

XA

-48;

c T

n201

3 is

a c

lass

I Tn

with

the

follo

win

g st

ruct

ure:

ISEc

p1-b

laO

XA

-181

-Δly

sR- Δ

ere-

IRR

2 (r

ecog

nize

d by

ISEc

p1),

in b

laO

XA

-232

ISEc

p1 is

trun

cate

d; d Δ

Tn12

5 de

rive

d fr

om T

n125

, a c

lass

I Tn

from

A. b

aum

anni

i, w

hich

pre

sent

s the

follo

win

g st

ruct

ure

amon

g E.

col

i and

K. p

neum

onia

e is

olat

es: Δ

ISAb

a25-

bla N

DM

-ble

MBL

; em

ainl

y re

port

ed a

mon

g K

. pne

umon

iae

isol

ates

.

TABL

E 7.

MGE

asso

ciate

d wi

th th

e mos

t wid

espr

ead

bla

gene

s con

ferr

ing r

esist

ance

to ex

tend

ed-s

pect

rum

β-la

ctam

s am

ong E

. col

i and

K. p

neum

onia

e fro

m

hum

an so

urce

s.

bla

gene

IS

and

/or t

rans

poso

ns in

volv

ed

in th

e m

obili

zatio

n of

bla

Ep

idem

ic p

lasm

ids (

Inc

Gro

up)

Ref

eren

ces

E. c

oli

K. p

neum

onia

e

bla S

HV-

12

IS26

(com

posit

e Tn)

In

cI1,

IncX

3, In

cFII

-(Inc

FIA-

IncF

IB),

IncH

I2, I

ncN

, Inc

L/M

In

cX3,

IncN

, Inc

HI2

, Inc

FII K

, In

cI1,

IncL

/M, I

ncR

(67)

bla C

TX-M

-15

ISEc

p1, I

S26,

IS26

-ΔIS

Ecp1

, ΔIS

Ecp1

In

cFII

-(In

cFIA

-Inc

FIB)

, Inc

N,

IncI

1, In

cA/C

, Inc

L/M

In

cR, I

ncFI

I K, I

ncFI

I, In

cA/C

, In

cL/M

(5

1,73,

77,3

16,3

18,3

34)

bla C

TX-M

-14

ISEc

p1 o

r ISC

R1 cl

ass I

inte

gron

as

socia

ted

IncK

, Inc

FII-

(Inc

FIA-

IncF

IB),

IncI

1 In

cFII

K (5

1,90)

bla C

TX-M

-27

ISEc

p1, I

S26-ΔI

SEcp

1, ΔI

SEcp

1 In

cFII

-(In

cFIA

-Inc

FIB)

N

R (7

4,25

0)

bla K

PC-2

Tn44

01a

a Tn

4401

b Tn

4401

c N

TEKP

C-Ia

IncF

, Inc

FII K

2, In

cFII

K1

IncN

In

cN, I

ncH

I2

IncN

IncF

IIK

2, In

cFII

K1, C

olE1

, Inc

X3

IncN

In

cN

IncF

IIK2

-Inc

R

(129

,345

,346

)

bla K

PC-3

Tn

4401

a Tn

4401

b Tn

4401

d

IncF

IIK

2, In

cFII

In

cN

IncF

IA, I

ncFI

I

IncF

IIK

2

IncI

2, C

olE1

In

cFIA

, Inc

N

(254

,345

,346

)

bla O

XA-

48

Tn19

99 b

IncL

(1

29,3

05)

bla O

XA-

181

ISEc

p1/T

n201

3 c

ColE

-like

, Inc

L, In

cA/C

, Inc

FII K

, IncX

3 (1

29,2

05,3

47)

bla O

XA-

232

ΔISE

cp1/

Tn20

13 c

ColE

-like

(1

76,3

48)

bla N

DM

ΔT

n125

d In

cA/C

, Inc

X3,

IncH

I1, I

ncN

, Inc

L/M

, Inc

Re , In

cHIB

-Inc

FIB

e (1

29,2

15,3

05)

bla V

IM-1

-

like

class

I in

tegr

on

IncN

2, In

cHI2

, Inc

A/C,

IncI

1, In

cRe ,

IncF

IIK

e (2

13,3

49)

bla C

MY-

2 IS

Ecp1

In

cI1,

IncK

, Inc

A/C

(pre

dom

inan

tly E

. col

i) (3

49)

bla D

HA-

1 IS

26, I

SCR1

cIas

se I

inte

gron

as

socia

ted

IncL

/M, I

ncH

I2, I

ncR

, Inc

FIA

(pre

dom

inan

tly K

. pne

umon

iae)

(3

49) (

Ribe

iro T

et a

l.,

unpu

blish

ed re

sults

)


48

Table 7 summarizes the principal MGE involved in the dissemination of main

extended-spectrum acquired β-lactamases identified among E. coli and K. pneumoniae,

with the plasmids associated and evolution of its characterization being detailed below. The

development of a PCR-based replicon typing (PBRT) scheme in 2005 boosted plasmid

typing studies among Enterobacteriaceae populations, including ABR plasmids (343). This

scheme is based on the determination of eighteen Inc groups (IncFIA, IncFIB, IncFIC,

IncHI1, IncHI2, IncI1, IncL/M, IncN, IncP, IncW, IncT, IncA/C, IncK, IncB/O, IncX, IncY,

IncFrepB, and IncFIIS) (343). The wide application of this scheme to large populations of

different Enterobacteriaceae species unveiled a high heterogeneity among certain plasmid

groups (e.g. IncF plasmids), variants within certain plasmid families (e.g. IncI1 or IncHI2)

or the need for revising plasmid classifications. For these reasons, improvements on PBRT

targets and resolution were proposed: i) discrimination of IncF plasmids from E. coli

(IncFII, IncFIC), K. pneumoniae (IncFIIK), Salmonella spp. (IncFIIS) and Yersinia spp.

(IncFIIY) (350); ii) increased resolution of plasmid variants from certain plasmid families

with the design of pMLST (plasmid MLST) schemes for IncI1, IncA/C, IncHI2, IncN and

IncF (https://pubmlst.org/plasmid/primers/); iii) revision of classification of IncL/M and

IncX plasmids (351,352); iv) enlargement of plasmid types coverage with description of

primers for IncR (353). In fact, IncFIIK plasmids from K. pneumoniae were previously not

detected or were wrongly classified as IncFIIs (Salmonella spp. plasmids) by the first PBRT

scheme published, and for this reason their precise definition and contribution to ABR in

this species have been poorly addressed.

The main ABR plasmids circulating in E. coli and K. pneumoniae can be generally

divided in two main groups – IncF and IncR plasmid families, which are narrow-host range

plasmids; and IncA/C, IncL, IncM and IncN plasmids, that are broad-host range, with

ability to transfer among different species (305,349) (Table 7). These plasmid families are

those frequently carrying clinically relevant bla genes, but also other ABR genes as

aminoglycosides, trimethoprim, sulphonamides and/or genes conferring tolerance to

metals such as copper, silver, arsenic, mercury and tellurium (349,354,355). ABR plasmids

belonging to the heterogeneous IncF family are particularly abundant among E. coli and K.

pneumoniae and a portion of them (particular IncFII or IncFIIk) are particularly amplified

in certain clones/lineages within these species carrying specific genetic determinants of

resistance. The revised scheme for IncF plasmids typing (350) and sequencing of FII or FIIK

replicons allowed increased resolution of plasmid variants that were unnoticed till then

(350). IncFII plasmids [with variable presence of FIA and/or FIB replicons; mainly F2:A-

:B- (pEK516, GenBank accession number EU935738) and F2:A1:B- (pEK499, GenBank

accession number EU935739)] have been strongly linked to the worldwide dissemination of

CTX-M-15, especially among B2-ST131 H30-Rx lineage (249,350,356,357). On the other


49

hand, CTX-M-15 pandemic in K. pneumoniae is mostly associated with IncFIIK5/K7 and IncR

plasmids, showing the implication of different plasmid families well adapted to each of

these species in the dissemination of blaCTX-M-15. IncFIIK2/K1 plasmids (with variable presence

of FIB replicon) have also been associated mainly with the dissemination of KPC-2/-3

among K. pneumoniae ST258 clades (249,301,303,350). IncR plasmids have also been

linked to the expansion of DHA-1 and different carbapenemases (VIM, NDM) (Table 7). In

fact, these IncR plasmids seem to be well adapted to K. pneumoniae populations (although

they have also been sporadically described in E. coli and Salmonella spp.). Regarding KPC

producers, besides IncFIIK plasmids, other important plasmids, such as IncN, IncFIA and

IncI2 have also been reported, including among E. coli isolates (345). Concerning other

carbapenemases and plasmid-acquired AmpCs, plasmids identified are mainly broad-host

range, circulating in both species (e.g. OXA-48 and IncL; NDM and IncA/C, IncX3, IncN;

VIM-1-like and IncN; CMY-2 and IncI1 or IncA/C, DHA-1 and the former IncL/M) (Table

7). IncI1, IncA/C and IncN plasmids are frequently linked to the dissemination of ESBL and

plasmid-acquired AmpCs, and less frequently to carbapenemases, mainly among E. coli

isolates in non-human sources (food-production animals and the environment), suggesting

the role of non-human hosts as reservoirs of ABR (72,349,358). Mobilizable plasmids, such

as ColE-like, have been linked to the recent emergence of OXA-181 and OXA-232 enzymes

(347,348).

Along with the increasing use of WGS in ABR populations, the number of plasmid

sequences, and particularly those involved in ABR, greatly increased in public databases.

The plasmidome linked to main high-risk ABR clones has been explored mainly for E. coli

ST131 and K. pneumoniae CG258 (345,356,357), being needed a deeper knowledge of the

circulating plasmids in other high-risk clones. However, the plasmidome study is limited

mainly due to the scarcity of bioinformatics tools available for plasmid extraction from

WGS data [PLAsmid Constellation NETwork (PLACNET), plasmidSPAdes] and the level of

required expertise required for their application, which is particularly difficult in large

collections of isolates (357,359). Thereat, most of the studies using WGS characterize only

the replicon content of the bacterial strains using databases as PlasmidFinder

(https://cge.cbs.dtu.dk/services/PlasmidFinder/), which is insufficient nowadays to fully

understand the role of plasmids in the adaptation, evolution and maintenance of MDR

lineages in different populations of species, clones and subclones.


50

1.2. Overview of typing methods for Escherichia coli and Klebsiella pneumoniae: exploring the potential of omics approaches

As previously discussed in the subsections 1.1.3.1 and 1.1.3.2, MDR E. coli and K.

pneumoniae clones/lineages correspond to a small proportion of all population of these

species, which have been identified by different typing methods that have evolved over the

years towards higher resolution levels. Bacterial typing, or the analysis at infraspecies level

[see Box 7 to review concepts associated with bacterial typing], can be used to generate

discriminatory strain-specific fingerprintings or datasets, which can be applied to a wide

variety of contexts. These contexts include the assessment of epidemiological relatedness

and identification of emerging pathogenic strains or clones within a species, study of the

population structure and phylogeny of a given species, clarification of bacterial

transmission patterns, detection of outbreaks and of reservoirs or sources of human

pathogens (Figure 21) (229,360). Accurate, rapid, low-cost, reproducible and user-friendly

typing methods are crucial nowadays, mainly in the prompt detection of outbreaks, to

improve the definition of index cases and to notify infection control commissions and public

health services, in order to improve guidelines and strategies for the prevention and control

of future outbreaks (223,229).

FIGURE 21. Applications of bacterial typing.

Population structure,

dynamics and phylogeny

Outbreak investigation and

control

Infectious disease ecology and

pathogenicity

Surveillance of infectious diseases


51

BOX 7. Glossary of concepts commonly used in bacterial typing.

Isolate: a population of bacterial cells in pure culture derived from a single colony. In terms of clinical

microbiology, isolates are usually derived from the primary culture of a clinical specimen obtained from a

single patient (233,361). Strain: the descendants of single isolation in pure culture (usually resulting from a succession of cultures

originated from the single initial colony). A strain might be considered an isolate or group of isolates

exhibiting common phenotypic and/or genotypic characteristics that differ from other isolates from the same

genus or species (233,361).

Clone: an isolate or a group of isolates descending from a common ancestor and exhibiting identical or

closely similar phenotypic or genotypic traits, which are characterized by strain-typing methods and as

belonging to the same group (229).

Type: a given bacterial isolate or group of bacterial isolates might be allocated to a type according to an

existing typing scheme (e.g. isolate X belongs to capsular type 2 according to the scheme used to define the

capsular types) (229).

Phylogeny: study of the evolutionary relationships among members of the same taxon (e.g. species, strains,

clones) (229). Phylogenetic tree: a diagram that depicts the hypothetical evolutionary history of the taxa under study.

The points at which lineages split represent ancestor taxa to the descendant taxa appearing at the terminal

points of the tree (229).

Lineage: group of isolates sharing essential characteristics due to a common ancestor. Lineages are

generally inferred from a phylogenetic tree (229).

Clade: a monophyletic taxon; a group of organisms which includes the most recent common ancestor of all

of its members and all of the descendants of that most recent common ancestor. Clades are depicted by

evolutionary trees (http://www.ucmp.berkeley.edu/glossary/glossary.html).

Population dynamics: the study of factors that interfere with the variability of bacterial populations over

time and space, including the interactions of these two factors (229).

Population genetics: the study of natural bacterial genetic diversity (variation in genes) arising from evolutionary processes among a group of particular bacterial strains (229).

Endemic clones: refers to the constant presence and/or usual prevalence of a particular clone in a

population within a giving setting (229,233).

Epidemic clones: refers to an increase, often sudden, in the number of cases involving a particular clone

above what is normally expected in that population in that area (229,233). Clonal outbreak: carries the same definition of epidemic, but is often used for a more limited geographic

area (229,233).

Pandemic clone: refers to an epidemic clone that has spread over several countries or continents, usually

affecting a large number of people (e.g. ST131 E. coli) (229,233).

Fingerprint: a particular pattern (e.g. DNA banding pattern; peak patterns) or a set of markers scores (e.g.

absorbance values) exhibited by an isolate on application of one or more typing methods, which can be used

to asses epidemiological relatedness among bacterial isolates. Phenotyping: refers to the discrimination of bacterial strains based on the observable characteristics (e.g.

morphology of colonies on various culture media, biochemical tests, serology, killer toxin susceptibility,

pathogenicity, and antibiotic susceptibility) (362).

Genotyping: refers to the discrimination of bacterial strains based on their genetic content (362). Omic technologies: technologies that measure some characteristic of a large family of cellular molecules,


52

such as genes, proteins, or small metabolites, have been named by appending the suffix “-omics,” (e.g.

genomics). The term “omics” refers to the collective technologies used to characterize and quantify the pools

of biological molecules that underpin the structure, function and dynamics of a microorganism or

microorganisms (363).

Genomics: refers to the study of the full genetic complement of an organism (the genome)

(http://www.nature.com/subjects/genomics). Core genome: set of genes shared by all of the genomes studied (e.g. genomes belonging to the same clone,

bacterial species, etc.). These genes are generally related with housekeeping and regulatory functions

(364,365). Accessory genome: set of genes shared by some organisms which are not present in all of the studied

organisms (these elements are generally associated with MGE) (364,365).

Pan-genome: refers to the total number of non-redundant genes that are present in a given dataset. It comprises three parts: i) core genome, ii) accessory genome and iii) species-specific or strain-specific genes,

which are those genes that are present in a single genome (Figure 22) (365).

FIGURE 22. Pan-genome schematic representation. Circles represent the genomes of three different strains.

Phylogenomics: is the result of the intersections of the fields of evolution and genomics. In fact,

phylogenomics use genomic data to infer phylogenetic relationships and gain insights into evolution reconstructions (366).

Proteomics: large-scale study of proteomes (set of proteins produced in an organism, system, or biological

context). Metabolomics: refers to the identification and quantification of the small molecule metabolic products

(metabolome) of a biological system (cell, tissue, organ, biological fluid, or organism) at a specific point in

time (http://www.nature.com/subjects/metabolomics).

Reproducibility: ability of a typing method to generate the same results upon repeating testing.

Discriminatory power: ability of a typing method to distinguish between epidemiologically unrelated

strains (229).

Typeability: The proportion of strains for which a type can be generated by a giving typing method (223).

Notwithstanding, the selection of an appropriate typing method relies on the

problem to be solved, the epidemiological, temporal and geographic contexts, as well as the

availability of technical and financial resources and experienced personnel. An overview of

Core Genes

Accessory Genes

Strain-specific genes


53

the evolution and applications of the typing methods used for E. coli and K. pneumoniae

populations and their advantages and disadvantages is presented in the following sections.

1.2.1. Phenotyping methods

Phenotyping methods group organisms according to their similarity in characters

that result from the expression of a genotype. They may comprise tests easy to perform,

such as analysis of colony morphology (e.g. the string test used for identification of

hypervirulent K. pneumoniae strains), odour, colour or other macroscopic features. On the

other hand, they might rely on traits that need specialized technology in order to be

documented (e.g. ability of isolates to grow on the presence of specific substances;

expression of specific molecules as surface antigens) and require strict standardization of

experimental conditions. In E. coli and K. pneumoniae the most common phenotyping

methods used were: serotyping, biotyping, phage and bacteriocin typing, and multilocus

enzyme electrophoresis (MLEE), the latter particularly relevant in E. coli.

Serotyping

Traditionally, serotyping methods adopted for E. coli and K. pneumoniae strains are

among the most important phenotypic methods developed from the early days of

microbiology.

Differentiation of E. coli isolates by traditional serotyping has been performed using

antisera against the highly polymorphic somatic (O), flagellar (H) and capsular (K) antigens

(367,368). Currently, 182 O-groups, 53 H-types and more than 80 K-types have been

recognized by traditional serotyping (369–371). However, due to the technical complexity of

capsular typing, serotyping based only in O- and H- antigens (antigenic formulae written as

O:H) became the gold standard technique for E. coli serotyping, with some specific

serotypes being recognized in pathogenic E. coli strains (e.g. ExPEC O25b:H4 ST131 clone

and EHEC O157:H7). The O-specific polysaccharide chain, also known as O-antigen, is part

of the tripartite structure of the LPS (which also includes the lipid A and the core

polysaccharide) (see subsection 1.1.3.2 for details) and is translocated across the outer-

membrane to the cell surface. The H-antigen, is a homopolymer filament composed by

repeated molecules of the protein flagellin that projects outside the cell wall, providing cell

motility, whereas K-antigen is constituted by surface polysaccharides external to the cell

wall (369). Capsular typing (K-typing) has been performed mainly in ExPEC strains, where

certain K-types have been associated with particular infections, such as K1 commonly found


54

in E. coli strains causing neonatal meningitis or K5 strongly associated with UTIs and

bacteraemia (371).

In K. pneumoniae, serotyping is based on the characterization of K- and O-antigens

(this species is non- flagellated being H-antigen is absent). Currently, 77 K-types and 9 O-

types have been recognized (279). The presence of heat-stable capsules difficults

identification of O-types, being necessary to generate non-capsulated strains for its correct

identification (372). As only 9 O-types have been described for K. pneumoniae, with O1, O2

and O3 being the most prevalent, the discriminatory power was considered less useful than

K-types in terms of epidemiological surveillance. However, with the increasing levels of

MDR strains, the interest in O-types as potential vaccine targets was renewed due to the low

level of diversification in these strains (mainly O1 and O2) (279,295). K-typing by serology

was established in 1926 and was further developed until the 1970s due to its high

discriminatory power, being still the reference method for serotyping (372,373). In fact, the

use of serology for K-typing was the first method allowing a discrimination between

hypervirulent and MDR K. pneumoniae strains, showing that K1 and K2 were frequently

linked to the hypervirulent strains (294).

Despite the high discriminatory potential in both species, serotyping is labour

intensive, time-consuming, subject to variations in interpretation (due to autoagglutination

and cross-reactions), and associated with a high cost of the reagents. For these reasons, its

application is restricted to reference centers, in the case of K. pneumoniae, limiting its

usefulness in terms of epidemiology and surveillance. These limitations led to an effort to

replace serological phenotyping by molecular serotyping, and more recently, in silico

serotyping based on WGS data. Different techniques were proposed for both species, which

will be addressed in the next sections (296,299,304,369,370,374,375).

Biotyping

Biotyping is a method of typing based on biochemical profiles that are known to vary

within a given species, and can be very useful in small laboratories where modern and more

accurate technology for epidemiological studies is not available (229,279,376). Typeability

in biotyping is usually excellent and it’s a method technically easy and with low-costs, with

data generated being typically simple to score and interpret (229,279). Biotyping was widely

used among E. coli strains as early as 1975 (377–380), subtypes being determined mainly

based on carbohydrate fermentation patterns tested on acid-base indicators (main

carbohydrates are adonitol, L-arabinose, D-cellobiose, dulcitol, n-inositol, lactose, maltose,

D-rafinose, L-rhamnose, salicin, D-sorbitol, L-sorbose, sucrose, D-trehalose, and D-xylose),

enzyme profiles (β-galactosidase, ornithine and lysine descarboxilase, arginine dehydrolase,


55

and tryptophanase) and the particularities of growth on specific agar mediums

(MacConkey, citrate and nutrient agar) (376–380). Among K. pneumoniae strains,

biotyping was mainly used to distinguish K. pneumoniae from other Klebsiella species,

being limited and based on probes for adonitol, citrate, dulcitol, mucate, malonate, L-

sorbose and D-tartrate, urease and lysine decarboxylase production, and Voges–Proskauer

test (279,280).

Biotyping can be performed using macrotube tests alone and/or by combining a

commercially available miniaturized system (e.g. API 20E system) (229,279). Nevertheless,

it is laborious and time-consuming when applied to multiple strains (376). Studies

comparing biotyping and serotyping among E. coli and K. pneumoniae strains showed that

while serotyping is more discriminatory, biotyping typeability is higher and associated with

easier interpretation of results (377). It has advantages considering that it covers

phenotypic features of the bacteria but, as for other phenotypic methods, it has been

replaced by genotyping methodologies.

Phage typing

Bacteriophages are viruses with capacity to invade and lyse specific bacterial cells.

The specificity of the phage is mediated by the proteins associated with the tails that attach

to target molecules in the bacterial cell surface. The variability in susceptibility of different

bacterial strains to different phages determines their phage type, which has been used as a

typing tool in outbreaks and epidemiological studies in different species, including E. coli

and less extensively in K. pneumoniae (279,376,381). The method is based in the spread of

the bacterial strain to be tested in the plate, which is then inoculated with different phages,

and after incubation (at a standardized temperature and period) the visualization of plaques

(clear zones) indicates lytic phage activity, and indicates the correspondent phage type

(Figure 23).

FIGURE 23. Scheme of the methodology applied in phage typing [reprinted with permission from (381)].


56

Regarding E. coli strains, phage typing was widely used since the 1960s, mainly

among diarrhoeagenic strains (including in the subtyping of EHEC O157:H7) (376,382–

384). Among K. pneumoniae strains, different phage typing systems have been proposed in

the 1970s with discrimination power similar to serotyping, however, with lower typeability

and reproducibility (279).

Nowadays, phage typing has a limited application as an epidemiological tool for

surveillance and characterization, mainly due to the high number of strains that are not

typeable (due to the lower sensitivity of phages or development of bacteriophage-insensitive

mutants). Besides, the difficulty to produce and maintain live cultures of phages under

laboratory conditions, the high costs associated and the expertise needed for interpretation

of results restricted its practice to a few reference laboratories (229,376).

Bacteriocin typing

Bacteriocins are a highly diverse and abundant toxins family produced by bacteria to

inhibit the growth of similar or closely related bacterial strains, which are usually named

according to the bacterial species of origin (e.g. colicins in E. coli, klebocins or klebecins in

Klebsiella spp.) (385). Two schemes have been used in bacteriocin typing; in the first

scheme, the bacteriocins produced by the test strains are detected by their activity (pattern

of growth inhibition) on a set of indicator strains (active test); in the second scheme, the

sensitivity to bacteriocins produced by a set of producer strains is evaluated in the test

strains (passive test) (279,385).

Bacteriocin typing in E. coli (mainly in uropathogenic strains) and K. pneumoniae

strains has been developed in the 1960s and has been used generally coupled with other

typing methods (386–389). The lack of typeability on many strains, poor reproducibility

and the fact of being available only at reference centres, has led to a practically disuse of it

as a typing tool (279).

Multilocus enzyme electrophoresis (MLEE)

MLEE consists in the study of polymorphic variants on a set of housekeeping

enzymes, encoded by different alleles of the same gene, using gel electrophoresis, thus

providing small but detectable variations in protein size and charge (390). MLEE was

widely used among E. coli strains between the 1980s and 1990s, and allowed discrimination

in different phylogenetic groups (A, B1, B2, D and E), which was extremely important in the

delineation of E. coli population structure (390–392). At that time, MLEE was also helpful

to establish relationships between phylogeny and pathogenicity (see subsection 1.1.3.1)


57

(393). In contrast to E. coli, MLEE has rarely been used for typing of K. pneumoniae

strains, mainly due to genetic diversity and recombination observed among this species

(279). MLEE is not a rapid and hence widely applied typing method, but it was extremely

important for clarifying the population biology of E. coli, and to provide a strong theoretical

basis and experimental validation for the development of MLST (see below) (229).

1.2.2. Genotyping methods

The rapid advances in molecular biology tools during the 1970s changed the course

of bacterial strain typing (229,362,394). Genotyping methods are based on the bacterial

genetic content, and have progressively replaced the phenotyping methods in

microbiological laboratories, mainly due to its higher accuracy, but also for being generally

less laborious and time-consuming. A recent revolution in instrumentation for increased

throughput and bioinformatics tools for data interpretation boosted a number of novel

approaches for strain typing, including DNA-based typing methods. Genotyping methods

can be classified in two important categories: DNA banding pattern- and DNA sequencing-

based methods.

In DNA banding pattern-based genotyping methods, the strains are classified based

on differences in the size of the DNA fragments generated either by cleavage of genomic

DNA with restriction enzymes or by amplification of genomic DNA, or both (362,394). In

DNA sequencing-based methods, strain discrimination is based on the number and content

of polymorphisms detected in partial (particular gene biomarkers) or total (the whole

genome) nucleotide sequences. The main advantage of DNA sequencing-based genotyping

is the non-ambiguity, portability and reproducibility of DNA sequence data, which boosted

the use of this typing approach (394).

The applicability and main characteristics of common genotyping methods used in

E. coli and K. pneumoniae strains are summarized in Table 8. A detailed overview of the

gold-standard typing methods for these species (such as PFGE, MLST, molecular serotyping

and WGS) is provided in this section.


58

TAB

LE 8

. Cha

ract

eris

tics a

nd a

pplic

atio

ns o

f the

mai

n ge

noty

ping

met

hods

use

d in

ana

lysi

s of E

. col

i and

K. p

neum

onia

e po

pula

tions

.

TABL

E 8.

Cha

ract

erist

ics an

d ap

plica

tions

of th

e mai

n ge

noty

ping

met

hods

use

d in

analy

sis of

E. c

oli a

nd K

. pne

umon

iae p

opul

atio

ns.

Geno

typi

ng m

etho

ds

Typi

ng m

echa

nism

ba

sis

Spec

ies

Dis

crim

inat

ion

leve

l D

iscr

imin

ator

y po

wer

a Ty

peab

ility

a Re

prod

ucib

ility

a Co

st

Ease

of u

se

Anal

ytic

al

com

plex

ity Re

fere

nce

Gene

-spe

cific

PCR

stx1,

stx2,

eaeA

, bf

pA, e

stA, e

ltB, a

stA, a

ggR,

pC

VD ia

l

PCR

Ec

pa

thot

ypes

(E

HEC

, EPE

C, E

TEC,

EA

EC, E

IEC)

++++

++

++

++++

lo

w sim

ple

easy

(3

95,3

96)

arpA

, chu

A, y

jaA,

Tsp

E4.C

2 PC

R

Ec

phylo

-typi

ng

++++

++

+ ++

++

low

simpl

e ea

sy

(232

)

ybbW

PC

R

Ec

subt

ypin

g of S

T131

(R

x clad

e)

++++

++

++

++++

lo

w sim

ple

easy

(2

37)

afa/

draB

C, ir

oN, s

at, i

beA,

pa

pGII,

cnf1,

hly

A, p

apGI

II,

cdtB

, neu

C-K1

PCR

Ec

su

btyp

ing o

f ST1

31

(viro

type

s)

++++

++

++

++++

lo

w sim

ple

easy

(2

39)

gyrA

/par

C S (P

CR +

sequ

encin

g)

Ec, K

p su

btyp

ing o

f ST1

31

phylo

-typi

ng of

Kp

com

plex

++++

++

++

++++

lo

w to

m

oder

ate

simpl

e ea

sy

(247

,397

)

CH ty

ping

S (P

CR +

sequ

encin

g)

Ec

subt

ypin

g of S

T131

(fu

mC/

fimH

allel

es)

++++

++

++

++++

lo

w to

m

oder

ate

simpl

e ea

sy

(236

)

Puls

ed-fi

eld

gel

elec

trop

hore

sis

(PFG

E)

P (DNA

restr

ictio

n +

electr

opho

resis

gel)

Ec, K

p Cl

ones

++++

++

++

++++

m

oder

ate

mod

erat

e,

labou

r int

ensiv

e co

mpl

ex

(233

,398

)

Ampl

ifica

tion

of fr

agm

ent

lengt

h po

lymor

phism

(AFL

P) P (D

NA re

strict

ion+

PCR

+

electr

opho

resis

gel)

Ec, K

p Ph

ylo-ty

ping

of K

p co

mpl

ex

Clon

es

+++

+++

+++

mod

erat

e m

oder

ate

ea

sy

(399

–402

)

Rand

om am

plifi

ed

polym

orph

ism D

NA (R

APD)

or

Arb

itrar

ily p

rimed

PCR

(A

P-PC

R)

P (PCR

+ el

ectro

phor

esis

gel)

Ec, K

p Ph

ylo-ty

ping

of K

p co

mpl

ex

Clon

es

++

+++

+ lo

w sim

ple

mod

erat

e (3

97,4

03,4

04)

Repe

titive

sequ

encin

g-ba

sed

PCR

(rep-

PCR)

P (P

CR +

elec

troph

ores

is ge

l)

Ec, K

p Cl

ones

++

++

+ ++

lo

w to

m

oder

ate

mod

erat

e Au

tom

ated

m

etho

d ea

sier

and

quick

ly -

Dive

rsiL

abb

simpl

e (4

00,4

05–

408)


59

TAB

LE 8

. Cha

ract

eris

tics a

nd a

pplic

atio

ns o

f the

mai

n ge

noty

ping

met

hods

use

d in

ana

lysi

s of E

. col

i and

K. p

neum

onia

e po

pula

tions

(con

t.).

Gen

otyp

ing

met

hods

Ty

ping

mec

hani

sm

basi

s Sp

ecie

s D

iscr

imin

atio

n le

vel

Dis

crim

inat

ory

pow

era

Type

abili

tya

Rep

rodu

cibi

litya

Cost

Ea

se o

f use

A

naly

tica

l co

mpl

exit

y R

efer

ence

Ribo

typi

ng

S (DN

A re

stric

tion

+ el

ectr

opho

resis

gel

+

hybr

idiz

atio

n w

ith 16

S an

d 23

S rR

NA

prob

es)

Ec, K

p Ph

ylo-

typi

ng o

f Ec a

nd

Kp co

mpl

ex

Clon

es

++

++++

++

+ lo

w

(man

ual)

mod

erat

ed

(aut

omat

ic)

mod

erat

e Au

tom

ated

m

etho

d ea

sier

and

quic

kly -

Ri

boPr

inte

r® c

easy

(few

fr

agm

ents

ge

nera

ted)

(397

,409

–412

)

Mul

tiple

-locu

s var

iabl

e-nu

mbe

r tan

dem

repe

at

anal

ysis

(MLV

A)

S (PCR

+ se

quen

cing

) Ec

, Kp

Clon

es

++++

++

+ ++

+ m

oder

ate

simpl

e ea

sy

(413

–416

)

Mul

tilo

cus

sequ

ence

ty

ping

(MLS

T)

S (PCR

+ se

quen

cing

) Ec

, Kp

Clon

es

++

++++

++

++

mod

erat

e to

high

(d

epen

ding

on

the

num

ber o

f iso

late

s)

mod

erat

e ea

sy

(onl

ine

data

base

s)

http

://m

lst.w

arw

ick.

ac.u

k/m

lst

/dbs

/Eco

li ht

tp:/

/big

sdb.

past

eur.f

r/ec

oli/

ecol

i.htm

l ht

tp:/

/big

sdb.

past

eur.f

r/kl

ebsie

lla/k

lebs

iell

a.ht

ml

Mol

ecul

ar s

erot

ypin

g O

-, H

- and

K-t

ypin

g PC

R Ec

, Kp

Sero

type

s or K

-type

s

++++

++

+ ++

++

low

sim

ple

easy

(2

35,2

95,4

17–

419)

C-pa

ttern

P

(P

CR +

DN

A re

stric

tion

+ el

ectr

opho

resis

gel

)

Kp

K-ty

pes

++

+ ++

++

+ lo

w

mod

erat

e,

labo

ur in

tens

ive

com

plex

(3

74)

rfb-

Rest

rictio

n Fr

agm

ent

Leng

th P

olym

orph

ism

(RFL

P)

P (PCR

+ D

NA

rest

rictio

n +

elec

trop

hore

sis g

el)

Ec

O-ty

pes

++

+ ++

++

+ lo

w

mod

erat

e,

labo

ur in

tens

ive

com

plex

(4

20)


60

P, D

NA

band

ing

patt

ern-

base

d m

etho

d; S

, DN

A se

quen

cing

-bas

ed m

etho

d; E

c, E

. col

i, K

p, K

. pne

umon

iae.

Bol

d ite

ms a

re d

iscu

ssed

in d

etai

l in

the

text

. a

Char

acte

rist

ics r

ange

d fr

om +

, whi

ch is

poo

r, to

+++

++, w

hich

is e

xcel

lent

. b Bio

Mér

ieux

, Mar

cy l'

Etoi

le, F

ranc

e; c Q

ualic

om, W

ilmin

gton

, USA

.

P, D

NA ba

ndin

g patt

ern-

base

d meth

od; S

, DNA

sequ

encin

g-ba

sed m

ethod

; Ec,

E. co

li, Kp

, K. p

neum

onia

e. Bo

ld ite

ms a

re di

scus

sed i

n deta

il in t

he te

xt.

a Char

acter

istics

rang

ed fr

om +

, whi

ch is

poor

, to +

++++

, whi

ch is

exce

llent

. b BioM

érieu

x, M

arcy

l'Eto

ile, F

ranc

e; c Qu

alico

m, W

ilmin

gton,

USA.

Geno

typi

ng m

etho

ds

Typi

ng m

echa

nism

ba

sis

Spec

ies

Disc

rimin

atio

n le

vel

Disc

rimin

ator

y po

wera

Type

abili

tya

Repr

oduc

ibili

tya C

ost

Ease

of u

se

Anal

ytic

al

com

plex

ity Re

fere

nce

fliC-

RFLP

P (PCR

+ D

NA

restr

iction

+

electr

opho

resis

gel)

Ec

H-typ

es

++

+ ++

++

+ low

m

oder

ate,

labou

r int

ensiv

e co

mple

x (4

21,42

2)

wzi

and

wzc

S (P

CR +

sequ

encin

g)

Kp

K-typ

es

++++

++

+ ++

++

low

simple

ea

sy

(298

,299)

WGS

SN

P an

alys

is S

Ec, K

p Li

neag

es, c

lades

++

+++

++++

++

++

high

co

mple

x (re

quire

s bio

infor

mati

c ex

perti

se)

com

plex

(283

,301,4

23)

core

geno

me (

cg) M

LST

S Kp

Cl

onal

Grou

ps

Line

ages

++

+++

++++

++

++

high

co

mple

x (re

quire

s bio

infor

mati

cs

expe

rtise

)

com

plex

(284

,297)

in si

lico

MLS

T S

Ec, K

p Cl

ones

++

+++

++++

++

++

high

ea

sy to

mod

erate

easy

ht

tps:/

/cge

.cbs

.dtu.d

k/se

rvice

s/M

LST/

#ref0

5 in

silic

o se

roty

ping

S Ec

, Kp

Sero

types

and K

-type

s

++++

+ ++

++

++++

hi

gh

easy

to m

oder

ate e

asy

(369

,424,4

25)

http

s://c

ge.cb

s.dt

u.dk/

serv

ices/

Sero

typeF

inde

r/

TAB

LE 8

. Cha

ract

eris

tics a

nd a

pplic

atio

ns o

f the

mai

n ge

noty

ping

met

hods

use

d in

ana

lysi

s of E

. col

i and

K. p

neum

onia

e po

pula

tions

(con

t.).


61

The development of DNA-based strain typing methods such as PFGE in the late

1990s (233,398) and MLST in the middle 2000s (426–429) contributed largely to the

bacterial strain typing metamorphose, being nowadays considered “gold-standard

methods” for E. coli and K. pneumoniae typing.

Although the high discriminatory power of PFGE, which is extremely useful in

outbreak investigations, insertions and deletions of MGE may result in changes in the

PFGE patterns, leading to an erroneous categorization of some strains. Besides, PFGE is

technically demanding, time-consuming and the analysis of the results is subjective and

operator-dependent, difficulting inter-laboratory reproducibility and comparability

(Table 8) (229,362,430). Interpretation of banding patterns has been facilitated by the

development of commercial software programs (e.g. Infoquest from BioRad;

BioNumerics from Applied Maths NV), helping to overcome some of those inter-

laboratory comparison problems.

MLST provides a uniform nomenclature owing to a standard reproducible system

and thus is suitable for large-scale epidemiology and has been extensively used in the

identification and characterization of main clones implicated in the dissemination of

MDR E. coli and K. pneumoniae strains worldwide (231,234,242,430,431). However, the

design of most MLST schemes currently in use (in some cases more than one per

species) was based on a low number of genomes and some of the housekeeping genes

used lack sufficient discriminatory power, leading to variable resolution according to the

species (228,234). Furthermore, it is expensive if we consider that for each bacteria

seven genes need to be sequenced, and might be labour intensive and time-consuming if

we have many isolates to test (234,362,430) (Table 8).

More recently, the development of molecular serotyping techniques for both

species revived K-typing, and brought serotyping to the spotlight of typing tools

(298,299,418). Besides the extended knowledge of the surface polysaccharides and

proteins of these species, it is extremely important to understand specific host-pathogen

interactions, and even to develop possible antibacterial targets.

Although the high diversity of existing DNA-based typing methods (Table 8),

most of them are limited to certain taxonomic levels and some (PFGE, MLST, K-typing)

have been critical to characterize MDR E. coli and K. pneumoniae populations. However,

the recent burst of WGS was crucial to provide a high-resolution of these populations,

and to explore factors responsible for its global expansion and maintenance

(284,301,423). In fact, the increased accessibility to WGS and the development of

bioinformatic tools for data analysis and interpretation allowed the characterization of

large representative collections in a very short time, and a deeper characterization and

evolutionary insight regarding MDR E. coli and K. pneumoniae populations.


62

WGS has been used in the phylogenetic analyses of core genome of E. coli and K.

pneumoniae, either by development of cgMLST schemes in the case of K. pneumoniae,

or by comparison of SNPs across the whole genome. Two cgMLST schemes (based on

634 or 1143 core genes) have been developed for K. pneumoniae, enabling a precise

delineation between hypervirulent and MDR K. pneumoniae strains, the identification of

globally distributed CGs, and the definition of distinct lineages within particular clones

delineated by MLST (284,297). Phylogenetic analyses using SNPs have also been

extremely important to establish evolutionary routes of major MDR E. coli [e.g. E. coli

ST131 (clade C-fimH30, clade B-fimH22 and clade A-fimH41)] and K. pneumoniae [e.g.

K. pneumoniae ST258 (clade I-wzi154 and clade II-wzi29/K41)] clones, although it has

been underexplored for many other high-risk clones in both species (301,303,423,432).

The development of in silico schemes for typing directly from whole-genome sequence

data (reads or assembled sequences) in both species (e.g. MLST, fimH for E. coli,

serotyping for E. coli and K. pneumoniae) has also been particularly useful to facilitate

data extraction (Table 8) (http://www.genomicepidemiology.org) (369,375,425).

Genomic data are currently growing at an extraordinary rate (Figure 24), and

despite its usefulness, the use of WGS in routine clinical laboratories for typing is still

costly (although the price of sequencing by genome has declined over the years) and

requires specific skills for data processing.

FIGURE 24. Number of bacterial and archaeal genomes sequenced each year (between 1995 and 2014) and submitted to NCBI [reprinted with permission from (433)].


63

1.2.3. Proteomics

Proteomics techniques are based in the study of the expression of genes, as well

as the structure and function of the resulting proteins. Among them, matrix-assisted

laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)

revolutionized in the last years the workflow of many clinical microbiology laboratories

for microbial species identification (363,434,435). MALDI-TOF MS is a high-throughput

analytical technique based on the ionization and separation of ions according to mass-to-

charge (m/z) ratio, yielding a characteristic fingerprint of a given microbial cell that

reflects mostly the content of peptides and small proteins (mainly ribosomal proteins)

(Figure 25) (435,436). Microbial identification is established by the comparison of the

mass spectra obtained (directly from the colony or after using adequate extraction

protocols) with those from known species included in a reference database. This method

is user-friendly, cost-effective, accurate and rapid, with the results obtained in a few

minutes (434,436).

The availability of MALDI-TOF MS in many routine laboratories worldwide

attracted increasing attention to its development as a bacterial strain typing tool (437).

While species identification relies on the biomarker peaks that are universally conserved

among all isolates within a specific species, high-resolution typing is based on the

identification of distinct peak biomarkers among isolates within the same species

(435,437). However, the use of MALDI-TOF MS as a proteotyping tool is still

controversial, mainly due to the wide diversity of analysis workflows (variations in

bacterial collections and growth conditions, extraction methods, matrix, mass

spectrometers and bioinformatics tools used for data analysis), hindering the desired

reproducibility (434,435,437,438). In fact, different resolution outcomes have been

observed for the same species by different research groups and additionally for different

clinically relevant Gram-positive (S. aureus, Enterococcus faecium, S. pneumoniae,

Streptococcus pyogenes, Listeria spp.) and Gram-negative (E. coli, K. pneumoniae, A.

baumannii, Salmonella spp., N. gonorrhoeae) species (435,439–444), which might

reflect a species-dependent discriminatory potential of MALDI-TOF MS. To overcome

these issues, studies with standardized workflows to evaluate long-term and inter-

laboratory reproducibility are needed (439,440,443,444,445).


64

FIGURE 25. Schematic representation showing the technical fundament of MALDI-TOF MS [adapted from (446)].

The levels of infraspecies discrimination using MALDI-TOF MS are distinct in the

case of E. coli and K. pneumoniae (Table 9). In the case of E. coli, different reports have

demonstrated the suitability of MALDI-TOF MS to discriminate E. coli clonal lineages

according to serotype, MLST or pathotypes, reporting the identification of different peak

biomarkers. Among K. pneumoniae few works have been performed and results obtained

could not be correctly correlated with the genotyping methods used for clonal

discrimination (PFGE, MLST, RAPD and rep-PCR) (Table 9).

TABLE 9. Proteotyping studies performed in E. coli and K. pneumoniae using MALDI-TOF MS.

Species No. of isolates tested

Mass spectrometer/ analysis software

Discrimination level Results Reference

E. coli 25 Voyager DE PRO mass spectrometer/Data Explorer (Applied Biosystems1)

Serotype (for O157:H7) +a (447)

E. coli 136 Autoflex III Smartbeam mass spectrometer (Bruker Daltonics2)/Bionumerics3

Pathotype +a,b (436)

E. coli 656 Microflex LT mass spectrometer/ClinProTools

Phylogenetic group +c,d (448)

Separation region


65

Species No. of isolates tested

Mass spectrometer/ analysis software

Discrimination level Results Reference

(Bruker Daltonik2)

E. coli 73

Microflex LT mass spectrometer/MALDI Biotyper (Bruker Daltonik2) and Matlab4 and PLS Toolbox5

Clone + (MLSTc,d,e) - (PFGE) (444)

E. coli 149 Microflex LT mass spectrometer/MALDI Biotyper (Bruker Daltonik2)

Clone/Serogroup + (MLSTc) + (serogroupf) (449)

E. coli 216 VITEK MS mass spectrometer /SARAMIS (BioMérieux6) Clone/Serogroup + (MLSTc)

- (serogroup) (450)

E. coli 109 Microflex LT mass spectrometer/ClinProTools (Bruker Daltonik2)

Clone +c,g (451)

E. coli 74 MALDI biotyper /Flexanalysis and ClinProTools (Bruker Daltonik2)

Clone +e,h (452)

E. coli 47 VITEK MS mass spectrometer /SARAMIS (BioMérieux6) and PAST7

Phylogenetic group Clone

+ (402)

E. coli

61 reference strains (53 H-types)

AutoFlex MALDI-TOF mass spectrometer/Autoflex III Smartbeam (Bruker Daltonik2)

H-typing + (453)

K. pneumoniae 10

Microflex LT mass spectrometer/ClinProTools (Bruker Daltonik2), SpecAlign8

and R software9

Clone - (454)

K. pneumoniae 17 AXIMA mass spectrometer (Saramis10)/SARAMIS (BioMérieux6)

Clone - (404)

K. pneumoniae 535 AutoFlex MALDI-TOF mass spectrometer/MALDI Biotyper (Bruker Daltonik2)

Biotype Clone

+ (biotype) - (clone) (455)

1Applied Biosystems, Foster City, California, USA; 2Bruker Daltonics, GmbH, Germany; 3Applied Maths,

Kortrijk, Belgium; 4MathWorks, Natick, MA, USA; 5Eigenvector Research, Manson, WA, USA; 6BioMérieux,

Marcy l'Etoile, France; 7 University of Oslo, Norway; 8Cartwright Group PTCL, University of Oxford; United

Kingdom; 9 http://www.R-project.org; 10Saramis; Shimadzu-Biotech, Corp., Kyoto, Japan. +, discrimination level pretended obtained; - the discrimination level pretended was not obtained.

aPeak absence at 9060 m/z (HdeB protein) in O157:H7 strains; bpeak presence at 6040 m/z in O157:H7

strains; cpeak shift from 9749 to 9716 m/z (HdeA protein) in B2 strains; dpeak presence at 4858 m/z in B2

strains; epeak presence ate 8349 m/z in B2-ST131 strains; fpeak presence at 11716 m/z in O16-B2-ST131

strains; gpeak presence at 6612 and 10474 m/z in B2-ST131 strains; hpeak shift at 4174, 7650 (YahO protein)

and peak presence at 10693 m/z in B2-ST131 strains.

Nowadays, MALDI-TOF MS is competing with WGS for typing routine

implementation, and in both methodologies important investments are needed, mainly

in order to automatize the analysis software for typing (435,456).


66

1.2.4. Metabolomics

The main purpose of metabolomics techniques is identifying/characterizing

certain small metabolic products of a biological system. Within metabolomics

techniques, vibrational spectroscopic such as Fourier transform infrared (FTIR) and

Raman, are recognized as rapid whole-organism fingerprinting methods that generate

metabolic fingerprints from a bacterial cell. They reflect the phenotype of the

microorganism under investigation, through the detection of a diversity of biomolecules

(lipids, polysaccharides, proteins and nucleic acids) (457,458). These technologies have

been widely used since the 1970s for bacterial identification, but it was only with the

recent development of highly sophisticated spectrometers (as happen with MALDI-TOF

MS) supporting high sample throughput and cost reductions, that it was possible to

improve the resolution and sensitivity in order to differentiate bacteria at different

taxonomic levels. Different sampling techniques such as transmittance diffuse

reflectance and attenuated total reflectance (ATR) in FTIR, or surface-enhanced Raman

spectroscopy (SERS) are used. These high-throughput methodologies are rapid, simple

(a minimal sample processing is required) and cost-effective. Variability between the

spectra needs to be interpreted using pattern-recognition tools of spectral data such as

multivariate data analysis (MDA). MDA or chemometrics uses mathematical methods to

analyse and extract relevant information from chemically complex data, as that

generated by FTIR, Raman or even MALDI-TOF MS spectra obtained from a bacterial

cell (458–461).

FTIR spectra result from the interaction of infrared radiation with the bacterial

isolate, providing a specific fingerprint that reflects the structure and composition of the

whole cell, but particularly of the components of the bacterial surface (Figure 26). The

spectral region corresponding to the phospholipids/DNA/RNA and polysaccharides

vibrations (1500-900 cm-1) is generally the most discriminatory one, and hence has been

the widely used for characterization (458,460).

FIGURE 26. FTIR bacterial characteristic spectrum.

lipid

s

prot

eins

fosf

olip

ids/

DNA/

RNA

poly

sacc

harid

es

finge

rprin

t

3000–2800 cm-1

1700–1500 cm-1

900–600 cm-1

1500–1185 cm-1

1185-900 cm-1


67

In fact, FTIR has already demonstrated an interesting potential for

discrimination at different taxonomic levels in E. coli (discrimination of main high-risk

clones; O157:H7 subtyping) (462–464) and many other different bacterial species (S.

aureus, S. pneumoniae, Listeria monocytogenes, Y. enterocolitica, Salmonella spp., A.

baumannii) (460,465–467). Moreover, the application of FTIR-based capsular typing

has shown good results for distinct species, such as S. aureus and S. pneumoniae

(466,467), enhancing the interest of this technique as a rapid capsular typing tool, which

has never been explored for K. pneumoniae.

Raman spectroscopy is based on the inelastic scattering of monochromatic light

by molecules, usually from a laser in the visible, near infrared, or near ultraviolet range.

The interaction of the laser with different biomolecules results in molecular vibrations

and changes in wavelength, which are molecule-specific and reflected in Raman

spectrum. The potential of Raman spectroscopy as a bacterial strain typing tool has also

been evaluated in some studies in different species (P. aeruginosa, Acinetobacter spp., S.

aureus, methicillin-resistant coagulase-negative Staphylococcus), although in a smaller

number compared to FTIR (468–471). In E. coli and K. pneumoniae, the few studies

regarding the typeability potential of Raman spectroscopy were performed using

SpectraCellRA (River Diagnostics, The Netherlands) and results analysed with a

commercial software, with this approach showing a discriminatory power comparable to

that obtained with PFGE in both species (472). In another study analysing only

Klebsiella spp. strains, no correlation was obtained between Raman and MLST-based

assignments (473). In these species, more studies using Raman spectroscopy are needed

in order to evaluate its potential as a typing tool.

An ideal typing method should be accurate, quick in sample preparation and

result analysis, low-cost and reproducible. Classical bacterial strain typing methods

based on serological or biochemical properties were historically important as typing

tools, but genotyping methods, such as PFGE and MLST, were crucial for defining the

population structure of E. coli and K. pneumoniae. However, these methodologies are

still time-consuming, expensive, and in the case of PFGE sample preparation is highly

laborious, whereas discriminatory power of MLST has been questioned. The

development of accessible WGS platforms revolutionized the high-resolution typing of

MDR E. coli and K. pneumoniae populations (Table 10). Nevertheless, its use as a typing

tool is also still expensive, time-consuming and requires some bioinformatics expertise,

mainly for phylogenetic analysis (Table 10). Alternatives, such as MALDI-TOF, FTIR or

Raman spectroscopy, could answer to the requirements of speed and low-cost methods

for bacterial typing. However, the routine implementation of these techniques as typing


68

tools still need efforts regarding the standardization of protocols according to the

species, automatization of the analytical process of the results and creation of robust

reference spectrum libraries (Table 10).

TABLE 10. Overview, advantages and disadvantages of the main bacterial strain typing methods currently used for E. coli and K. pneumoniae populations.

aCharacteristics ranged from +, which is low, to +++, which is high. bCharacteristics ranged from +, which is minimum sample processing, to +++, which is complexity of sample processing. cIt depends of the goal. Phylogenetic analysis (WGS) requires bioinformatic expertise, whereas in silico software tools for typing (e.g. MLST, serotyping) are easier to use.

Target molecules

Costa Timea Sample

processingb Analytical

complexitya DNA Proteins Whole

cellular content

PFGE + + +++ +++ +++ MLST + ++ ++ ++ + WGS + +++ ++ +++ +++c MALDI-TOF MS + + + + +++ FTIR/Raman spectroscopy

+ + ++ + +++

References

69

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“Imagination is more important than knowledge.”

Albert Einstein

Objectives and outline of the study

Chapter 2

Chapter 2 – Objectives and outline of the study

115

2.1. Statement of objectives

Antibiotic resistance (ABR) is a growing global threat and a significant social and

economic challenge. One of the main issues is the exponential rise of infections caused by

Escherichia coli and Klebsiella pneumoniae resistant to multiple clinically important

antibiotics for human medicine (extended-spectrum β-lactams, fluoroquinolones and/or

aminoglycosides)

(http://ecdc.europa.eu/en/publications/_layouts/forms/Publication_DispForm.aspx?Lis

t=4f55ad51-4aed-4d32-b960-af70113dbb90&ID=1637). Increasing resistance levels to

extended-spectrum β-lactams [extended-spectrum cephalosporins (ESC) and more

recently to carbapenems] and to other last resort antibiotics, such as fosfomycin or

colistin, results in a depletion of therapeutic options to treat infections caused by these

bacterial pathogens, threatening our ability to cure even the simplest infections.

Expansion of multidrug resistant (MDR) Escherichia coli and Klebsiella

pneumoniae strains has been greatly driven by the acquisition of genes conferring

resistance to extended-spectrum β-lactams [mainly by production of extended-spectrum

β-lactamases (ESBLs) or carbapenemases] and other co-determinants of resistance.

However, fluctuations were observed in the relative prevalence of these species

throughout the years, linked to variable molecular epidemiological traits. While before

2000s, K. pneumoniae resistant to ESC predominated among hospital acquired infections

linked most frequently to production of TEM- or SHV-type ESBLs, the remarkable

increase of E. coli resistant to ESC in either hospital or community-acquired infections

observed since 2000s was mainly driven by acquisition of CTX-M-type ESBL enzymes and

less frequently carbapenemases. More recently (from mid 2000s), we assisted to a global

expansion of ESC resistant K. pneumoniae, linked to production of ESBLs (mainly CTX-

M-15 and in a lesser extent SHV-type variants), and especially to the global expansion of

different carbapenemases, which rendered this species an increased noteworthiness

recognized by different world health organizations (ECDC, CDC, WHO). In Portugal, CTX-

M-producing E. coli are endemic since long time in different clinical settings, and among

non-human sources (food-producing, companion and wild animals or environment), but

comprehensive multilevel analysis of recent bacterial collections is lacking. Conversely,

the emergence and expansion of ESC resistant K. pneumoniae in the hospital setting is a

more recent event (since at least 2010) (https://www.dgs.pt/estatisticas-de-

saude/estatisticas-de-saude/publicacoes/portugal-controlo-da-infecao-e-resistencia-aos-

antimicrobianos-em-numeros-2015-pdf.aspx), justifying the poor characterization of the

molecular basis for this scenario.


116

In the last years, the large-scale application of multilocus sequence typing (MLST)

as a universal typing tool and more recently of whole genome sequencing (WGS) unveiled

global clonal expansion events of a small fraction of E. coli (e.g. ST131) or K. pneumoniae

[e.g. clonal group (CG)258] populations. Moreover, the increased resolution of WGS and

the availability of molecular tools for strain discrimination (e.g. identification of ST131

subclones or wzi/wzc-based capsular typing in K. pneumoniae) and plasmid typing (PCR-

based replicon typing) allowed a more precise definition of the dispersion drivers (clones,

lineages, plasmids) of ABR genes, particularly among isolates causing hospital infections.

But ABR is a global and borderless problem that needs to be tackled by a holistic

approach. For this reason, it is important to understand the contribution of other clinical

settings in close relationship with hospitals such as nursing homes (NHs) or long-term

care facilities (LTCFs), or the role of the human intestinal flora as reservoirs of particular

clinically relevant E. coli and K. pneumoniae drivers. However, they all have been

underexplored in Portugal.

Furthermore, the accurate and quick detection of E. coli and K. pneumoniae clones

or lineages with enhanced ability to spread and persist and exhibiting particular MDR

phenotypes is crucial for surveillance at local, regional, national or global levels, to guide

infection control measures in order to block transmission, and last but not least, to

underpin informed therapeutic decisions. Although the extremely important advances in

DNA-based molecular typing tools in the definition of E. coli and K. pneumoniae

population structures, most of these methods are still expensive, time-consuming and/or

require specialized skills, hindering their implementation in routine clinical laboratories.

Thus, alternative methods offering a high-throughput and a low-cost and quick

performance are highly demanded. Some progresses have been attained with Fourier

transform infrared (FTIR) spectroscopy for a few bacterial species, or matrix assisted laser

desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), already

thoroughly adopted in clinical microbiology laboratories for species identification,

including E. coli (Sousa et al., Sci Rep 2013, 20:3278; Novais et al., Eur J Clin Microbiol

Infect Dis. 2014, 33:1391-9), paving the way for further developments on K. pneumoniae

and envisioning a bright future for real-time application for typing. Since these

methodologies are based on different non-DNA-based principles and target different

biomolecules from the bacterial cell, they provide an opportunity to integrate

multidisciplinary data (genomics, proteomics, metabolomics), and can additionally

provide relevant insights into key molecular features that can be of utmost interest to

understand bacterial evolution, pathogenesis and host-pathogen interactions.


117

To accomplish these purposes, recent E. coli and K. pneumoniae isolates non-

susceptible to extended-spectrum β-lactams from different clinical (hospital, community,

LTCFs, NHs) and non-clinical (healthy volunteers) human settings mainly from the North

region of Portugal (2006-2016) were screened for the expression of extended-spectrum

acquired β-lactamases (ESBLs, carbapenemases and/or plasmid-acquired AmpCs), and

the drivers (clones, plasmids, transposons and integrons) associated with its emergence

and/or expansion were characterized. Furthermore, an international collection (Portugal,

Spain, Romania, Brazil, Poland, Greece; 2003-2015) representing MDR K. pneumoniae

clonal lineages globally disseminated and associated with the production of ESBLs,

carbapenemases and/or plasmid-acquired AmpCs, as well as K. pneumoniae CG14 and

CG15 genomes included in public databases (NCBI, BIGSdb), were used to assess the

potential of different high-throughput methodologies (FTIR, MALDI-TOF MS and WGS)

for typing and characterization of K. pneumoniae.

The main goals of this work are: I. To establish the current epidemiological scenario involving acquired resistance to

extended-spectrum β-lactams in recent collections of E. coli and K. pneumoniae

from different clinical and non-clinical human settings in Portugal by a

comprehensive and detailed characterization of clones and mobile genetic elements.

II. To explore the potential of different DNA- and non-DNA-based high-throughput

omics approaches for accurate and high-resolution typing of MDR K. pneumoniae

strains, and ultimately to understand key molecular features on the basis of the

discriminatory potential.

The hypothesis of this Thesis is that a detailed characterization of drivers (clones,

plasmids and antibiotic resistance genes) involved in the epidemiology of

contemporary E. coli and K. pneumoniae strains resistant to extended-spectrum β-

lactams in clinical and non-clinical human settings would contribute to better

understand host adaptation processes, as well as dissemination and persistence

abilities of resistant strains. Additionally, we also hypothesized that high-

throughput approaches based on different biomolecules of K. pneumoniae might

constitute alternative methods for rapid and cost-effective routine typing of this

species.


118

The specific aims of this Thesis are:

1. To assess recent epidemiological changes on ESBL-producing Enterobacteriaceae

identified in different Portuguese hospitals in two-time periods (n=200; 2006–

2007 and 2010).

2. To extend the knowledge on human reservoirs of E. coli and K. pneumoniae

producing different extended-spectrum acquired β-lactamases (ESBLs,

carbapenemases, plasmid-acquired AmpCs) in residents of Portuguese LTCFs and

NHs, and in healthy volunteers (n=47, n=20 and n=199 samples, respectively;

2013-2016).

3. To establish possible origins or transmission pathways of E. coli and K.

pneumoniae carrying extended-spectrum acquired β-lactamases between the

different clinical and non-clinical human niches by conducting a detailed and

accurate multilevel population analysis of clones/lineages and plasmids.

4. To monitor the emergence and to characterize the epidemiological features of E.

coli and K. pneumoniae with acquired resistance genes to carbapenems,

fosfomycin and colistin in Portuguese clinical institutions (hospitals, community

laboratories, LTCFs and NHs) (n=493; 2011-2015).

5. To assess the suitability of high-throughput spectroscopic (FTIR; n=158; 2003-

2015) and spectrometric (MALDI-TOF MS; n=83; 2003-2012) methods for

accurate, quick and low-cost typing of clinically relevant MDR K. pneumoniae

isolates.

6. To perform a comparative high-resolution genomic analysis of globally

disseminated MDR K. pneumoniae CG14 and CG15 isolates (n=99; 1986-2015), in

order to elucidate factors (phylogeny, cps evolution, plasmidome) that might have

contributed to its global expansion.


119

2.2. Outline of the thesis This Thesis is organized in four chapters as follows:

Chapter 1 which presents an overview of the state of the art regarding the topics of this

Thesis, and is subdivided in two sections. Section 1.1. includes an overview of the

problematic of ABR (and particularly of the increasing incidence of infections caused by

MDR E. coli and K. pneumoniae), contemporary molecular epidemiological trends

involving resistance to extended-spectrum β-lactams (ESC and carbapenems), fosfomycin

and colistin, and the clinical relevance, virulence, population structure and plasmids

involved in the current global scenario of MDR E. coli and K. pneumoniae. Section 1.2.

presents an overview of the diverse typing methods used for E. coli and K. pneumoniae

strains and its evolution throughout time, detailing the main achievements, advantages,

disadvantages and future perspectives.

Chapter 2 includes the objectives and outline of the Thesis.

Chapter 3 presents the findings that answer to the specific aims of the Thesis. Results

from the experimental research performed in this Thesis have been organized through

research articles (n=12; 7 publications in peer review journals and 5 manuscripts in

preparation). This chapter is divided in three sections:

Section 3.1. Multi-niche dispersal of contemporary antibiotic resistance genetic backgrounds associated with Escherichia coli from human origin. This section presents the occurrence of clinically relevant genes encoding acquired

resistance to extended-spectrum β-lactams (bla genes) among E. coli. Besides, a detailed

characterization of population structure and/or plasmids involved in the dissemination of

these genes in different clinical (hospitals, community) and non-clinical (healthy

volunteers) human settings in Portugal (2006-2014) was also performed, highlighting

recent changes in the molecular epidemiology of ESBL-producing E. coli. Moreover, the

first plasmid-mediated resistance gene to fosfomycin (fosA3) is described in Europe in a

CTX-M-15-producing E. coli isolate. Results were organized in the following research

articles:

• Rodrigues C, Machado E, Pires J, Novais Â, Peixe L. 2015. Increase of

widespread A, B1 and D Escherichia coli clones producing a high-diversity of CTX-

M-types in a Portuguese hospital. Future Microbiology. 10(7):1125-31.


120

• Rodrigues C, Machado E, Fernandes S, Leixe P, Novais Â. 2017. Occurrence of

extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Portuguese

nursing homes: the identification of different B2-ST131 subclones (fimH30,

fimH22) (manuscript final draft).

• Rodrigues C, Machado E, Fernandes S, Peixe L, Novais Â. 2016. An update on

faecal carriage of ESBL-producing Enterobacteriaceae by Portuguese healthy

humans: detection of the H30 subclone of B2-ST131 Escherichia coli producing

CTX-M-27. Journal of Antimicrobial Chemotherapy. 71(4):1120-2.

• Constança AC, Rodrigues C, Pires J, Amorim J, Ramos H, Novais Â, Peixe L.

2016. Importation of fosfomycin resistance fosA3 gene to Europe. Emerging

Infectious Diseases. 22(2):346-8.

Section 3.2. Elucidating the drivers for expansion of Klebsiella pneumoniae resistant to extended-spectrum β-lactams in different Portuguese clinical settings. The expansion of K. pneumoniae isolates producing ESBLs and/or carbapenemases in

different Portuguese clinical settings (hospitals, LTCFs and NHs) is analysed (2006-2016).

The contribution of clones/lineages and/or mobile genetic elements (plasmids,

transposons, integrons) in the dissemination of genes encoding acquired resistance to

extended-spectrum β-lactams is also assessed, pointing-out the existence of prevalent K.

pneumoniae lineages circulating in our country. Moreover, a new variant of VIM-1

carbapenemase, namely VIM-34, is firstly described in our country. The data obtained is

presented and organized in the following research articles:

• Rodrigues C, Machado E, Peixe L, Novais Â. 2014. Expansion of ESBL-producing

Klebsiella pneumoniae in hospitalized patients: a successful story of international

clones (ST15, ST147, ST336) and epidemic plasmids (IncR, IncFIIK). International

Journal of Medical Microbiology. 304(8):1100–08.

• Rodrigues C, Bavlovič J, Machado E, Amorim J, Peixe L, Novais Â. 2016. KPC-3-

Producing Klebsiella pneumoniae in Portugal Linked to Previously Circulating

Non-CG258 Lineages and Uncommon Genetic Platforms (Tn4401d-IncFIA and

Tn4401d-IncN). Frontiers in Microbiology. 7:1000.

• Rodrigues C, Novais Â, Machado E, Peixe L. 2014. Detection of VIM-34, a novel

VIM-1 variant identified in the intercontinental ST15 Klebsiella pneumoniae clone.

Journal of Antimicrobial Chemotherapy. 69(1):274-5.

• Rodrigues C, Mendes AC, Sigma F, Bavlovič J, Machado E, Novais Â, Peixe L.

2017. High rates of long-term care facilities (LTCFs) residents colonized with


121

multidrug resistant Klebsiella pneumoniae lineages frequently causing infections

in Portuguese clinical institutions (manuscript final draft).

Section 3.3. Detailing multidrug resistant (MDR) Klebsiella pneumoniae by omic approaches: from classical to high-throughput modern typing. The potential of high-throughput technologies, such as FTIR, MALDI-TOF MS and WGS

coupled with different bioinformatic tools for delineation and characterization of clinically

relevant MDR K. pneumoniae strains is evaluated in an international collection of isolates

(Portugal, Spain, Romania, Brazil, Poland, Greece; 2003-2015), and also in isolates from

public databases (NCBI, BIGSdb). Results obtained in the context of the application of

FTIR in the rapid and accurate detection of an outbreak of KPC-producing K. pneumoniae

are also presented. Results achieved are presented in the following research articles:

• Rodrigues C, Sousa C, Lopes JA, Novais Â, Peixe L. 2017. Congruence between

capsular genotypic and phenotypic features of multidrug-resistant (MDR)

Klebsiella pneumoniae clones: a step-forward on K-typing by Fourier Transform

Infrared (FTIR) spectroscopy (manuscript final draft).

• Silva L, Rodrigues C, Sousa C, Lira A, Leão M, Mota M, Lopes P, Lameirão Â,

Abreu G, Lopes JA, Novais Â, Peixe L. 2017. Fourier Transform Infrared (FTIR)

spectroscopy based-typing for “real-time” analysis of an outbreak by carbapenem-

resistant Klebsiella pneumoniae isolates (manuscript final draft).

• Rodrigues C, Novais Â, Sousa C, Ramos H, Coque TM, Cantón R, Lopes JA, Peixe

L. 2017. Elucidating constraints for differentiation of major human Klebsiella

pneumoniae clones by MALDI-TOF MS. European Journal of Clinical

Microbiology and Infectious Diseases. 36(2):379-86.

• Rodrigues C, Lanza VF, Peixe L, Novais Â, Coque TM. 2017. High-resolution

analysis of the globally disseminated multidrug-resistant Klebsiella pneumoniae

Clonal Groups 14 and 15 (manuscript final draft).

Highlighted results from these research studies are reflected in the General Conclusions depicted in Chapter 4.

“A person who never made a mistake never tried anything new.”

Albert Einstein

Chapter 3

Results and discussion

Chapter 3 – Results and discussion

125

3.1. Multi-niche dispersal of contemporary antibiotic resistance genetic backgrounds associated with Escherichia coli from human origin

3.1.1. Increase of widespread A, B1 and D Escherichia coli clones producing a

high-diversity of CTX-M-types in a Portuguese hospital.

3.1.2. Occurrence of extended-spectrum β-lactamase (ESBL)-producing

Escherichia coli in Portuguese nursing homes: the identification of

different B2-ST131 subclones (fimH30, fimH22).

3.1.3. An update on faecal carriage of ESBL-producing Enterobacteriaceae by

Portuguese healthy humans: detection of the H30 subclone of B2-ST131

Escherichia coli producing CTX-M-27.

3.1.4. Importation of fosfomycin resistance fosA3 gene to Europe.

08 Fall


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Increase of widespread A, B1 and D Escherichia coli clones producing a

high-diversity of CTX-M-types in a Portuguese hospital

Carla Rodrigues1, Elisabete Machado1,2, João Pires1, Helena Ramos3,

Ângela Novais1, Luísa Peixe1*

1UCIBIO/REQUIMTE. Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2CEBIMED/FP-ENAS, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa,

Porto, Portugal; 3Centro Hospitalar do Porto - Hospital de Santo António, Porto, Portugal.

Future Microbiology 2015; 10(7):1125-31

Future Medicine Ltd has authorized the reproduction of the final published PDF version of this

paper in this thesis through the License Agreement Number 4074090243990 on March 1, 2017.


128


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SHORT COMMUNICATION Rodrigues, Machado, Pires, Ramos, Novais & Peixe

future science group

two million people) and comprising 12 months of two different time periods (June 2006–June 2007 [n = 43] and January–December 2010 [n = 69]) were characterized. They correspond to all ESBL-producing E. coli isolates identified in this hospital in both time periods, where only one isolate per patient and/or antibiotic resistant pat-tern was characterized. ESBL-producing E. coli isolates were mostly recovered from outpatients (49% in 2006–2007 and 70% in 2010) but also from medical (35% in 2006–2007 and 23% in 2010) or surgical wards (9% in 2006–2007 and 1% in 2010) and intensive care units (0% in 2006–2007 and 3% in 2010), and most of the isolates were identified in urine samples (78%). Bacterial identification and antimicrobial suscep-tibility testing for β-lactams and non-β-lactams were performed by automated systems (VITEK) and disk diffusion [12]. All intermediate-suscep-tible strains were considered resistant strains. Isolates resistant to extended-spectrum cepha-losporins were screened for ESBL production by the standard double disk synergy test (DDST) using cefotaxime, ceftazidime and amoxicillin-clavulanate on Mueller–Hinton agar plates with and without cloxacillin (250 mg/l) (Sigma-Aldrich, St Quentin-Fallavier, France) [12]. Presence of blaCTX-M, blaSHV and blaTEM genes was confirmed by PCR and sequencing [9]. Population structure characterization included identification of E. coli phylogenetic groups and detection of O25b-ST131 by PCR, XbaI-PFGE, MLST and Fourier transform infrared spectros-copy (FTIR) [8,13–15]. Allelic profiles of differ-ent sequence types (ST) were compared with

eBURST-like minimum spanning tree (MStree) method in Phyloviz 1.0 [16]. The content in 38 virulence factors was investigated by PCR in representative B2-ST131 isolates (n = 13, one per PFGE-type), as described [13]. Statistical significance for comparison of proportions was calculated by the χ2 or Fisher exact test using IBM SPSS Statistics 22.0 software (p-values of < 0.05 were considered statistically significant).

Results & discussionWe observed a significant change in the popula-tion structure of ESBL-producing E. coli, with an increase in the proportion of A, B1 and D phylogroups (from 21 to 54%, p < 0.001), pro-ducing most frequently diverse CTX-M enzymes (CTX-M-1, -2, -9, -14, -32, -79) (Tables 1 & 2). These enzymes were mainly identified in par-ticular clones belonging to widespread clonal complexes within A (ST10, ST23), B1 (ST155) and D (ST117, ST648) phylogenetic groups (Table 2 & Supplementary Figure 1). It is also of note that despite B2-ST131 E. coli (producing almost exclusively CTX-M-15) prevails as the predomi-nant clone in our collection, the proportion of this worldwide spread clone decreased signifi-cantly (from 79 to 45%; p < 0.001) throughout time (Table 1). SHV-12 and TEM-52 ESBLs were less frequently detected (Table 1). We cannot con-firm if this scenario is extended to other clinical institutions or will persist in time, but similar results were observed in recent (2011–2013) ESBL E. coli producers from the same hospital and also from a community laboratory from the north of Portugal (diversity of CTX-M enzymes,

Table 1. Temporal shifts in the composition of Escherichia coli phylogenetic groups and extended-spectrum β-lactamase types.

Number of isolates (%)

2006–7 (n = 43) 2010 (n = 69) p-value†

Escherichia coli phylogenetic groups (n/%) B2 (66/58.9%) 34 (79.1%) 32 (46.4%) 0.001B2-ST131 (65/58.0%) 34 (79.1%) 31 (44.9%) 0.001D (13/11.6%) 1 (2.3%) 12 (17.4%) 0.016A (16/14.3%) 3 (7.0%) 13 (18.8%) 0.142B1 (17/15.2%) 5 (11.6%) 12 (17.4%) 0.578A+B1+D (46/41.1%) 9 (20.9%) 37 (53.6%) 0.001

ESBLs (n/%) CTX-M-15 (67/59.8%) 35 (81.4%) 32 (46.4%) 0.001Other CTX-M (34/30.4%) (CTX-M-1, -2, -9, -14, -32, -79) 5 (11.6%) 29 (42.0%) 0.001SHV-12 (9/8.0%) 2 (4.7%) 7 (10.1%) 0.478TEM-52 (2/1.8%) 1 (2.3%) 1 (1.5%) 1.000†p-values are shown in bold when p < 0.05.

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Increase of E. coli clones producing CTX-M-types in a Portuguese hospital SHORT COMMUNICATION

future science group www.futuremedicine.com

Table 2. Epidemiological data of ESBL-producing E. coli recovered from a general Portuguese hospital in two different time periods (2006–2007 and 2010).

Year of isolation (number)

PhG† (number)

Sequence type (ST)/clonal complex (CC) (number of isolates/number of PFGE types)

ESBL (number) Non-β-lactam resistance phenotype‡

Medical units (number)

2006–7 (n = 43)

B2 (34) ST131/CC131 (34/2) CTX-M-15 (33) (AMK), CIP, (CLO), (GEN), KAN, NAL, (NET), (SUL), (STR), (TET), (TMP), TOB

Outpatients (14), medical wards (13), surgical wards (4), unknown (2)

CTX-M-32 (1) NAL, CIP, SUL, STR, TET, TMP Medical ward D (1) ST3177/CC648 (1/1) CTX-M-15 CIP, NAL, SUL, STR, TMP Outpatient A (3) ST410/CC23 (1/1) CTX-M-15 CIP, GEN, KAN, NAL, NET, STR, SUL,

TET, TMP, TOBUnknown

ST88 /CC23 (1/1) TEM-52 STR Outpatient ST10/CC10 (1/1) CTX-M-2 CIP, NAL, SUL, STR, TET, TMP Outpatient B1 (5) ST155/CC155 (2/1) CTX-M-1 CIP, (CLO), (KAN), NAL, STR, SUL,

TMPOutpatients

ST58/CC155 (1/1) SHV-12 AMK, CIP, KAN, NAL, NET, SUL, STR, TET

Outpatient

ST448/CC448 (1/1) CTX-M-14 CIP, KAN, NAL, SUL, STR, TET, TMP Outpatient ST212/- (1/1) SHV-12 CLO, NAL, SUL, STR, TET Medical ward2010 (n = 69)

B2 (32) ST131/CC131 (31/5) CTX-M-15 (29) (AMK), CIP, (CLO), (GEN), (KAN), NAL, (NET), (SUL), (STR), (TET), (TMP), (TOB)

Outpatients (19), medical wards (6), intensive care units (2), unknown (2)

CTX-M-1 (1) GEN, NAL, SUL, TET, TMP, TOB Medical ward CTX-M-14 (1) NAL, TET Outpatient ST4504/CC28 (1/1) CTX-M-2 SUL, STR, TET, TMP Outpatient D (12) ST117/CC117 (2/1) CTX-M-1 (NAL), KAN, (SUL), STR, TET, TMP Outpatient (1), medical

ward (1) ST2974/CC117 (1/1) SHV-12 CIP, NAL, SUL Outpatient ST57/CC350 (3/2) CTX-M-14 (2) CIP, CLO, NAL, (NIT), SUL, STR, TET,

(TMP)Surgical ward (1), medical ward (1)

CTX-M-1 (1) SUL, TET, TMP Outpatient ST354/CC354 (1/1) CTX-M-14 CIP, GEN, KAN, NAL, NET, STR, TOB Outpatient ST648/CC648 (1/1) TEM-52 CIP, NAL, GEN, STR, TET, TMP Outpatient ST1011/- (2/2) CTX-M-1 CIP, NAL, NIT, SUL, STR, TET, TMP Medical ward CTX-M-32 CIP, NAL, SUL, STR, TMP Outpatient ST457/- (1/1) CTX-M-79 CIP, GEN, NAL, SUL, STR, TET, TMP,

TOBMedical ward

ST363/-(1/1) CTX-M-1 KAN, NAL, SUL, TET Outpatient

A (13) ST10/CC10 (2/1) CTX-M-15 CIP, (GEN), KAN, NAL, (NIT), SUL, STR, (TET), TMP, TOB

Outpatients

ST167/CC10 (1/1) CTX-M-1 CIP, CLO, NAL, SUL, STR, TET, TMP Outpatient ST744/CC10 (1/1) SHV-12 CLO, CIP, KAN, NAL, SUL, STR, TET,

TMPmedical ward

ST4270/CC23 (2/1) CTX-M-14 (KAN), SUL, STR, TET Outpatient (1), medical ward (1)

ST93/CC168 (3/1) CTX-M-14 (1) CIP, KAN, NAL, SUL, STR, TET, TMP Outpatient CTX-M-32 (1) CIP, CLO, GEN, NAL, SUL, STR, TET,

TMP, TOBOutpatient

†PhG, E. coli phylogenetic group.‡Variable presence of resistance phenotype is indicated by parenthesis.AMK: Amikacin; CLO: Chloramphenicol; CIP: Ciprofloxacin; GEN: Gentamicin; KAN: Kanamycin; NAL: Nalidixic acid; NET: Netilmicin; NIT: Nitrofurantoin; STR: Streptomycin; SUL: Sulphonamides; TOB: Tobramycin; TET: Tetracycline; TMP: Trimethoprim;

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40–53% of B2-ST131 and 47–60% A, B1 and D E. coli), suggesting a broader extension [17,18]. Similar phylogroup proportions in ESBL-producing E. coli identified in recent reports from other countries suggest that this situation might be more common than assumed, possibly masked by the overall predominance of a single clone (B2-ST131) [19,20].

Antibiotic resistance profiles to non-β-lactams were variable according to each phylogenetic group, with high resistance rates observed for nalidixic acid (75–99%), ciprofloxacin (69–96%), tetracycline (65–92%), sulphonamides (64–94%) and trimethoprim (69–82%). B2 iso-lates were more frequently resistant to kanamy-cin and tobramycin (92%), gentamycin (81%), netilmicin (71%) and amikacin (62%), while A, B1 and D isolates were commonly resistant to streptomycin (85–100% vs 67% in B2 isolates) (Table 2).

B2-E. coli were much more frequently observed in 2006–2007 (79%) than in 2010 (46%), mostly associated in both time periods with the production of CTX-M-15 (n = 62/94%; n = 33 in 2006–2007 and n = 29 in 2010), and rarely other CTX-M types (n = 4/6%; n = 1 in 2006–2007

and n = 3 in 2010; CTX-M-1, -2, -14, -32) (Tables 1 & 2). They mainly belonged to diverse ST131 (n = = 65/66, 6 PFGE-types) (Table 2 & Supplementary Figure 1). To the extent of our knowledge this is the first study highlighting a decrease in the pro-portion of CTX-M-15-producing ST131 E. coli over time in a given clinical context. ST131 vari-ants (differing in PFGE-type and virulence gene content) were unevenly distributed throughout time. Most isolates (n = 60/65, 1 PFGE-type) corresponded to the arbitrarily designated viro-type C (fimH-iha-traT-usp-sat-malX-fyuA-iutA-ompT-kpsMTII-K5) circulating since 2006–2007, which corresponds to the most disseminated ST131 variant worldwide [1,13]. Variants to this profile were identified more frequently in 2010 (n = 9 isolates; 6 PFGE-types), namely virotype A (including afa/draBC), virotype B (including iroN) or virotype D5 (including ibeA, cnf1 and hlyA) [1]. Although nosocomial transmission of ST131 E. coli isolates could have occurred, the diversity of PFGE-types, virotypes and blaCTX-M genes and the increasing proportion of ST131 iso-lates from outpatients (from 41% in 2006–2007 to 65% in 2010) suggests multiple entries from community reservoirs.

Year of isolation (number)

PhG† (number)

Sequence type (ST)/clonal complex (CC) (number of isolates/number of PFGE types)

ESBL (number) Non-β-lactam resistance phenotype‡

Medical units (number)

2010 (n = 69) (cont.)

CTX-M-1 (1) NAL, SUL, TET, TMP Outpatient

ST1594/CC168 (1/1) SHV-12 STR Outpatient ST4269/- (2/1) CTX-M-32 CIP, (KAN), NAL, (SUL), STR, TET,

(TMP), TOBOutpatients

ST4484/- (1/1) CTX-M-15 CIP, KAN, NAL, SUL, STR, TMP Outpatient B1 (12) ST453/CC86 (2/2) CTX-M-14 SUL, STR, TET, TMP Outpatient SHV-12 CIP, CLO, NAL, SUL, STR, TET, TMP Medical ward ST58/CC155 (1/1) CTX-M-14 SUL, STR, TMP Medical ward ST2229/CC101 (1/1) CTX-M-14 CLO, SUL, STR, TET, TMP Medical ward ST205/CC205 (1/1) CTX-M-1 CIP, KAN, NAL, SUL, STR, TMP Outpatient ST224/- (2/2) SHV-12 CIP, CLO, NAL, SUL, STR, TET, TMP Outpatient CTX-M-9 CIP, NAL, SUL, STR, TET, TMP Outpatient ST1196/- (2/1) SHV-12 CIP, CLO, (KAN), NAL, SUL, STR,

TET, TMPOutpatients

ST1642/- (2/1) CTX-M-14 CIP, CLO, GEN, KAN, NAL, SUL, STR, (TET), TMP

Outpatients

ST4476/- (1) CTX-M-14 CIP, NAL, STR Outpatient†PhG, E. coli phylogenetic group.‡Variable presence of resistance phenotype is indicated by parenthesis.AMK: Amikacin; CLO: Chloramphenicol; CIP: Ciprofloxacin; GEN: Gentamicin; KAN: Kanamycin; NAL: Nalidixic acid; NET: Netilmicin; NIT: Nitrofurantoin; STR: Streptomycin; SUL: Sulphonamides; TOB: Tobramycin; TET: Tetracycline; TMP: Trimethoprim;

Table 2. Epidemiological data of ESBL-producing E. coli recovered from a general Portuguese hospital in two different time periods (2006–2007 and 2010) (cont.).



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E. coli isolates from phylogroups A, B1 and D were genetically diverse (n = 13, n = 14 and n = 12 PFGE-types, respectively) and produced a high diversity of ESBLs (CTX-M-1, -2, -9, -14, -15, -32, -79, TEM-52 or SHV-12), with the exception of phylogroup B1 that produced mainly CTX-M-14 or SHV-12 (n = 13/17, 77%) (Table 2 & Supplementary Figure 1). Interestingly, these isolates were mainly recovered from out-patients (n = 34/74%), which is in line with the identification of these enzymes (CTX-M-1, -14, -32; SHV-12 and TEM-52) over the years in diverse nonclinical niches in Portugal (Supplementary Figure 2) and elsewhere [4,21]. Thus, it seems reasonable to hypothesize that the clonal expansion of B2-ST131 E. coli since the beginning of 2000’s might have masked the role of nonclinical reservoirs in the landscape of ESBL-producing E. coli in the clinical setting.

E. coli belonging to phylogroup D, which sig-nificantly increased in 2010, were linked to wide-spread ST117 (2 ST117, 1 ST2974), ST350 (3 ST57) and ST648 (1 ST648, 1 ST3177) clonal complexes (Table 2 & Supplementary Figure 1), which are first described in Portugal. Interestingly, these STs, primarily associated with nonhuman hosts [3–4,21–22], have recently been reported in

human clinical isolates in different countries (mainly ST648) associated with ESBLs and/or carbapenemases [2,6–7,23–25], suggesting frequent and continuous intercompartment flux (hospital-ized or community patients, healthy humans, ani-mals and environment). The absence of D-ST69, D-ST393 and D-ST405 E. coli clones, which are often observed in clinical samples from other geo-graphic regions, including in Spain, a neighboring country, was also noted [5,8].

Among E. coli phylogroups A and B1, most of the isolates (n = 23/70%) belonged to clones or clonal complexes (ST10, ST23, ST168, ST155, ST224, ST101, ST448) repeatedly described in dif-ferent studies and niches, most of them identified after 2006–2007 (Table 2 & Supplementary Figure 1). ESBL-producing A and B1 E. coli (and particularly the main clonal complexes identified in this work) have been more frequently associated with food-producing animals and food products, including in Portugal (Supplementary Figure 2) [3,4], but also with ESBL producers from hospitals and human community settings worldwide [2,7,19–20,24,26].

Conclusion & future perspectiveIn conclusion, we observed a significant change in the population structure of ESBL-producing

EXECUTIVE SUMMARYBackground

● The phylogenetic group composition of ESBL-producing E. coli isolates (i.e., the relative frequency of the most frequent phylogroups B2, D, A and B1) in different settings is unbalanced.

● The B2-ST131 high-risk E. coli clone has been implicated in most nosocomial or community extraintestinal infections worldwide.

● Particular clones from A, B1 or D phylogroups (e.g., ST10, ST117 or ST648 clonal complexes) are more commonly detected in isolates from nonhuman sources.

Results

● The population structure of clinical ESBL-producing E. coli changed between 2006–2007 and 2010.

● Although CTX-M-15-producing B2-ST131 remains the predominant clone, its proportion decreased significantly throughout time.

● The proportion of clones from A (ST10, ST23), B1 (ST155) and D (ST117, ST648) phylogroups producing diverse CTX-M enzymes increased.

● CTX-M-1, CTX-M-32, CTX-M-14 or SHV-12 were more commonly detected in the recent period.

Conclusion

● The significant change in the population structure of ESBL-producing E. coli was linked to the amplification of diverse CTX-M-producing A, B1 and D clonal complexes.

● The distribution of these ESBLs and clones in Portuguese nonclinical niches unveils a role for these reservoirs in the landscape of ESBL-producing E. coli in the clinical setting.

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E. coli in the hospital studied, linked to an increase of diverse CTX-M enzymes other than CTX-M-15 and E. coli clones from A, B1 and D phylogroups. Most of them are widely distributed in nonclinical niches, and have long been identi-fied in diverse Portuguese nonclinical settings, unveiling a role for these reservoirs in the land-scape of ESBL-producing E. coli in the clinical setting. Future studies should be performed in order to evaluate the persistence of this epide-miological change in time and in other clinical institutions.

Financial & competing interests disclosureThis work received financial support from the European Union (FEDER funds) through Programa Operacional Factores de Competitividade – COMPETE and QREN, and from National Funds (Fundação para a Ciência e Tecnologia [FCT]) through grant numbers EXPL/DTP-EPI/0196/2012, FCOMP-01-0124-FEDER-027745,

PEst-C/EQB/LA0006/2013, UID/Multi/04378/2013 and NORTE-07-0124-FEDER-000066 and from an ESCMID research grant 2012. C Rodrigues was supported by a fellowship from FCT (SFRH/BD/84341/2012). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of researchThe authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addi-tion, for investigations involving human subjects, informed consent has been obtained from the participants involved.

ReferencesPapers of special note have been highlighted as:

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2 Park SH, Byun JH, Choi SM et al. Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli in the community and hospital in Korea: emergence of ST131 producing CTX-M-15. BMC Infect. Dis. 12, 149 (2012).

3 Manges AR, Johnson JR. Food-borne origins of Escherichia coli causing extraintestinal infections. Clin. Infect. Dis. 55(5), 712–719 (2012).

Escherichia coli

4 Ewers C, Bethe A, Semmler T, Guenther S, Wieler LH. Extended-spectrum beta-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin. Microbiol. Infect. 18(7), 646–655 (2012).

Escherichia coli

5 Blanco J, Mora A, Mamani R et al. National survey of Escherichia coli causing extraintestinal infections reveals the spread of drug-resistant clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 with high virulence gene content in Spain. J. Antimicrob. Chemother. 66(9), 2011–2021 (2011).

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8 Matsumura Y, Yamamoto M, Nagao M et al. Emergence and spread of B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups among extended-spectrum-beta-lactamase-producing Escherichia coli in Japan. J. Antimicrob. Chemother. 67(11), 2612–2620 (2012).

9 Machado E, Coque TM, Cantón R et al. High diversity of extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae from Portugal.

J. Antimicrob. Chemother. 60(6), 1370–1374 (2007).

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12 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Tweenty-first Informational Supplement. CLSI document M100-S21. PA, USA (2011).

13 Novais Â, Pires J, Ferreira H et al. Characterization of globally spread Escherichia coli ST131 isolates (1991 to 2010). Antimicrob. Agents Chemother. 56(7), 3973–3976 (2012).

E. coli

14 Sousa C, Novais Â, Magalhães A, Lopes J, Peixe L. Diverse high-risk B2 and D Escherichia coli clones depicted by Fourier transform infrared spectroscopy. Sci. Rep. 3, 3278 (2013).



134



15 University of Warwick. MLST Databases at UoW. http://mlst.ucc.ie/mlst/dbs/Ecoli

16 PHYLOViZ. www.phyloviz.net/wiki/

17 Pires J, Rodrigues C, Campainha R et al. ESBL-producing E. coli and K. pneumoniae: is the community a reservoir of widespread clones or clonal variants? Presented at: 24th European Congress of Clinical Microbiology and Infectious Diseases. Barcelona, Spain, 10–13 May 2014.

18 Novais Â, Rodrigues C, Pires J, Peixe L. Comparison of Escherichia coli ST131 H30 subclone with other ST131 variants and B2-non-ST131 isolates. Presented at: 24th European Congress of Clinical Microbiology and Infectious Diseases. Barcelona, Spain, 10–13 May 2014.

19 Peirano G, Asensi MD, Pitondo-Silva A et al. Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli from Rio de Janeiro, Brazil. Clin. Microbiol. Infect. 17(7), 1039–1043 (2011).

20 Rodriguez-Bano J, Mingorance J, Fernandez-Romero N et al. Virulence profiles of bacteremic extended-spectrum beta-lactamase-producing Escherichia coli: association with epidemiological and clinical features. PLoS ONE 7(9), e44238 (2012).

21 Liebana E, Carattoli A, Coque TM et al. Public health risks of enterobacterial isolates producing extended-spectrum beta-lactamases or AmpC beta-lactamases in food and food-producing animals: an EU perspective of epidemiology, analytical methods, risk factors, and control options. Clin. Infect. Dis. 56(7), 1030–1037 (2013).

22 Ewers C, Bethe A, Stamm I et al. CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J. Antimicrob. Chemother. 69(5), 1224–1230 (2014).

23 Hu YY, Cai JC, Zhou HW et al. Molecular typing of CTX-M-producing Escherichia coli isolates from environmental

water, swine feces, specimens from healthy humans, and human patients. Appl. Environ. Microbiol. 79(19), 5988–5996 (2013).

24 Oteo J, Diestra K, Juan C et al. Extended-spectrum beta-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2. Int. J. Antimicrob. Agents 34(2), 173–176 (2009).

25 Mushtaq S, Irfan S, Sarma JB et al. Phylogenetic diversity of Escherichia coli strains producing NDM-type carbapenemases. J. Antimicrob. Chemother. 66(9), 2002–2005 (2011).

26 Valverde A, Cantón R, Garcillan-Barcia MP et al. Spread of bla (CTX-M-14) is driven mainly by IncK plasmids disseminated among Escherichia coli phylogroups A, B1, and D in Spain. Antimicrob. Agents Chemother. 53(12), 5204–5212 (2009).

10.2217/FMB.15.38


135

CTX-M-15

CTX-M-1 CTX-M-14

CTX-M-2

TEM-52 SHV-12

CTX-M-32

CTX-M-79 CTX-M-9 2006-7 (n=43) 2010 (n=69)

Figure S1: Population structure of ESBL-producing E. coli. The minimum spanning tree shown was constructed on the basis of MLST allelic profiles by using Phyloviz1.0. Each circle corresponds to one sequence-type (ST), being its designation and the number of isolates shown inside it. Colors inside the circles represent different ESBL-types identified in the respective ST, as indicated in the upper right corner. STs belonging to known ST clonal complexes are surrounded by gray shading and designated in black. The links between circles indicated the number of allelic mismatches between STs.

B2-ST131

A-ST10

D-ST354

B2-ST28

A-ST168

B1-ST101

B1-ST205

A-ST23

B1-ST86

B1-ST155 D-ST648

D-ST117

(31)

(1)

(1) (2)

(2)

A

(1)

A

(2)

(1)

(3)

(1)

(1)

(1)

D

(1)

(2)

(2)

D

(1)

(1)

(2)

B1 (2)

B1

D

(1)

(1)

(1)

(2)

B1

(1)

B1

(2)

(3)

D-ST350

(1)

(34)

(1)

(1)

(1)

(1)

B1

(1)

(1)

(1)

(1)

(2)

(1)

(1)

D-ST648

B2-ST131

B1-ST155

A-ST23

B1-ST448

A-ST10

(1)

(34)

(1)

(1)

0

20

40

60

80

100

120

140

160

180

2001-04 2005-08 2009-12

Num

ber

of is

olat

es r

epor

ted

CTX-M-9

CTX-M-14

CTX-M-15

CTX-M-32

CTX-M-1

SHV-5

SHV-12

TEM-20 or -153

TEM-52

Figure S2. ESBL-producing E. coli isolates reported in non.clinical niches in Portugal in three time periods (2001-04, 2005-08 and 2009-12). E. coli ST clonal complexes identical to those identified in this study are shown (available only for 34/354 isolates in 6/25 studies). a One isolate co-producing TEM-20 (2005-08); b Two isolates co-producing TEM-52 (2001-04) or SHV-12 (2009-12).

a

b

B2-ST131 (n=5)

A-ST10 (n=6)

A-ST10 (n=3)

B2-ST131 (n=1) B2-ST131 (n=1)

A-ST10 (n=11) B1-ST155 (n=4) A-ST23 (n=1)

B1-ST155 (n=2)

D-ST354 (n=1) B1-ST101 (n=1) D-ST354 (n=1)

B1-ST101 (n=1)


136

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spectrum beta-lactamases in fecal Escherichia coli isolates of captive ostrich in Portugal.

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Escherichia coli strains from healthy pets in Portugal. J Antimicrob Chemother.54(5), 960-1

(2004).

4. Costa D, Poeta P, Saenz Y et al. Detection of Escherichia coli harbouring extended-spectrum

beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in

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lactamases in Escherichia coli isolates of pigs from a Portuguese intensive swine farm.

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137

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139

Occurrence of extended-spectrum β-lactamase (ESBL)-producing

Escherichia coli in Portuguese nursing homes: the identification of different B2-ST131 subclones (fimH30, fimH22)

Carla Rodrigues1, Elisabete Machado1,2, Sofia Fernandes1, Luísa Peixe1, Ângela Novais1*

1UCIBIO/REQUIMTE. Laboratório de Microbiologia, Faculdade de Farmácia,

Universidade do Porto, Porto, Portugal; 2FP-ENAS/CEBIMED. Faculdade de Ciências da

Saúde, Universidade Fernando Pessoa, Porto, Portugal.

Running title: CTX-M-producing B2-ST131 E. coli subclones in Portuguese nursing

homes

Keywords: colonization, CTX-M, B2-ST131, fimH subtypes, virotypes

*Corresponding author:

Ângela Novais REQUIMTE Researcher (Associate Laboratory).

Faculty of Pharmacy, University of Porto.

Rua Jorge Viterbo Ferreira 228, 4050-313 Porto Portugal.

Direct: (+351) 220 428 580;

Mobile: (+351) 966 228 110.

E-mail: [email protected]

Manuscript Final Draft


140

Sir,

Nursing homes (NHs) residents are known to be reservoirs of multidrug-resistant (MDR)

bacteria, mainly due to their frequent hospitalizations, recurrent use of invasive medical devices and

antibiotic consumption (1). The rates of intestinal colonization with extended-spectrum β-lactamase

(ESBL)-producing Enterobacteriaceae (ESBL-E) among NH residents vary greatly depending on the

geographic region analysed (6-47%), while carbapenemase-producing Enterobacteriaceae (CPE)

have rarely been reported (0.05-0.4%) (2–8). CTX-M-producing Escherichia coli Sequence Type

(ST) 131 clone dominates by far the population of MDR Enterobacteriaceae colonizing the intestine

of NH residents (6), but detailed analysis of ST131 subclonal structure has been scarce. In Portugal,

ESBL-E (and particularly E. coli or Klebsiella pneumoniae clones producing CTX-M-15) are

endemic for several years (9–11), whereas CPE are quickly penetrating in diverse clinical settings,

and especially on susceptible populations (12). The aim of this work was to perform a pilot study to

assess the current faecal carriage rate of ESBL-E or CPE, and the corresponding clonal and subclonal

structure, among NH residents in Portugal.

Fresh rectal swabs from 20 residents (10 females and 10 males) at 4 NHs (5 residents per

NH) located in the North of Portugal (situated at median distance of 5-6 Km between them) were

collected in July 2014 and analysed. Eighty-five percent of residents were ≥65 years old, 70% were

previously hospitalized and all of them received antibiotic treatment during the three months

preceding sampling. Samples were suspended in 2 mL of saline and screened for Enterobacteriaceae

resistant to third-generation cephalosporins and/or carbapenems by seeding 0.2 mL of the suspension

on CHROMagar™ Orientation plates supplemented with vancomycin (4 mg/L) plus ceftazidime (1

mg/L) or ertapenem (0.25 mg/L), respectively, and further incubation (37ºC/24h) (13). Presumptive

Enterobacteriaceae isolates (oxidase negative, each different morphotype per plate) were selected for

further studies. ESBLs and/or carbapenemases were identified by DDST and Blue-Carba test,

respectively, PCR and sequencing (13). Susceptibility testing to non-β-lactam antibiotics was

performed by the disk diffusion method (http://www.eucast.org/clinical_breakpoints/) and

presumptive E. coli ESBL producers were identified by species-specific PCR (13). The clonal

structure of ESBL-producing E. coli was analysed by identification of E. coli phylogenetic groups and


141

MLST (http://mlst.ucc.ie/mlst/dbs/Ecoli) (13). Subclonal typing of B2-ST131 isolates was performed

by PCR or PCR and sequencing of markers for O25b:H4, O16:H5, fimHTR allele and virulence genes

(ibeA-iroN-sat-afa/draBc-papG allele II/III-cnf1-hlyA-cdtB-K1) (13). Plasmid analysis included

replicon typing and subtyping (IncF plasmids) by PCR

(http://pubmlst.org/plasmid/primers/incF.shtml).

Intestinal colonization by ESBL-E was detected in 4/20 (20%) of the residents (Table 1).

Despite the low sample size, it is of relevance to point out the absence of CPE and an overall ESBL-E

colonization rate similar to that (24.5%) reported previously in our country in a larger sample

including NHs and long-term care facilities (11). However, the asymmetry observed between

institutions might uncover a different scenario (rates of colonization varied from 0% in NH1/NH2,

20% in NH3 and 60% in NH4). The four positive NH residents possessed recognized risk factors for

ESBL-E carriage such as previous antibiotic exposure (amoxicillin and clavulanic acid), and previous

hospitalization (at least two of them in the same hospital, L12 and L19). The overall rate of

colonization detected (20%) was higher than that reported in some European countries (6.2% in

Belgium and 9.9% in France), being only comparable to rates found in The Netherlands (20.6%) (2–

4).

All the ESBL producers were identified as E. coli producing CTX-M-15 (n=2; 2 samples) or

CTX-M-14 (n=2; 2 samples) from different residents (Table 1). The species and the ESBL-types

detected in our study are in line with the recent epidemiological trends in Portuguese hospitals (9),

and with other works conducted among NHs in different countries, including in Portugal (2,3,5,11).

All ESBL-producing E. coli identified belonged to the pandemic B2-ST131-O25b:H4 clone and

different lineages thereof (fimH30, fimH22) (Table 1). The fimH30 subclone producing CTX-M-15

(n=2) was identified in two residents from the same institution (NH4). It belonged to virotype C (sat),

presented a MDR pattern and harbored only N plasmid replicon (Table 1). This subclone corresponds

to the most worldwide disseminated within E. coli B2-ST131, including in Portugal (Novais Â,

unpublished results) (6). The fimH22 subclone producing CTX-M-14 was identified in residents from

NH3 and NH4, belonged to virotype D5 (ibeA, iroN, cnf1, hlyA), was resistant to different antibiotics

and carried a higher diversity of plasmid replicons (I1, HI2, ColE), including an F2:A-:B1 virulence


142

plasmid (resembling pAPEC-O2-ColV, GenBank accession number AY545598) (Table 1) (14). This

subclone, less frequent than fimH30 and usually linked to community-acquired infections (6), is firstly

described in our country. Interestingly, B2-ST131-fimH30 virotypes A and B, previously associated

with NH residents, were not detected in our sample (6).

In summary, this pilot study among NH residents in our country confirmed the high carriage

rates (20%) of CTX-M-producing E. coli B2-ST131 in this setting, and highlighted colonization by

different subclones (O25b:H4-CTX-M-15-fimH30-virotype C and O25b:H4-CTX-M-14-fimH22-

virotype D5). Further studies exploring the dimension of this problem in other institutions and

including subclonal characterization of ST131 isolates are necessary to assess transmission dynamics

of the different ST131 subclones ultimately to support adequate public health measures.

Acknowledgments: We are grateful to the residents, the healthcare personnel and the management

board of the nursing homes that participated in this study.

Funding: This work received financial support from the European Union (FEDER funds

POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e

Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020

UID/MULTI/04378/2013. CR and ÂN were supported by fellowships from FCT

(SFRH/BD/84341/2012 and SFRH/BPD/104927/2014, respectively).

Transparency declarations: Nothing to declare.

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145

Tabl

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Portu

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2014

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; b PhG,

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, Seq

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T, te

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; TM

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metho

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-type

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Resid

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(n=4

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) NH

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147

An update on faecal carriage of ESBL-producing Enterobacteriaceae by Portuguese healthy humans: detection of the H30 subclone of B2-

ST131 Escherichia coli producing CTX-M-27

Carla Rodrigues1, Elisabete Machado1,2, Sofia Fernandes1, Luísa Peixe1, Ângela Novais1*

1UCIBIO/REQUIMTE. Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2FP-ENAS/CEBIMED. Faculdade de Ciências da Saúde, Universidade Fernando Pessoa,

Porto, Portugal.

Journal of Antimicrobial and Chemotherapy 2016; 71(4):1120-2.

Oxford University Press has authorized the reproduction of the final published PDF version of this

paper in this thesis through the License Agreement Number 4091970612880 on April 18, 2017.


148

J Antimicrob Chemotherdoi:10.1093/jac/dkv443

An update on faecal carriage of ESBL-producing Enterobacteriaceae byPortuguese healthy humans: detectionof the H30 subclone of B2-ST131Escherichia coli producing CTX-M-27

Carla Rodrigues1, Elisabete Machado1,2,Sofia Fernandes1, Luısa Peixe1 and Angela Novais1*

1UCIBIO@REQUIMTE, Faculdade de Farmacia, Universidade doPorto, Porto, Portugal; 2FP-ENAS/CEBIMED, Faculdade de Ciencias daSaude, Universidade Fernando Pessoa, Porto, Portugal

*Corresponding author. Tel: +351-220-428-580; Fax: +351-222-003-977;E-mail: [email protected]

Sir,Multiple studies have documented increasing rates of intestinalcolonization by ESBL-producing Enterobacteriaceae (ESBL-E)in the community, with remarkable heterogeneity according tothe geographical region [Asia (2%–70%) and Africa (7%–31%)versus Europe (2%–12%)].1 CTX-M enzymes (mainly CTX-M-15or CTX-M-14) are predominant and observed mainly in diverseEscherichia coli genetic backgrounds.1 However, detailed charac-terization of E. coli isolates at clonal and subclonal levels is miss-ing, which is important to understand ecological and hostcolonization differences of specific pathogenic and/or MDR E. colilineages.2,3 In Portugal, a study conducted in healthy humans10 years ago revealed an ESBL-E carriage rate of 1.8%.4 In the con-text of current endemicity of ESBLs (especially CTX-M-15) and emer-gence of different carbapenemases in clinical settings,5,6 we aimedto assess the current faecal carriage rate and provide detailedmolecular data (clonal and plasmid subtyping) of ESBL- and/orcarbapenemase-producing Enterobacteriaceae in Portuguesehealthy humans.

Faecal samples from 199 randomly selected healthyhumans (aged .18 years) living in northern, central and south-ern regions of Portugal were collected between December 2013and May 2014. All participants provided written informed consentand completed a standardized questionnaire (Table S1, availableas Supplementary data at JAC Online). Samples (200 mL of a2 mL saline suspension of the rectal swab) were seeded ontoCHROMagarTM Orientation plates supplemented with vancomycin(4 mg/L) plus ceftazidime (1 mg/L) or ertapenem (0.25 mg/L) andincubated (378C for 24 h). All presumptive Enterobacteriaceae iso-lates (oxidase negative, approximately one to five unique morpho-types per plate) were further characterized. ESBL or carbapenemaseproduction was assessed by the double-disc synergy test orBlue-Carba, respectively, and further characterized by PCR andsequencing.5 Susceptibility to non-b-lactam antibiotics was

determined by the disc diffusion method (http://www.eucast.org/clinical_breakpoints/) and presumptive E. coli ESBL producers wereconfirmed by species-specific PCR. Population structure analysisperformed on representative ESBL-producing E. coli (according tomorphotype and antibiotic susceptibility pattern) included identifica-tion of E. coli phylogroups and virulence gene markers [extraintestinalE. coli and enteroaggregative E. coli (EAEC)] by PCR, XbaI-PFGE, MLST(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli)5,7,8 and subclonal typingof B2-ST131 isolates (screening of O25b:H4, O16:H5, fimHTR alleleand Rx clade by PCR or PCR and sequencing).5,7,9 Plasmid analysisincluded replicon typing and subtyping (IncF plasmids), S1/I-CeuI-PFGE and hybridization (http://pubmlst.org/plasmid/primers/incF.shtml).4

ESBL producers were detected in 2% (4/199) of the samples andidentified as E. coli producing CTX-M-14 (n¼4) and CTX-M-27(n¼2), with the latter being reported for the first time in our coun-try, whereas carbapenemase producers were not detected. Theywere recovered from volunteers presenting potential risk factorsfor the acquisition of ESBL-E (Table S1).1 Interestingly, our studyrevealed a similar carriage rate of ESBL-E to that observed10 years ago in our country (1.8%, 2/113),4 in contrast to theincreasing proportions reported in other follow-up studies in neigh-bouring European countries [e.g. 2-fold in Spain (2003 versus 2007)or 11-fold in France (2006 versus 2011)].1,2 The CTX-M typesidentified in our study have been more frequently associated withclinical settings or human intestinal carriage in Asian countries andSpain,1,10 but they are also increasingly being detected in clinicalsettings (!20% of all ESBL-producing E. coli between 2010 and2013 versus ,5% in previous years) or non-clinical niches in ourcountry (data not shown).4,5

CTX-M-14-producing E. coli belonged to phylogroups A0 (n¼3,two samples) or D1 (n¼1, one sample) (Table 1). A0-E. coli isolateswere assigned to ST226, a clone previously associated with theEAEC pathotype (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli), butspecific marker genes (astA, aggR and pCVD) were not detected.Its identification in two non-related participants suggests thatthis clone might be common in the human commensal flora(Table 1). The D1-E. coli isolate was assigned to ST59, exhibiting asimilar virulence gene content (fimH-papGI-papGII-sat-iutA-kpsMTII-K1-traT-malX-usp) and non-b-lactam resistance pattern(streptomycin) to that observed among CTX-M-14-producingE. coli of clinical origin from Spain.10 CTX-M-14 producers harbouredfour to seven different replicons with blaCTX-M-14 being locatedwithin an 80 kb IncK plasmid, a common platform circulating indifferent countries for years, including in Portugal (Table 1).4

CTX-M-27-producing E. coli isolates (n¼2, one sample)belonged to the worldwide-disseminated non-Rx H30 B2-ST131O25b:H4 subclone from virotype C (fimH-iha-traT-usp-sat-malX-fyuA-iutA-ompT-kpsMTII-K5)7,9 and presented an MDR pat-tern (Table 1). The blaCTX-M-27 gene was located in a 120 kbF1:A2:B20 plasmid, identified recently in commensal or clinicalST131 isolates in different countries.3,11 A high identity (PFGE,antibiotic resistance or virulence profiles, plasmids) was observedbetween these isolates and CTX-M-27 non-Rx H30 ST131 produ-cers recently emerging in different hospitals and communityclinical settings in our country (data not shown), suggesting a

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highly successful penetration of this ST131 subclone into ourpopulation. Highly homogeneous non-Rx H30 ST131-CTX-M-27isolates are also being increasingly reported in other Europeanand Asian countries, suggesting a wide diffusion probablyfavoured by globalization of human travel, globalization ofgoods and foods trade, and other unrecognized reasons.3,11,12

The absence of CTX-M-15-producing ST131 (and particularly theH30 Rx variant) in our sample might be explained by its apparentpreferential linkage to clinical settings in Portugal andelsewhere.5,7,9

Our study revealed an unexpected steady carriage rate of ESBLproducers in healthy humans in a 10 year period in our country,but a noteworthy enrichment of the human commensal flora incontemporary worldwide spread ESBLs (CTX-M-14-like enzymesencoded by common platforms) and clones (non-Rx H30 ST131,ST59). The dominance of particular E. coli subclones withenhanced transmissibility potential in the human intestinal com-mensal flora is worrying and needs to be monitored.

AcknowledgementsWe are very grateful to all persons who took part in this investigation,either as sample providers or recruiters. We are also grateful to LilianaSilva for the support in sample processing.

FundingThis work received financial support from the European Union (FEDER funds)through Programa Operacional Factores de Competitividade—COMPETE andQREN, and from National Funds (FCT) through projects PEst-C/EQB/LA0006/2013, UID/Multi/04378/2013 and NORTE-07-0124-FEDER-000066. C. R. andA. N. were supported by fellowships from FCT (SFRH/BD/84341/2012 andSFRH/BPD/104927/2014, respectively).

Transparency declarationsNone to declare.

Supplementary dataTable S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

References1 Woerther PL, Burdet C, Chachaty E et al. Trends in human fecal carriageof extended-spectrum b-lactamases in the community: toward the glo-balization of CTX-M. Clin Microbiol Rev 2013; 26: 744–58.

2 Nicolas-Chanoine MH, Gruson C, Bialek-Davenet S et al. 10-Fold increase(2006–11) in the rate of healthy subjects with extended-spectrumb-lactamase-producing Escherichia coli faecal carriage in a Parisiancheck-up centre. J Antimicrob Chemother 2013; 68: 562–8.

3 Zhong YM, Liu WE, Liang XH et al. Emergence and spread of O16-ST131and O25b-ST131 clones among faecal CTX-M-producing Escherichia coli inhealthy individuals in Hunan Province, China. J Antimicrob Chemother2015; 70: 2223–7.

4 Machado E, Coque TM, Canton R et al. Commensal Enterobacteriaceaeas reservoirs of extended-spectrum b-lactamases, integrons, and sulgenes in Portugal. Front Microbiol 2013; 4: 80.Ta

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5 Rodrigues C, Machado E, Pires J et al. Increase of widespread A, B1 and DEscherichia coli clones producing a high diversity of CTX-M-types in aPortuguese hospital. Future Microbiol 2015; 10: 1125–31.

6 Manageiro V, Ferreira E, Almeida J et al. Predominance of KPC-3 in a sur-vey for carbapenemase-producing Enterobacteriaceae in Portugal.Antimicrob Agents Chemother 2015; 59: 3588–92.

7 Nicolas-Chanoine MH, Bertrand X, Madec JY. Escherichia coli ST131, anintriguing clonal group. Clin Microbiol Rev 2014; 27: 543–74.

8 Campos J, Gil J, Mourao J et al. Ready-to-eat street-vended food as apotential vehicle of bacterial pathogens and antimicrobial resistance:an exploratory study in Porto region, Portugal. Int J Food Microbiol2015; 206: 1–6.

9 Banerjee R, Robicsek A, Kuskowski MA et al. Molecular epidemiology ofEscherichia coli sequence type 131 and its H30 and H30-Rx subclonesamong extended-spectrum-b-lactamase-positive and -negative E. coli

clinical isolates from the Chicago region, 2007 to 2010. AntimicrobAgents Chemother 2013; 57: 6385–8.

10 Mora A, Blanco M, Lopez C et al. Emergence of clonal groupsO1:HNM-D-ST59, O15:H1-D-ST393, O20:H34/HNM-D-ST354, O25b:H4-B2-ST131 and ONT:H21,42-B1-ST101 among CTX-M-14-producingEscherichia coli clinical isolates in Galicia, northwest Spain. Int JAntimicrob Agents 2011; 37: 16–21.

11 Blanc V, Leflon-Guibout V, Blanco J et al. Prevalence of day-care centrechildren (France) with faecal CTX-M-producing Escherichia coli comprisingO25b:H4 and O16:H5 ST131 strains. J Antimicrob Chemother 2014; 69:1231–7.

12 Matsumura Y, Johnson JR, Yamamoto M et al. CTX-M-27- andCTX-M-14-producing, ciprofloxacin-resistant Escherichia coli of the H30subclonal group within ST131 drive a Japanese regional ESBL epidemic.J Antimicrob Chemother 2015; 70: 1639–49.

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Importation of fosfomycin resistance fosA3 gene to Europe

Ana Constança Mendes1,2, Carla Rodrigues1, João Pires1, José Amorim3,

Helena Ramos2, Ângela Novais1,4*, Luísa Peixe1,4*

1UCIBIO/REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2Centro Hospitalar do Porto, Porto, Portugal; 3Botelho Moniz Análises Clínicas (BMAC),

Santo Tirso, Portugal

Emerging Infectious Diseases 2016; 22(2):346-8.

Open Access Journal.


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Appendix Table, http://wwwnc.cdc.gov/EID/article/22/2/ 15-1292-Techapp1.pdf).). P. falciparum infection was de-tected in 2 Anopheles species: 1 (12.5%) of 8 An. ininii and 1 (5.0%) of 19 An. nuneztovari s.l. mosquitoes collected; P. vivax infection was found in 1 (5.5%) of 19 An. nunezt-ovari s.l. mosquitoes.

In September 2013, another malaria outbreak occurred 3 weeks after the deployment of 15 soldiers in Dagobert (4.06028°N, -53.70667°E; Figure). The attack rate among these soldiers was 53.3% (8/15): 7 P. vivax infections and 1 co-infection with P. vivax and P. falciparum. Mosqui-toes were collected 3 months later by using human landing catches during 5 consecutive days. The area had been free of illegal gold mining activities since the 15 soldiers were deployed. A total of 321 Anopheles mosquitoes were col-lected in this location; 95.6% were identified as the same 4 species as in the Eau Claire mosquito collection (on-line Technical Appendix Table). Only 1 specimen (0.4%, 1/282), An. darlingi mosquito, was infected with P. vivax.

These results suggest a high level of malaria transmis-sion involving An. darlingi and other Anopheles species as primary vectors of malaria in the rainforest. The findings probably highlight malaria hyperendemicity in communi-ties of undocumented gold miners, who are often mobile and pose a challenge for controlling malaria and other in-fectious diseases in the region. Indeed, these gold miners could reintroduce malaria in areas where competent vectors exist in the coastal part of French Guiana and in Surinam and Brazil, which border French Guiana. This potential for transmission could seriously threaten the success of ma-laria elimination programs in the Guiana Shield. Further studies are needed to better evaluate malaria epidemiology in these undocumented populations to determine how best to adapt strategies to control malaria transmission in this subregion of South America.

AcknowledgmentsWe thank military physicians who participated in malaria epidemiologic surveillance in French Guiana and France during 2008–2014, especially E. de Parseval, N. Barthes, J.-P. Boudsocq, C. Ilcinkas, P.-A. Poutou, G. Samy, E. Martinez, F.-X. Le Flem, and C. Marchand. We also thank P. Gaborit, R. Carinci, and J. Issaly for their support in the entomologic studies.

References 1. Ardillon V, Carvalho L, Prince C, Abboud P, Djossou F.

Bilans 2013 et 2014 de la situation du paludisme en Guyane. Bulletin de veille sanitaire Antilles–Guyane. 2015 [cited 2015 Jul 15]. p. 16–20. http://www.invs.sante.fr/fr/Publications-et-outils/Bulletin-de-veille-sanitaire/Tous-les-numeros/Antilles-Guyane/Bulletin-de-veille-sanitaire-Antilles-Guyane.-n-1-Janvier-2015

2. Musset L, Pelleau S, Girod R, Ardillon V, Carvalho L, Dusfour I, et al. Malaria on the Guiana Shield: a review of the situation in French Guiana. Mem Inst Oswaldo Cruz. 2014;109:525–33. http://dx.doi.org/10.1590/0074-0276140031

3. Carme B. Substantial increase of malaria in inland areas of eastern French Guiana. Trop Med Int Health. 2005;10:154–9. http://dx.doi.org/10.1111/j.1365-3156.2004.01365.x

4. Berger F, Flamand C, Musset L, Djossou F, Rosine J, Sanquer MA, et al. Investigation of a sudden malaria outbreak in the isolated Amazonian village of Saul, French Guiana, January–April 2009. Am J Trop Med Hyg. 2012;86:591–7. http://dx.doi.org/10.4269/ajtmh.2012.11-0582

5. Migliani R, Pradines B, Michel R, Aoun O, Dia A, Deparis X, et al. Malaria control strategies in French armed forces. Travel Med Infect Dis. 2014;12:307–17. http://dx.doi.org/ 10.1016/j.tmaid.2014.05.008

6. Queyriaux B, Texier G, Ollivier L, Galoisy-Guibal L, Michel R, Meynard JB, et al. Plasmodium vivax malaria among military personnel, French Guiana, 1998–2008. Emerg Infect Dis. 2011;17:1280–2. http://dx.doi.org/10.3201/eid1707.100009

7. Floch H, Abonnenc E. Anophèles de la Guyane Française. Arch Inst Pasteur Guyane. 1951;236:1–92.

8. Beebe NW, Saul A. Discrimination of all members of the Anopheles punctulatus complex by polymerase chain reaction– restriction fragment length polymorphism analysis. Am J Trop Med Hyg. 1995;53:478–81.

9. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993;61:315–20. http://dx.doi.org/10.1016/ 0166-6851(93)90077-B

10. Vezenegho SB, Adde A, Gaborit P, Carinci R, Issaly J, Pommier de Santi V, et al. Mosquito magnet® liberty plus trap baited with octenol confirmed best candidate for Anopheles surveillance and proved promising in predicting risk of malaria transmission in French Guiana. Malar J. 2014;13:384. http://dx.doi.org/10.1186/1475-2875-13-384

Address for correspondence: Vincent Pommier de Santi, Military Center for Epidemiology and Public Health, Camp Militaire de Sainte Marthe,

BP 40026, 13568 Marseille CEDEX 02, France; email: [email protected]

Importation of Fosfomycin Resistance fosA3 Gene to Europe

Ana C. Mendes, Carla Rodrigues, João Pires, José Amorim, Maria Helena Ramos, Ângela Novais,1 Luísa Peixe1

Author affiliations: Universidade do Porto, Porto, Portugal (A.C. Mendes, C. Rodrigues, J. Pires, Â. Novais, L. Peixe); Centro Hospitalar do Porto, Porto (A.C. Mendes, M.H. Ramos); Botelho Moniz Análises Clínicas, Santo Tirso, Portugal (J. Amorim)

DOI: http://dx.doi.org/10.3202/eid2202.151301

To the Editor: The wide spread of Enterobacteriaceae resistant to last-resource therapeutic options, including

346 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 2, February 2016

LETTERS

1These authors contributed equally to this article.


153

extended-spectrum β-lactams, fluoroquinolones, and ami-noglycosides, has re-ignited interest in old antimicrobial drugs, such as fosfomycin (1). Fosfomycin resistance rates are generally low (<10%) but substantially higher when carbapenemase producers are considered (15%–34%) (1–3). Resistance phenotypes have been more thoroughly investigated in Escherichia coli and linked to chromosom-al mutations in the target (murA) or transporter (glpT and uhpT) genes or less frequently to plasmid-mediated fosfo-mycin resistance genes (fosA, fosB, fosC) encoding gluta-thione S-transferases that inactivate the drug (4). fosA3 is the most prevalent gene variant, disseminated mainly in E. coli isolates from clinical and nonclinical origins (healthy persons, companion and food animals) in countries in Asia (China, South Korea, and Japan) (2–6) and only recently in a migratory bird in Europe (7). We investigated the oc-currence and molecular features of 43 fosfomycin-resis-tant Enterobacteriaceae isolates (21 E. coli, 21 Klebsiella pneumoniae, and 1 Morganella morganii). These isolates were identified among 461 third-generation cephalosporin-resistant Enterobacteriaceae isolates from a community laboratory in northern Portugal during a 13-month period (August 2012–August 2013).

We screened for carriage of plasmidborne fosfomycin resistance genes (fosA, fosA3, fosB, fosC2) by PCR and sequencing (2,5). Chromosomal mutations in murA, glpT, and uhpT were investigated for 9 representative E. coli isolates (8) and 7 representative K. pneumoniae isolates with variable MICs to fosfomycin (>64 mg/L) by PCR and comparison of sequences with reference wild-type strains (E. coli ATCC25922 and K. pneumoniae type strain JCM1662) (8; this study). Fosfomycin-resistant isolates represented 9.3% (43/461) of the collection surveyed dur-ing the study period, which is in line with rates reported for clinical isolates from other countries (2,3). Bacterial identification and antimicrobial drug susceptibility testing to β-lactams and non–β-lactams were performed by auto-mated methods and further confirmed by disk diffusion and agar dilution (for fosfomycin, MIC cutoff 32 mg/L) according to European Committee on Antimicrobial Sus-ceptibility Testing guidelines (http://www.eucast.org). We screened blaESBL genes (blaCTX-M, blaTEM, blaSHV) by PCR and sequencing (9).

One (2.3%) of 43 E. coli isolates carried fosA3, blaCTX-M-15, and blaTEM-1 and contained mutations in GlpT (L297F, T348N, Q443E, E444Q) and UhpT (E350Q) (GenBank accession nos. KT832798 and KT832797, re-spectively), most of which were previously associated with fosfomycin resistance (8). This isolate was detected in a urine sample from a 61-year-old man who had a clini-cal history of chronic prostatitis and was associated with a urinary tract infection (UTI) acquired after travel to Asia (China, Philippines). aac-6’-lb-cr, blaOXA-I, and rmtB

genes were negative by PCR. This isolate exhibited fos-fomycin MIC >256 mg/L and was concomitantly resistant to cefotaxime, cefepime, aztreonam, ciprofloxacin, gen-tamicin, kanamycin, netilmycin, streptomycin, sulphon-amide, tetracycline, tobramycin, and trimethoprim but not to carbapenems, amoxicillin/clavulanic acid, or cefoxi-tin. In other E. coli isolates, fosfomycin resistance phe-notypes were linked to mutations in transporter proteins UhpT (8 isolates, E350Q) and GlpT (3 isolates, premature stop codons resulting in truncated proteins of 45, 134, or 442 aminoacids [GenBank accession nos. KT832799, KT832800, and KT832801, respectively]); however, no amino acid changes were detected in K. pneumoniae iso-lates. The detection of fosA3 in a clinical E. coli isolate in Europe is alarming because of its association with blaCTX-M-15, which is highly disseminated in Portugal and other European Union countries (9), whereas fosfomycin is increasingly being used to treat UTIs caused by extend-ed-spectrum β-lactams–producing E. coli (1).

Strain typing (identification of E. coli phylogroups and multilocus sequence typing; http://mlst.warwick.ac.uk/mlst/) revealed that this isolate belonged to phylogenetic group D1 and the sequence type 393 clone (9). This clone was not previously detected among fosA3-carrying isolates (3,4), but it is distributed worldwide (including Asia) linked to community-acquired UTI and multidrug resistance patterns (9).

Conjugative assays (solid/broth mating at 24°C/37°C using E. coli Hb101 azide and kanamycin resistant as recipient) and plasmid typing assessed by PCR-based replicon typing, IncFII typing formula (FAB), I-CeuI pulsed-field gel electrophoresis, and hybridization (5) showed that both fosA3 and blaCTX-M-15 were co-located in a conjugative F2:A-:B- plasmid (transconjugant MIC to fosfomycin >256 mg/L). Moreover, the genetic en-vironment of fosA3 was assessed by PCR mapping and sequencing (2,6), showing a composite transposon con-taining an insertion sequence 26 323 bp upstream fosA3; the orf1, orf2, and orf3 genes (homologous to regulatory ones in K. pneumoniae 342); and an insertion sequence (IS) 26 downstream (GenBank accession no. KT734860). The genetic platform (IS26 composite transposon) and the IncFII plasmid variant (F2:A-:B-) are main vehicles for disseminating fosA3 among clinical isolates, companion and food animals in Asian countries (3,5,6), or blaCTX-M-15 worldwide (10). Thus, epidemiologic and molecular data suggest that the detection of fosA3 in a clinical isolate in Europe is associated with a travel-related infection ac-quired after international travel to Asia. The acquisition of fosA3 by a successful clone and an efficient resistance plasmid, which might entail subsequent dissemination and alerts to the need of close monitoring of fosfomycin resistant isolates, is of particular concern.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 2, February 2016 347

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154

This work received financial support from European Union FEDER (Fundo Europeu de Desenvolvimento Regional) funds through COMPETE (Programa Operacional Fatores de Competi-tividade), and National Funds (Fundação para a Ciência e Tec-nologia) through project UID/Multi/04378/2013. The work also received financial support from the European Union (FEDER funds) under the framework of QREN (Quadro de Referência Estratégica Nacional through project NORTE-07-0124-FED-ER-000066. C.R. and Â.N. were supported by fellowships from Fundação para a Ciência e Tecnologia (SFRH/BD/84341/2012 and SFRH/BPD/104927/2014, respectively).

References 1. Giske CG. Contemporary resistance trends and mechanisms

for the old antibiotics colistin, temocillin, fosfomycin, mecillinam and nitrofurantoin. Clin Microbiol Infect. 2015;21:899–905. http://dx.doi.org/10.1016/j.cmi.2015.05.022

2. Lee SY, Park YJ, Yu JK, Jung S, Kim Y, Jeong SH, et al. Prevalence of acquired fosfomycin resistance among extended-spectrum beta-lactamase–producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Korea and IS26-composite transposon surrounding fosA3. J Antimicrob Chemother. 2012;67:2843–7. http://dx.doi.org/10.1093/jac/dks319

3. Ho PL, Chan J, Lo WU, Lai EL, Cheung YY, Lau TC, et al. Prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates. J Med Microbiol. 2013;62:1707–13. http://dx.doi.org/ 10.1099/jmm.0.062653-0

4. Sato N, Kawamura K, Nakane K, Wachino J, Arakawa Y. First detection of fosfomycin resistance gene fosA3 in CTX-M-producing Escherichia coli isolates from healthy individuals in Japan. Microb Drug Resist. 2013;19:477–82. http://dx.doi.org/10.1089/mdr.2013.0061

5. Hou J, Huang X, Deng Y, He L, Yang T, Zeng Z, et al. Dissemination of the fosfomycin resistance gene fosA3 with CTX-M beta-lactamase genes and rmtB carried on IncFII plasmids among Escherichia coli isolates from pets in China. Antimicrob Agents Chemother. 2012;56:2135–8. http://dx.doi.org/10.1128/AAC.05104-11

6. Ho PL, Chan J, Lo WU, Law PY, Li Z, Lai EL, et al. Dissemination of plasmid-mediated fosfomycin resistance fosA3 among multidrug-resistant Escherichia coli from livestock and other animals. J Appl Microbiol. 2013;114:695–702. http://dx.doi.org/ 10.1111/jam.12099

7. Villa L, Guerra B, Schmoger S, Fischer J, Helmuth R, Zong Z, et al. IncA/C plasmid carrying blaNDM-1, blaCMY-16, and fosA3 in a Salmonella enterica serovar Corvallis strain isolated from a migratory wild bird in Germany. Antimicrob Agents Chemother. 2015;59:6597–600. http://dx.doi.org/10.1128/AAC.00944-15

8. Takahata S, Ida T, Hiraishi T, Sakakibara S, Maebashi K, Terada S, et al. Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli. Int J Antimicrob Agents. 2010;35:333–7. http://dx.doi.org/10.1016/j.ijantimicag.2009.11.011

9. Rodrigues C, Machado E, Pires J, Ramos H, Novais Â, Peixe L. Increase of widespread A, B1 and D Escherichia coli clones producing a high-diversity of CTX-M-types in a Portuguese hospital. Future Microbiol. 2015;10:1125–31. http://dx.doi.org/ 10.2217/fmb.15.38

10. Coque TM, Novais A, Carattoli A, Poirel L, Pitout J, Peixe L, et al. Dissemination of clonally related Escherichia coli strains express-ing extended-spectrum beta-lactamase CTX-M-15. Emerg Infect Dis. 2008;14:195–200. http://dx.doi.org/10.3201/eid1402.070350

Address for correspondence: Ângela Novais, UCIBIO/REQUIMTE Researcher, Laboratory of Microbiology, Faculty of Pharmacy, University of Porto. Rua Jorge Viterbo Ferreira no. 228 4050-313, Porto, Portugal; email: [email protected]

Mycoplasma pneumoniae Monoclonal P1 Type 2c Outbreak, Russia, 2013

Inna Edelstein, Svetlana Rachina, Arabella Touati, Roman Kozlov, Nadège Henin, Cécile Bébéar, Sabine PereyreAuthor affiliations: Smolensk State Medical University of Ministry of Health of Russian Federation, Smolensk, Russian Federation (I. Edelstein, R. Kozlov); Inter-regional Association for Clinical Microbiology & Antimicrobial Chemotherapy, Smolensk (S. Rachina); University of Bordeaux, Bordeaux, France (A. Touati, N. Henin, C. Bébéar, S. Pereyre); Institut National de la Recherche Agronomique, Bordeaux (A. Touati, N. Henin, C. Bébéar, S. Pereyre); Bordeaux University Hospital, Bordeaux (C. Bébéar, S. Pereyre)

DOI: http://dx.doi.org/10.3201/eid2202.151349

To the Editor: Mycoplasma pneumoniae is a major cause of respiratory infections among children and young adults and is responsible for up to 40% of all community-acquired pneumonia. In 2011, an epidemic of M. pneu-moniae infection was reported in several countries in Eu-rope and Asia and in Israel that primarily involved adhesin P1 type 1 strains and only a few P1 type 2 strains (1,2). The spread of M. pneumoniae was polyclonal (1–3), except in a few semiclosed settings, such as schools (4). Recently, a new adhesin P1 type 2 variant, named 2c, was reported (5,6) and accounted for 8.3% of 96 M. pneumoniae–posi-tive samples in Germany (7).

In 2013, an increase in the number of community-acquired pneumonia cases was reported in children and their adult contacts from 2 towns in Russia separated by 45 km, Ozerniy and Duchovshina, during Janu-ary–March and October–November, respectively. To characterize the outbreak in Ozerniy, we collected 13 throat swabs from 9 symptomatic children and 4 asymp-tomatic adults who were the parents or grandparents of the affected children. All children attended the same school and were treated in the same district hospital as inpatients or outpatients. In Duchovshina, throat swab samples were collected from 17 children and 2 adults. The children attended the same school, and the pre-school-aged children visited the same daycare center 1 km away. One adult patient was the first aid driver who

348 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 2, February 2016

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155

3.2. Elucidating the drivers for expansion of Klebsiella pneumoniae resistant to extended-spectrum β-lactams in different Portuguese clinical settings

3.2.1. Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized

patients: a successful story of international clones (ST15, ST147, ST336)

and epidemic plasmids (IncR, IncFIIK).

3.2.2. KPC-3-Producing Klebsiella pneumoniae in Portugal linked to previously

circulating non-CG258 lineages and uncommon genetic platforms

(Tn4401d-IncFIA and Tn4401d-IncN).

3.2.3. Detection of VIM-34, a novel VIM-1 variant identified in the

intercontinental ST15 Klebsiella pneumoniae clone.

3.2.4. High rates of long-term care facilities (LTCFs) residents colonized with

multidrug resistant Klebsiella pneumoniae lineages frequently causing

infections in Portuguese clinical institutions.


157

Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized

patients: a successful story of international clones (ST15, ST147, ST336) and epidemic plasmids (IncR, IncFIIK)

Carla Rodrigues1,2, Elisabete Machado1,2, Helena Ramos3, Luísa Peixe1, Ângela Novais1*

1REQUIMTE. Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; 2CEBIMED/FP-ENAS, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa,

4249-004 Porto, Portugal; 3Centro Hospitalar do Porto - Hospital Geral de Santo António,

4099-001 Porto, Portugal.

International Journal of Medical Microbiology 2014; 304(8):1100-8

Elsevier has authorized the reproduction of the final published PDF version of this paper in this

thesis through the License Agreement Number 4091970737220 on April 18, 2017.


158

International Journal of Medical Microbiology 304 (2014) 1100–1108

Contents lists available at ScienceDirect

International Journal of Medical Microbiology

j ourna l ho me page: www.elsev ier .com/ locate / i jmm

Expansion of ESBL-producing Klebsiella pneumoniae in hospitalizedpatients: A successful story of international clones (ST15, ST147,ST336) and epidemic plasmids (IncR, IncFIIK)

Carla Rodriguesa,b, Elisabete Machadoa,b, Helena Ramosc, Luísa Peixea, Ângela Novaisa,∗

a REQUIMTE, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugalb CEBIMED/FP-ENAS, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, 4249-004 Porto, Portugalc Centro Hospitalar do Porto – Hospital Geral de Santo António, 4099-001 Porto, Portugal

a r t i c l e i n f o

Article history:Received 3 June 2014Received in revised form 31 July 2014Accepted 4 August 2014

Keywords:Multi-level epidemiologyMLSTHigh-risk clonesCTX-M-15SHV ESBLTEM-10

a b s t r a c t

The aim of this study was to characterize by a multi-level approach extended-spectrum !-lactamase(ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eightESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiellaoxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitalslocated in the North (A) or Centre (B, C) regions during two time periods (2006–7 and 2010) were analyzed.Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL character-ization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis.Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2,-5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations.The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with theamplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species inHospitals B and C in 2006–7. Particular plasmid types were linked to blaCTX-M-15 (IncR or non-typeableplasmids), blaSHV-12 (IncR or IncHI2), blaSHV-28/-55/-106 (IncFIIK1 or IncFIIK5), blaTEM-10 (IncL/M) or blaTEM-24

(IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-typeESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15,ST147, ST336) and plasmid types (IncR, IncFIIK). Furthermore, we highlight the potential of IncFIIK plas-mids (here firstly associated with blaSHV-2/-28/-55/-106) to disseminate as antibiotic resistance plasmids.

© 2014 Elsevier GmbH. All rights reserved.

Introduction

Extended-spectrum-!-lactamase (ESBL)-producing Enterobac-teriaceae are amongst the most frequent pathogens involved innosocomial infections worldwide, with Enterobacteriaceae speciesother than Escherichia coli (especially Klebsiella pneumoniae) beingincreasingly reported in the last years (http://www.cdc.gov/drugresistance/threat-report2013/; http://www.ecdc.europa.eu/en/publications/Publications/healthcare-associated-infections-antimicrobial-use-PPS.pdf). Studies on molecular epidemiology

∗ Corresponding author at: REQUIMTE, Faculty of Pharmacy, University of Porto,Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Tel.: +351 220 428 580;mobile: +351 966 228 110.

E-mail address: [email protected] (Â. Novais).

of ESBL-producing isolates from Enterobacteriaceae species otherthan E. coli are needed, particularly under a multi-level epidemi-ological perspective, which is considered essential to understandtrans-hierarchical evolutionary changes in antibiotic resistantpopulations and ultimately to establish appropriate controlinterventions (Baquero and Coque, 2011). Most of the studieson antibiotic resistant K. pneumoniae isolates are focused onthe characterization of carbapenemase producers (KPC, NDM,VIM and OXA-48), where different clonal groups or clonalcomplexes (e.g. CG15, CG17, CG258 or CC147) and epidemicplasmids (IncF, IncN, IncA/C, IncL/M) have been implicated intheir worldwide spread (Baraniak et al., 2013; Breurec et al.,2013; Carattoli, 2013). For other non-E. coli species producingESBLs, molecular epidemiological data are mostly focused onparticular outbreak situations (Coque et al., 2008; Novais et al.,2010).

http://dx.doi.org/10.1016/j.ijmm.2014.08.0031438-4221/© 2014 Elsevier GmbH. All rights reserved.


159

C. Rodrigues et al. / International Journal of Medical Microbiology 304 (2014) 1100–1108 1101

Tab

le

1Ep

idem

iolo

gica

l dat

a

of

ESB

L-p

rod

uci

ng

Ente

roba

cter

iace

ae

oth

er

than

E.

coli

reco

vere

d

from

Port

ugu

ese

hos

pit

als

in

two

dif

fere

nt

tim

e

per

iod

s

(200

6–

7

and

201

0).

ESB

L

(no.

)

Spec

ies

(no.

)

PFG

E-ty

pe/

MLS

T(n

o.)

Plas

mid

con

ten

t

[siz

e

(kb

)

(In

c

fam

ily)

]

Hos

pit

ala

Isol

atio

nPe

riod

bSa

mp

le

(no.

)

Non

-!-L

acta

m

Res

ista

nce

Phen

otyp

ec,d

Ass

ocia

ted

wit

hbl

a ESB

L

Oth

er

CTX

-M-1

5(4

1)

K. p

neu

mon

iae

(38

)

Kp

1/S

T33

6

(31

)

70–

26

0e(N

T)

90–

16

0

(FII

K8) +

35

eA

2

Uri

ne

(27

),b

lood

(3),

exu

dat

e

(1)

(AM

K),

(CIP

),

(CLO

),

(GEN

),

(KA

N),

(NA

L), (

NET

),

(NIT

),

STR

, SU

L,

TMP,

(TO

B)

Kp

2/S

T15

(5)

60

(R)

300

eA

1, 2

Uri

ne

(4),

un

know

n

(1)

CIP

, (C

LO),

GEN

, KA

N, N

AL,

(NET

),

(NIT

),ST

R, S

UL,

(TET

),

TMP,

TOB

Kp

14

/ST1

4

(1)

70

(R)

19

0

(FII

K)

A

2

Exu

dat

e

CIP

, CLO

, GEN

, NA

L,

STR

, SU

L,

TET,

TMP

Kp

13

/–

(1)

85

(FII

)

200

(FII

K8) +

45

A

2

Uri

ne

CIP

, GEN

, KA

N, N

AL,

NIT

, STR

, SU

L,

TET,

TMP,

TOB

E.

cloa

cae

com

ple

x

(2)

Ecl3

/–

–f

80

(FII

) +

19

0

+

33

0

B, C

1

Uri

ne

CIP

, GEN

, KA

N, N

AL,

NET

, TET

, TO

BK

. oxy

toca

(1)

Ko1

/–

100

(FII

)

200

+

50

A

2

Spu

tum

CIP

, GEN

, KA

N, N

AL,

NIT

, SU

L,

STR

, TET

,TM

P,

TOB

SHV

-12

(26

)

K. p

neu

mon

iae

(15

)

Kp

6/S

T14

7

(9)

70

(R)

50

A

1, 2

Uri

ne

(5),

blo

od(2

),

un

know

n(2

)

(AM

K),

CIP

, (C

LO),

(GEN

),

(KA

N),

NA

L,(N

ET),

(NIT

),

STR

, (SU

L), (

TET)

, (TM

P),

(TO

B)

Kp

5/S

T15

(1)

70

(R)

16

0

+

13

5

A

2

Spu

tum

CIP

, NA

L,

STR

, SU

L,

TMP

Kp

9/–

(1)

70

(R)

50

A

1

Un

know

n

CIP

, CLO

, GEN

, KA

N, N

AL,

NET

, STR

,SU

L,

TET,

TMP,

TOB

Kp

7/–

(2)

38

0

(HI2

)

–

B

1

Uri

ne,

spu

tum

(AM

K),

CIP

, (C

LO),

GEN

, KA

N, N

AL,

(STR

),

SUL,

(TET

),

TOB

Kp

8/–

(1)

35

0

(HI2

)

–

B

1

Spu

tum

AM

K, C

IP, C

LO, G

EN, K

AN

, NA

L,

STR

,SU

L,

TET,

TMP,

TOB

Kp

10/

–

(1)

18

0

(HI2

)

25

0

+

14

0

(FII

K5)

B

1

Exu

dat

e

KA

N, S

UL,

TOB

E.

cloa

cae

com

ple

x

(11

)Ec

l1/–

(7)

400

(HI2

)

12

0

(I1

) +

50

(R)

B

1

Spu

tum

(4),

uri

ne

(3)

(AM

K),

(CIP

),

(CLO

),

GEN

, KA

N, (

NA

L),

(NET

),

(SU

L), T

ET, T

OB

Ecl2

/–

(3)

400

(HI2

)

–

B

1

Exu

dat

e

(2),

Uri

ne

(1)

AM

K, (

CIP

),

CLO

, GEN

, KA

N, N

AL,

NET

,ST

R, S

UL,

TET,

TOB

Ecl4

/–

(1)

400

(HI2

)

13

0

+

60

+

40

B

1

Spu

tum

CIP

, CLO

, GEN

, KA

N, N

AL,

SUL,

TET,

TOB

SHV

-106

(4)

K. p

neu

mon

iae

(4)

Kp

3/S

T15

22

0

(FII

K1)

–

A

2

Uri

ne

(3),

spu

tum

(1)

CIP

, CLO

, (G

EN),

(KA

N),

NA

L,

(NET

),(N

IT),

(STR

),

SUL,

TET,

TMP,

(TO

B)

SHV

-28

(3)

K. p

neu

mon

iae

(3)

Kp

4/S

T15

70

(FII

K1)

80e

+

40

+

35

A

2

Uri

ne

(2),

blo

od(1

)A

MK

, CIP

, (C

LO),

GEN

, KA

N, N

AL,

NET

,(N

IT),

STR

, SU

L,

TET,

TMP,

TOB

SHV

-55

(3)

K. p

neu

mon

iae

(3)

Kp

3/S

T15

(2)

22

0

(FII

K1)

–

A

2

Uri

ne

CIP

, CLO

, GEN

, NA

L,

STR

, SU

L,

TET,

TMP

Kp

17

/–

(1)

22

0

(FII

K5)

–

A

2

Spu

tum

CIP

, CLO

, GEN

, NA

L,

STR

, SU

L,

TOB

SHV

-2

(2)

K. p

neu

mon

iae

(2)

Kp

5/S

T15

(1)

200

(FII

K1)

55

(R) +

35

A

2

Uri

ne

CIP

, CLO

, GEN

, KA

N, N

AL,

NIT

, STR

, SU

L,TM

PK

p1

5/–

(1)

300

(R

+

FII K

1)

–

A

1

Un

know

n

CIP

, NA

L,

STR

, SU

L,

TMP

SHV

-5

(1)

K. p

neu

mon

iae

(1)

Kp

16

/–

55

(ND

)

14

0 C

1

Uri

ne

–

TEM

-10

(5)

K. p

neu

mon

iae

(2)

Kp

11

/–

65

(L/M

)

38

0

B

1

Spu

tum

GEN

, KA

N, N

ET, T

ET, T

OB

Kp

12

/–

65

(L/M

)

22

0

+

13

0

(FII

K1) +

55

B

1

Uri

ne

AM

K, C

LO, G

EN, K

AN

, NA

L,

NET

, STR

,TE

T,

TOB

K. o

xyto

ca

(1)

Ko2

/–

65

(L/M

)

16

0

B

1

Uri

ne

GEN

, KA

N, N

AL,

NET

, SU

L,

TMP,

TOB

S.

mar

cesc

ens

(1)

Sm1

/–

65

(L/M

) –

B

1

Spu

tum

GEN

, KA

N, N

ET, S

TR, T

ET, T

OB

E.

asbu

riae

(1)

Eas1

/–

16

0

(L/M

) 8

0

B

1

Spu

tum

GEN

, KA

N, N

ET, T

ET, T

OB


160

1102 C. Rodrigues et al. / International Journal of Medical Microbiology 304 (2014) 1100–1108

Tab

le

1

(Con

tin

ued

)

ESB

L

(no

.)

Spec

ies

(no

.)

PFG

E-t

ype/

MLS

T(n

o.)

Pla

smid

con

ten

t [si

ze

(kb

) (In

c

fam

ily)

]

Ho

spit

ala

Iso

lati

on

Per

iod

bSa

mp

le

(no

.)

No

n-!

-Lac

tam

Res

ista

nce

Ph

eno

typ

ec,d

Ass

oci

ated

wit

hbl

a ESB

L

Oth

er

TEM

-24

(2)

K. p

neu

mon

iae

(1)

Kp

19

/–

18

0

(A/C

)

33

0

(FII

K1)

A

1

Un

kno

wn

AM

K, C

IP, C

LO, K

AN

, NA

L,

NE

T,

STR

,SU

L,

TMP

, TO

BK

. oxy

toca

(1)

Ko

3/–

18

0

(A/C

)

15

0

+

10

0

A

2

Spu

tum

CIP

, CLO

, KA

N, N

AL,

SUL,

STR

, TE

T,

TMP

,TO

B

TEM

-19

9g

(1)

P.

mir

abil

is

(1)

Pm

1/–

18

0

(A/C

)

–

C

1

Blo

od

AM

K, C

IP, C

LO, G

EN

, KA

N, N

AL,

NE

T,ST

R, S

UL,

TET,

TMP

, TO

B

ND

, no

t

do

ne;

NT,

no

n

typ

able

.a

Ho

spit

al

A

is

loca

ted

at

the

No

rth

regi

on

of

Po

rtu

gal;

Ho

spit

als

B

and

C

are

loca

ted

at

the

Cen

tre

regi

on

of

Po

rtu

gal.

b1

, iso

late

s

reco

vere

d

in

20

06

–7

;

2, i

sola

tes

reco

vere

d

in

20

10

.c

AM

K,

amik

acin

;

CIP

,

cip

rofl

oxa

cin

;

CLO

,

chlo

ram

ph

enic

ol;

GE

N,

gen

tam

icin

;

KA

N,

kan

amyc

in;

NA

L,

nal

idix

ic

acid

;

NE

T,

net

ilm

icin

;

NIT

,

nit

rofu

ran

toin

;

STR

,

stre

pto

myc

in;

SUL,

sulp

ho

nam

ides

;

TET,

tetr

acyc

lin

e;

TMP

,tr

imet

ho

pri

m;

TOB

, to

bra

myc

in.

dV

aria

bil

ity

amo

ng

iso

late

s

is

sho

wn

in

par

enth

esis

.e

Var

iab

ilit

y

in

the

pla

smid

con

ten

t

was

ob

serv

ed

amo

ng

iso

late

s.f

bla

loca

ted

on

chro

mo

som

e.g

New

TEM

vari

ant

(Gen

Ban

k

acce

ssio

n

nu

mb

er

JX0

50

17

8).

In Portugal, last surveys including non-E. coli Enterobacteriaceaeproducing blaESBL date from more than 10 years ago (1999 and2002–2004) and reported a high diversity of blaESBL (mainly SHV-and TEM-types) and the emergence of CTX-M enzymes (Machadoet al., 2007; Mendonc a et al., 2009). However, they had scarcelyexplored the role of particular clones and/or mobile genetic ele-ments in the dissemination and persistence of blaESBL genes amongnon-E. coli ESBL producers. The aim of this study is to character-ize a collection of ESBL-producing Enterobacteriaceae isolates otherthan E. coli identified in different Portuguese hospitals during twodifferent time periods (2006–7 and 2010), by using a multi-levelepidemiological approach.

Material and methods

Bacterial isolates and epidemiological background

Eighty-eight ESBL-producing Enterobacteriaceae clinical isolatesother than E. coli (69 K. pneumoniae, 13 Enterobacter cloacae com-plex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis,1 Serratia marcescens) recovered in two different time periods(2006–7 and 2010) were characterized. Isolates from the firstperiod (n = 30, 2006–7) corresponded to all ESBL-producing non-E. coli Enterobacteriaceae recovered from one general hospital fromthe North (Hospital A, n = 6) and two local hospitals from the Cen-tre (Hospitals B and C, n = 24) of Portugal. They represented 9%(Hospital A), 30% (Hospital B) and 25% (Hospital C) of the total ESBL-producing Enterobacteriaceae identified in those hospitals in thatperiod. In the second period only isolates from Hospital A were ana-lyzed (n = 58, 2010), since it experienced a 5-fold increase (9%–45%)in the proportion of ESBL-producing non-E. coli Enterobacteriaceae(mainly K. pneumoniae). Isolates were mostly recovered from urine(n = 55/63%) or sputum (n = 17/19%) samples, and from patients atmedical wards (n = 41/47%) or outpatients (n = 29/33%) (Table 1).

Bacterial identification, antimicrobial susceptibility and ESBLcharacterization

Bacterial identification and preliminary susceptibility test-ing to !-lactam antibiotics were performed using the auto-mated PHOENIX (BD Diagnostic Systems, Sparks, MD) orVITEK (BioMérieux, Marcy l’Étoile, France) systems. Antibioticresistance profiles to aminoglycosides (amikacin, gentamicin,kanamycin, netilmicin, streptomycin and tobramycin), chloram-phenicol, nitrofurantoin, quinolones (nalidixic acid, ciprofloxacin),sulphonamides, tetracycline and trimethoprim (Oxoid Ltd., Bas-ingstoke, United Kingdom) were determined by the standard discdiffusion method according to Clinical and Laboratory StandardsInstitute (CLSI) guidelines and breakpoints (CLSI, 2011). All inter-mediate isolates were considered as resistant. ESBL production wasconfirmed by the standard double disc synergy test (DDST) (CLSI,2011) on Mueller–Hinton agar plates with and without cloxacillin(250 mg/L) (Sigma–Aldrich, St Quentin-Fallavier, France) and char-acterization of blaESBLs was performed by PCR (blaTEM, blaSHV,blaCTX-M) and sequencing (Novais et al., 2012).

Clonal relationships

Relatedness among isolates was assessed by pulsed-field gelelectrophoresis (PFGE), using XbaI as restriction enzyme and thefollowing experimental conditions: 10–40 s pulses for 20 h, 14 ◦C,6 V/cm2 (Novais et al., 2010). Representative K. pneumoniae isolates(according to PFGE-types) were also characterized by multi-locussequence typing (MLST) according to the Pasteur Institute


161


Fig. 1. Trends in ESBL-types among Enterobacteriaceae isolates other than E. coli recovered in Portuguese hospitals in two time periods (2006–7 and 2010). Footnote: Thenumber of epidemic K. pneumoniae clones identified in each period is indicated. *Other species (no.): E. cloacae complex (12), E. asburiae (1), K. oxytoca (2), P. mirabilis (1) andS. marcescens (1).

protocol (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html).

Plasmid analysis and location of blaESBL genes

Plasmid content (number, size, identity) was investigated by S1-PFGE, PCR-based replicon typing, sequencing and hybridization aspreviously described (Novais et al., 2012; Villa et al., 2010). Thelocation of bla genes (plasmidic/chromosomal) was assessed byhybridization of S1- or I-CeuI-digested genomic DNA with intra-genic !-lactamase (blaCTX-M, blaSHV, blaTEM), replicon (FII, FIIk, R,L/M, A/C, HI2) and 16S rDNA probes, as previously described (Novaiset al., 2012).

Nucleotide sequence accession numbers

The sequence of a new blaESBL gene (blaTEM-199) identified in thisstudy was deposited in the GenBank database (GenBank accessionnumber JX050178).

Statistical analysis

Data were analyzed using IBM SPSS Statistics 22.0 software(International Business Machines Corp., New York, USA). Statisti-cal significance for comparison of proportions was calculated bythe !2 test or Fisher exact test (P values of <0.05 were consideredstatistically significant).

Results

High diversity of ESBLs, with a significant increase of CTX-M-15and SHV-type ESBLs

A high diversity of ESBLs was identified in this study, withmarked differences in their local and/or temporal distribution. Inhospitals B and C, SHV and TEM-type ESBLs were identified in

different non-K. pneumoniae species (2006–7), whereas in hospi-tal A, CTX-M-15 and diverse SHV ESBL were mainly detected in K.pneumoniae in 2010 (Fig. 1).

The isolates produced mostly CTX-M-15 (47%) and SHV-12(30%), and less frequently other SHV (15%; SHV-2, SHV-5, SHV-28, SHV-55, SHV-106) or TEM (9%; TEM-10, TEM-24, TEM-199)ESBL-types (Fig. 1 and Table 1). CTX-M-15 was detected in all hos-pitals analyzed and in different species (mostly K. pneumoniae),representing 66% of the isolates recovered in 2010 versus 10% ofthose recovered during 2006–7 (P < 0.0000008). SHV-12 produc-ers were identified among K. pneumoniae and E. cloacae complexin different hospitals and represented 60% of the ESBL-producingisolates recovered in 2006–7 versus 14% in 2010 (P < 0.000007),whereas other SHV variants were identified exclusively among K.pneumoniae isolates recently recovered in Hospital A [19% in 2010versus 10% in 2006–7; statistically not significant]. TEM-type ESBLswere identified among diverse Enterobacteriaceae species most fre-quently obtained from Hospitals B and C (23% in 2006–7 versus2% in 2010, P < 0.002). The novel TEM-199 (containing mutationsQ39K, E104K, M155I and G238S) ESBL-type is firstly reported inthis study.

Identification of epidemic and international clones

Three multidrug-resistant (MDR) K. pneumoniae epidemic lin-eages represented 90% of K. pneumoniae isolates identified inHospital A, some of them detected since 2006–7 (Table 1):(i) Sequence Type (ST) 15 producing CTX-M-15 or diverseSHV-types (n = 1, 2006; n = 15 March–November 2010; 4 PFGE-types) (Table 1); (ii) ST147 encoding SHV-12 (n = 2, 2007; n = 7,February–December 2010; 1 PFGE-type); (iii) ST336 producingCTX-M-15 (n = 31, 1 PFGE-type, February–December 2010) affect-ing mostly patients under kidney transplantation or with urinarytract disorders (n = 25, 81%) (Table 1). Other sporadic K. pneumo-niae clones (12 PFGE-types) were linked to SHV (SHV-2, -5, -12,


162


Tab

le

2Fe

atu

res

of

IncR

and

IncF

IIK

pla

smid

s

asso

ciat

ed

wit

h

K. p

neu

mon

iae

iso

late

s

fro

m

wo

rld

wid

e

clin

ical

and

no

n-c

lin

ical

sett

ings

.

Pla

smid

Inc.

Gro

up

aP

lasm

id

nam

eSi

ze

(kb

)

Ass

oci

ated

resi

stan

cege

nes

bSe

qu

ence

typ

e

(ST)

of

the

bac

teri

al

ho

stC

ou

ntr

y

Yea

r(s)

of

iso

lati

on

Sou

rce

Gen

Ban

k

acce

ssio

n

no

. (re

fere

nce

)

IncR

pK

PN

5

89

bla T

EM

-1ST

38

(MG

H7

85

78

)U

SA

19

94

Hu

man

CP

00

06

50

pK

24

5

98

bla S

HV

-2, b

laTE

M-1

, qn

rS1

–

Taiw

an

20

02

Hu

man

DQ

44

95

78

(Ch

en

et

al.,

20

06

)p

ST1

-2, -

3;

pST

32

6-1

, -3

55

–9

0bl

a CTX

-M-1

5, a

ac(6

′ )-I

b-cr

ST1

, ST3

26

Spai

n

20

07

–0

8

Hu

man

–

(Co

elh

o

et

al.,

20

10

)p

KP

17

80

50

bla V

IM-1

ST1

47

Gre

ece

20

09

Hu

man

JX4

24

61

4

(Pap

agia

nn

itsi

s

et

al.,

20

13

)p

KP

S77

46

bla S

HV

-12, b

laTE

M-1

ST1

5

Fran

ce

20

08

Hu

man

KF9

54

15

0

(Co

mp

ain

et

al.,

20

14

)p

KP

S30

61

bla D

HA

-1, b

laO

XA

-1,

aac(

6′ )

-Ib-

cr, q

nrB

4ST

11

Fran

ce

20

08

Hu

man

KF7

93

93

7

(Co

mp

ain

et

al.,

20

14

)

pK

PC

-LK

30

87

bla K

PC

-2, b

laSH

V-1

1ST

11

Taiw

an

20

12

Hu

man

KC

40

56

22

(Ch

en

et

al.,

20

14

b)

–

33

–1

95

bla C

TX-M

-15,

aac(

6′ )

-Ib-

cr, q

nrB

6an

d/o

r

qnrS

1

ST3

41

, ST4

31

, ST4

33

Spai

n

20

03

–1

0

Hu

man

–

(Ru

iz

et

al.,

20

12

)

–

50

–9

0bl

a CTX

-M-1

5ST

15

Po

rtu

gal

20

10

Hu

man

–

(No

vais

et

al.,

20

12

)–

60

–7

0

bla C

TX-M

-15

ST1

5, S

T14

Po

rtu

gal

20

06

–2

01

0

Hu

man

–

This

stu

dy

–

70

bla S

HV

-12

ST1

5, S

T14

7P

ort

uga

l

20

06

–2

01

0H

um

an

–

This

stu

dy

IncF

IIK

1p

KP

N3

17

6

pco

A-E

,S;

silP

, R, S

, E;

arsA

, CST

38

(MG

H7

85

78

)U

SA

19

94

Hu

man

CP

00

06

48

pK

PN

4

10

8

bla S

HV

-12

ST3

8(M

GH

78

57

8)

USA

19

94

Hu

man

CP

00

06

49

pB

K3

21

79

16

5

bla K

PC

-2ST

25

8

USA

20

06

Hu

man

JX4

30

44

8

(Ch

en

et

al.,

20

13

)p

ST1

-1

19

0

bla C

TX-M

-15

ST1

Spai

n

20

05

Hu

man

–

(Co

elh

o

et

al.,

20

10

)p

KP

N-I

T

20

8

pco

A, B

, D, S

;

cop

A;

silE

,P;

cusB

;

arsB

, HST

25

8

Ital

y

20

10

Hu

man

JN2

33

70

4

(Gar

cía-

Fern

ánd

ez

et

al.,

20

12

)

pK

PN

10

1-I

T

10

8

bla K

PC

-2ST

10

1

Ital

y

20

11

Hu

man

JX2

83

45

6

(Fra

sso

n

et

al.,

20

12

)–

20

0–

22

0

bla S

HV

-2o

r

bla S

HV

-55

or

bla S

HV

-10

6

ST1

5

Po

rtu

gal

20

10

Hu

man

–

This

stu

dy

–

70

bla S

HV

-28

ST1

5

Po

rtu

gal

20

10

Hu

man

–

This

stu

dy

–

–

–

ST1

, ST3

7, S

T32

1,

ST3

27

Spai

n

20

05

–0

7

Hu

man

–

(Co

elh

o

et

al.,

20

10

)

IncF

IIK

2p

KF3

-94

94

bla C

TX-M

-15, b

laTE

M-1

–

Ch

ina

20

06

Hu

man

FJ8

76

82

6

(Zh

ao

et

al.,

20

10

)p

Kp

QIL

11

4

bla K

PC

-3, b

laTE

M-1

ST2

58

Isra

el

20

05

Hu

man

GU

59

51

96

(Lea

vitt

et

al.,

20

10

)p

Kp

QIL

-IT

11

5

bla K

PC

-3, b

laTE

M-1

ST2

58

Ital

y

20

10

Hu

man

JN2

33

70

5

(Gar

cía-

Fern

ánd

ez

et

al.,

20

12

)p

KP

HS2

11

1

bla K

PC

-2, b

laTE

M-1

ST1

1

Ch

ina

20

11

Hu

man

CP

00

32

24

(Liu

et

al.,

20

12

)p

13

70

–

bla K

PC

-2, b

laTE

M-1

ST1

60

Gre

ece

20

08

Hu

man

–

(pM

LST)

p1

51

6

–

bla K

PC

-2, b

laTE

M-1

ST1

7

Gre

ece

20

08

Hu

man

–

(pM

LST)

pK

P1

50

4-k

pc

11

3

bla K

PC

-2, b

laTE

M-1

ST2

58

Gre

ece

20

08

Hu

man

KF8

74

49

6

(pM

LST)

pK

P1

78

0-k

pc

11

3

bla K

PC

-2, b

laTE

M-1

ST1

47

Gre

ece

20

09

Hu

man

KF8

74

49

7

(pM

LST)

pK

pQ

IL-1

0/-

03

11

3

bla K

PC

-2, b

laTE

M-1

ST2

58

USA

20

04

–1

0

Hu

man

KJ1

46

68

7

(Ch

en

et

al.,

20

14

a,b

)p

Kp

QIL

-04

11

3

bla K

PC

-3, b

laTE

M-1

ST2

58

USA

20

03

Hu

man

–

(Ch

en

et

al.,

20

14

a)p

Kp

QIL

-23

4

11

4

bla K

PC

-2, b

laTE

M-1

ST2

34

USA

20

10

Hu

man

KJ1

46

68

9

(Ch

en

et

al.,

20

14

a)p

SSL

–

bla K

PC

-3–

Spai

n

20

13

Hu

man

–

(pM

LST)

pSL

MT

21

bla K

PC

-2–

–

–

Hu

man

HQ

58

93

50

pC

15

-k

95

bla C

TX-M

-15, b

laTE

M-1

–

Ch

ina

–

Hu

man

HQ

20

22

66

p5

34

41

–

–

–

Ind

ia

20

10

Hu

man

–

(pM

LST)

pR

YC

E1

1

94

bla T

EM

-4ST

13

, ST1

4, S

T28

, ST3

7,

ST3

48

, ST5

33

Spai

n

19

95

–2

00

4

Hu

man

–

(Co

qu

eTM

, 20

13

)

–

90

bla C

TX-M

-15, b

laTE

M-1

–

Ch

ina

20

07

–0

8

Hu

man

–

(Zh

uo

et

al.,

20

13

)

IncF

IIK

3p

GSH

50

0

–

–

–

–

–

–

AJ0

09

98

0

IncF

IIK

4p

KP

91

91

–

–

–

–

Co

rn

CP

00

09

66

(Fo

uts

et

al.,

20

08

)

IncF

IIK

5p

UU

H2

39

.2

22

0

bla C

TX-M

-15, b

laTE

M-1

,aa

c(6

′ )-I

b-cr

, bla

OX

A-1

ST1

6

Swed

en

20

05

–1

0

Hu

man

CP

00

24

74

(San

deg

ren

et

al.,

20

12

)

pK

P0

90

85

21

3

bla C

TX-M

-15, b

laTE

M-1

,aa

c(6

′ )-I

b-cr

, bla

OX

A-1

ST4

8

Ko

rea

20

08

Hu

man

KF7

19

97

0

(Sh

in

and

Soo

Ko

, 20

13

)


163


pKP0

2022

200

bla C

TX-M

-15,

aac(

6′ )-Ib

-cr,

bla O

XA

-1

ST15

Kor

ea

2008

Hum

an

KF7

1997

2

(Shi

n an

d So

o

Ko,

2013

)

pKP0

07

246

bla C

TX-M

-15, b

laTE

M-1

,aa

c(6′ )-

Ib-c

r,

bla O

XA

-1

ST23

Kor

ea

2008

Hum

an

KF7

1997

1 (S

hin

and

Soo

Ko,

2013

)

pND

M-1

sait

ama0

1

49

bla N

DM

-1, !

bla D

HA

-1,

arm

AST

42

Japa

n

2012

Hum

an

AB7

5969

0 (H

ishi

num

a

et

al.,

2013

)

–

220

bla C

TX-M

-15

–

Port

ugal

2010

Hum

an

– Th

is

stud

yp1

7829

–

–

ST37

Ital

y

2008

Hum

an

HM

7518

80

(Vill

a

et

al.,

2010

)p1

7830

–

–

ST37

Ital

y

2008

Hum

an

–

(Vill

a

et

al.,

2010

)p1

7834

–

–

ST37

Ital

y

2008

Hum

an

–

(Vill

a

et

al.,

2010

)p7

1697

–

–

ST37

Ital

y

2008

Hum

an

–

(Vill

a

et

al.,

2010

)

IncF

II K7

pKD

O1

127

bla C

TX-M

-15, b

laTE

M-1

,aa

c(6′ )-

Ib-c

r,

bla O

XA

-1

ST41

6

Czec

h

Repu

blic

2009

Hum

an

JX42

4423

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164


-55), TEM (TEM-10, -24) or CTX-M-15 ESBLs in different hospitals(Table 1).

ESBL-producing K. oxytoca, E. cloacae complex, E. asburiae, P.mirabilis or S. marcescens isolates were highly diverse (n = 19, 8PFGE-types, different ESBLs) and identified mostly in Hospitals Band C in the first period analyzed. Two epidemic E. cloacae complexclones produced SHV-12 (Table 1).

Location of blaESBL in epidemic plasmids or in the chromosome ofdifferent Enterobacteriaceae species

Isolates showed a variable number of plasmids (1–4 plas-mids) with different sizes (35–400 kb) (Table 1). K. pneumoniaeclones harboured frequently IncFIIK1(pKPN3), IncFIIK5(p17829)or IncFIIK8(pKPN CZ) plasmid variants (according to thehttp://pubmlst.org/plasmid database), associated or not withblaESBL genes. The different blaESBL genes were found in a lowdiversity of plasmid families (IncR, IncFIIk, IncHI2, IncL/M, IncA/C),linked to particular ESBL-types and clones circulating in eachhospital studied. The blaCTX-M-15 gene was mostly identified withina 60–70 kb IncRpK245 plasmid in ST15 or ST14 K. pneumoniae or a70 kb non-typeable plasmid in the epidemic ST336 K. pneumoniaeclone, and only sporadically within 85–100 kb IncFIIpC15-a plas-mids or the chromosome of different Enterobacteriaceae species(Table 1). The blaSHV-12 gene was located at a 70 kb IncRpK245plasmid in ST15 and ST147 K. pneumoniae clones circulating inHospital A, or within 180–400 kb IncHI2 plasmids in K. pneumoniaeand E. cloacae complex from Hospital B. blaSHV-2/-28/-55/-106 weremostly located on 70–220 kb IncFIIK1 and IncFIIK5 plasmid variantsin diverse ST15 K. pneumoniae isolates. The blaTEM-10 gene wasfound within 65–160 kb IncL/M plasmids among diverse Enterobac-teriaceae species, while blaTEM-24 was identified in 180 kb-IncA/Cplasmids in K. pneumoniae and K. oxytoca (Table 1).

Discussion

In this study, we used a multilayered approach to explaingeographical and temporal shifts in clinical ESBL-producingEnterobacteriaceae isolates other than E. coli. The increase in ESBL-producing K. pneumoniae isolates and CTX-M-15 is in line withreports from multiple countries (Breurec et al., 2013; Coque et al.,2008; Lee et al., 2011; Peirano et al., 2012), although with a lowerdiversity of CTX-M types than those observed in these studies (CTX-M-2, -9, -14 or -32) or even in clinical or non-clinical E. coli isolatesfrom Portugal (Rodrigues et al., unpublished results) (Machadoet al., 2013). It is of interest to highlight that preliminary char-acterization of recent (2012–2013) ESBL producers from differentPortuguese clinical institutions (including Hospitals A and C) sug-gests a similar scenario (increase in ESBL-producing K. pneumoniaeisolates, more than 50% of them encoding CTX-M-15) (unpublisheddata).

The amplification of CTX-M-15 and persistence of diverse SHVESBL-types among K. pneumoniae was mainly linked to a few epi-demic and international clones (ST15, ST147, ST336). ST15 (thecentral genotype of CG15) has previously been identified produc-ing CTX-M-15 or VIM-34 in nosocomial or community infections inPortugal (Novais et al., 2012; Rodrigues et al., 2014) (Pires J et al.,unpublished results), suggesting endemicity in either clinical ornon-clinical settings. This pandemic clone has also been involvedin the spread of CTX-M-15 and different carbapenemases (OXA-48, VIM, NDM-1) in other European, Asian and African countries(Baraniak et al., 2013; Breurec et al., 2013; Damjanova et al., 2008;Lee et al., 2011; Österblad et al., 2012; Poirel et al., 2011; Sánchez-Romero et al., 2012). The high diversity of PFGE- and ESBL-typesidentified among isolates belonging to the ST15 K. pneumoniae

clone in this study is remarkable and suggests a high genomicdiversification probably reflecting the acquisition of diverseESBL-encoding genetic platforms and/or the insertion of multipleclones from an unknown reservoir (Tofteland et al., 2013; Viau et al.,2012). The ST147 K. pneumoniae clone (belonging to CC147), asso-ciated with the spread of SHV-12 in our study, seems to be morecommonly linked to the worldwide spread of different carbapene-mases (OXA-48, NDM-1, VIM-1 and KPC-2) (Giakkoupi et al., 2011;Lascols et al., 2013; Samuelsen et al., 2011) or CTX-M-15 (Baraniaket al., 2013; Damjanova et al., 2008). To the best of our knowledge,we report for the first time the dissemination of a CTX-M-15-producing ST336 K. pneumoniae MDR clone affecting mostly kidneytransplant patients, who due to frequent subsequent hospitaliza-tions or medical visits, could contribute for further dissemination ofthis clone both at the hospital and community settings. This clonebelongs to the CG17 (including ST16, ST17 and ST20), which hasbeen frequently associated with the worldwide spread of CTX-M-15 among clinical or commensal isolates (Baraniak et al., 2013; Leeet al., 2011; Oteo et al., 2009; Peirano et al., 2012).

Besides clonal spread, plasmid transmission has also con-tributed to the dissemination of ESBLs among different non-E. coliEnterobacteriaceae clones and species in our country. The spreadof CTX-M-15 and SHV-12 in K. pneumoniae isolates belonging todifferent clones (ST15, ST147, ST336) was mainly driven by IncRplasmids. These plasmids have recently been described amongdiverse K. pneumoniae isolates harbouring different ESBLs (CTX-M-15, SHV-2, SHV-12) or carbapenemases (VIM-1), including inPortugal (Table 2). The non-typeable CTX-M-15-encoding plasmididentified in the ST336 clone coexisted with an IncFIIK8 plasmid.Although unrecognized plasmid types or variants cannot be dis-carded, the possible coexistence of two closely related plasmids, asobserved previously by Dolejska et al. (Dolejska et al., 2013) couldimpair plasmid identification. Furthermore, our results corroborateprevious studies suggesting that different plasmid types seem tobe responsible for the dissemination of CTX-M-15 in E. coli (mostlyIncFII variants) and K. pneumoniae (mostly IncFIIK variants or IncR)(Coelho et al., 2010; Shin et al., 2012). IncFIIK1 and IncFIIK5 plasmidvariants were for the first time associated with the spread of closelyrelated SHV-2, SHV-28, SHV-55 or SHV-106 ESBLs, suggesting localacquisition and possibly in vivo evolution. Finally, our study alsostrengthens previous data suggesting that IncFIIK plasmids, con-sidered as virulence plasmids (Dolejska et al., 2013; Villa et al.,2010), are highly frequent among K. pneumoniae and are quicklyevolving by replicon diversification and acquisition of antibioticresistance traits (Table 2) (Carattoli, 2013; Coelho et al., 2010; Villaet al., 2010).

Conclusion

In summary, we demonstrate that the dominance of CTX-M-15and of diverse SHV-type ESBLs among non-E. coli Enterobacteria-ceae isolates from Portuguese hospitals is largely influenced by theamplification of epidemic and international MDR K. pneumoniaeclones (ST15, ST147, ST336) and particular plasmid types (IncR,IncFIIk). This work also enlarges the diversity of ESBLs linked toIncFIIK plasmids (here firstly associated with blaSHV-2, blaSHV-28,blaSHV-55 or blaSHV-106), highlighting their potential to disseminateas antibiotic resistance and virulence plasmids.

Funding

This work received financial support from the EuropeanUnion (FEDER funds) through Programa Operacional Factoresde Competitividade – COMPETE, from National Funds (FCT,Fundac ão para a Ciência e a Tecnologia) through grant numbers


165


EXPL/DTP-EPI/0196/2012, FCOMP-01-0124-FEDER-027745 andPEst-C/EQB/LA0006/2013 and from Fundac ão Ensino e CulturaFernando Pessoa. This work also received financial support fromthe European Union (FEDER funds) under the framework of QRENthrough Project NORTE-07-0124-FEDER-000066. Carla Rodrigueswas supported by a fellowship from FCT (SFRH/BD/84341/2012).

Competing interests

None declared.

Ethical approval

Not required.

Acknowledgements

We are grateful to José Luís Graneda e Conceic ão Faria (Cen-tro Hospitalar Cova da Beira, EPE), and Servic o de Patologia Clínicaof Centro Hospitalar Tondela-Viseu, EPE for the clinical isolatesincluded in this study.

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167

KPC-3-Producing Klebsiella pneumoniae in Portugal linked to

previously circulating non-CG258 lineages and uncommon genetic platforms (Tn4401d-IncFIA and Tn4401d-IncN)

Carla Rodrigues 1, Jan Bavlovič 2,3, Elisabete Machado 1,4, José Amorim 5,

Luísa Peixe 1*, Ângela Novais 1*

1UCIBIO-REQUIMTE. Laboratório de Microbiologia, Faculdade de Farmácia,

Universidade do Porto, Porto, Portugal; 2Faculty of Pharmacy in Hradec Králové, Charles

University, Prague, Czech Republic.; 3Faculty of Military Health Sciences, University of

Defense, Brno, Czech Republic; 4FP-ENAS/CEBIMED. Faculdade de Ciências da Saúde,

Universidade Fernando Pessoa, Porto, Portugal; 5Botelho Moniz Análises Clínicas

(BMAC), Santo Tirso, Portugal.

Frontiers in Microbiology 2016; 7:1000

Open Acess Journal


168

ORIGINAL RESEARCHpublished: 28 June 2016

doi: 10.3389/fmicb.2016.01000

Frontiers in Microbiology | www.frontiersin.org 1 June 2016 | Volume 7 | Article 1000

Edited by:

John W. A. Rossen,University of Groningen, University

Medical Center, Netherlands

Reviewed by:

Kai Zhou,University Medical Center Groningen,

NetherlandsÁkos Tóth,

National Center for Epidemiology,Hungary

*Correspondence:

Luísa [email protected];Ângela Novais

[email protected]

Specialty section:

This article was submitted toInfectious Diseases,

a section of the journalFrontiers in Microbiology

Received: 06 April 2016Accepted: 13 June 2016Published: 28 June 2016

Citation:

Rodrigues C, Bavlovic J, Machado E,Amorim J, Peixe L and

Novais Â (2016) KPC-3-ProducingKlebsiella pneumoniae in PortugalLinked to Previously Circulating

Non-CG258 Lineages andUncommon Genetic Platforms

(Tn4401d-IncFIA and Tn4401d-IncN).Front. Microbiol. 7:1000.

doi: 10.3389/fmicb.2016.01000

KPC-3-Producing Klebsiellapneumoniae in Portugal Linked toPreviously Circulating Non-CG258Lineages and Uncommon GeneticPlatforms (Tn4401d-IncFIA andTn4401d-IncN)Carla Rodrigues 1, Jan Bavlovic 2, 3, Elisabete Machado1, 4, José Amorim5, Luísa Peixe 1*and Ângela Novais 1*

1 UCIBIO/REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal,2 Faculty of Pharmacy in Hradec Králové, Charles University, Prague, Czech Republic, 3 Faculty of Military Health Sciences,University of Defense, Brno, Czech Republic, 4 FP-ENAS/CEBIMED, Faculdade de Ciências da Saúde, UniversidadeFernando Pessoa, Porto, Portugal, 5 Botelho Moniz Análises Clínicas, Santo Tirso, Portugal

KPC-3-producing bacteria are endemic in many countries but only recently became

apparent their wide distribution in different Portuguese hospitals. The aim of this study

is to characterize genetic backgrounds associated with blaKPC−3 among Klebsiellapneumoniae isolates recently identified on non-hospitalized patients in Portugal. Twenty

KPC-producing K. pneumoniae identified between October 2014 and November 2015

in three different community laboratories were characterized. Isolates were mainly from

patients from long-term care facilities (n = 11) or nursing homes (n = 6), most of

them (75%) previously hospitalized in different Portuguese hospitals. Standard methods

were used for bacterial identification and antibiotic susceptibility testing. Carbapenemase

production was assessed by the Blue-Carba test, and identification of bla genes was

performed by PCR and sequencing. Epidemiological features of KPC-producing K.pneumoniae included population structure (XbaI-PFGE, MLST and wzi sequencing),genetic context (mapping of Tn4401), and plasmid (replicon typing, S1-PFGE, and

hybridization) analysis. All K. pneumoniae isolates produced KPC-3, with two MDR

K. pneumoniae epidemic clones representing 75% of the isolates, namely ST147

(wzi64/K14.64, February–November 2015) and ST15 (two lineages exhibiting capsular

types wzi19/K19 or wzi93/K60, July-November 2015). Other sporadic clones were

detected: ST231 (n= 3;wzi104), ST348 (n= 1;wzi94) and ST109 (n= 1,wzi22/K22.37).blaKPC−3 was identified within Tn4401d in all isolates, located in most cases (80%) on

cointegrated plasmids (repAFIA+repAFII+oriColE1;105-250 kb) or in 50 kb IncN plasmids.

In conclusion, this study highlights a polyclonal structure of KPC-3-producing K.pneumoniae and the predominance of the ST147 clone among non-hospitalized patients

in Portugal, linked to platforms still unnoticed in Europe (blaKPC−3-Tn4401d-IncFIA) or


169

Rodrigues et al. KPC-3 Producers among Non-Hospitalized Patients in Portugal

firstly reported (blaKPC−3-Tn4401d-IncN). This scenario underlines the recent penetration

of successful mobile genetic elements in previously circulating MDR K. pneumoniaelineages (mainly ST147 and ST15) in Portugal, rather than the importation of the global

lineages from clonal group 258.

Keywords:multidrug resistance, carbapenemases, international clones, ST15, ST147, cointegrated plasmids, ColE

INTRODUCTION

In the last years, carbapenem-resistant Enterobacteriaceaehave spread globally, being responsible for high ratesof morbidity and mortality among healthcare-associatedinfections, mainly due to the depletion of effectivetherapeutic options (WHO, 2014; Albiger et al., 2015,http://www.cdc.gov/drugresistance/threat-report-2013/). Afterthe first strain identified in 1996 in a North Carolina hospital(USA; Yigit et al., 2001), Klebsiella pneumoniae carbapenemases(KPCs) have exploded worldwide predominantly among K.pneumoniae isolates (Munoz-Price et al., 2013; Chen et al.,2014b). To date, 23 KPC variants (KPC-2 to KPC-24) havebeen described (http://www.lahey.org/Studies/other.asp#table1), being KPC-2 and KPC-3 the most widespread variantswith variable geographic distribution (Munoz-Price et al.,2013; Nordmann and Poirel, 2014). While in some countries(USA, Colombia, Italy, and Israel) both KPC-2- and KPC-3-producing bacteria are endemic, in others (Argentina, Brazil,Greece, Poland, and China) KPC-2 producers are predominant(Munoz-Price et al., 2013; Albiger et al., 2015).

The blaKPC genes are commonly located on Tn4401, a10 kb Tn3-like transposon delimited by two 39-bp imperfectinverted repeat sequences harboring blaKPC, transposase andresolvase genes, and insertion sequences ISKpn7 (upstreamblaKPC) and ISKpn6 (downstream blaKPC; Chen et al., 2014b).It is recognized as a highly active transposon enhancing thespread of blaKPC genes to different plasmid scaffolds (Cuzonet al., 2011). To date, six Tn4401 isoforms have been describedwith variable deletions between ISKpn7 and blaKPC providingdifferent promoter regions to the gene (a,−99 bp; b, no deletion;c, −215 bp; d, −68 bp; e, −255 bp; g, equal to isoform cbut with one single nucleotide mutation on P2 promotor),and consequently different expression levels of the blaKPC gene(Naas et al., 2012; Chmelnitsky et al., 2014). Besides its geneticenvironment, other factors are known to have greatly contributedto the spread of KPC producers in many countries, leading to anincreasing challenge in the design of effective infection controlmeasures. First, the introduction and subsequent expansionof blaKPC−2 and blaKPC−3 on multidrug resistant (MDR) K.pneumoniae lineages from clonal group (CG) 258 [sequencetypes (ST) 11, 258, 512] (Munoz-Price et al., 2013; Chenet al., 2014b), followed in a few countries (e.g., Israel, Italy,Colombia) by subsequent dispersion to other clonal backgrounds(Baraniak et al., 2015; Bonura et al., 2015; Ocampo et al., 2015).Second, the acquisition of blaKPC by plasmids from differentincompatibility groups (IncFIIK2, IncFIA, IncI2, IncN, IncX3,ColE), favored a quick intra- and inter-species dissemination(Chen et al., 2014b).

In Portugal, KPC-2 was identified only in an environmentalEscherichia coli isolate in 2010 (Poirel et al., 2012), while KPC-3 producers were first detected in 2009 in a central hospital(Machado et al., 2010). However, only recently became evidentthe widespread distribution of KPC-3 among K. pneumoniaeisolates in different Portuguese hospitals (Silva et al., unpublisheddata; Manageiro et al., 2015). In this study, we aim to trace thelandscape of KPC-3-producing K. pneumoniae isolates recentlyidentified outside hospital boundaries in Portugal by detailedcharacterization of clonal and plasmid genetic backgrounds.

MATERIALS AND METHODS

Bacterial Isolates and EpidemiologicalDataThirty K. pneumoniae isolates showing reduced susceptibilityto carbapenems were identified between October 2014 andNovember 2015 in three different community laboratories in theNorth of Portugal, one of them receiving samples from all overthe country. Twenty of them were identified as KPC producersand further characterized in this study. They were detected inurine samples (n = 19) or sputum (n = 1) of patients between61 and 89 years old (mean age = 83; 16 females, 4 males;Table 1). Most of these patients were institutionalized in long-term care facilities (LTCFs) [n = 11; 55%; six different LTCFs(A–F)] or nursing homes (NH) [n = 6; 30%; five different NH(A–E)], while some were identified in ambulatory (n = 3; 15%;Table 1). Most of them (n= 13; 65%) had been hospitalized in theprevious month in different hospitals from the North or Centreof Portugal, although in three cases no hospitalization or olderhospitalization events (4–9 months) were detected (Table 1).Travel history abroad was discarded for 60% of the patients (n =

12/20), or considered improbable for the remaining patients dueto their clinical conditions (impaired mobilization and chronicunderlying diseases).

Antibiotic Resistance Phenotypes andGenotypesBacterial identification and preliminary antibiotic susceptibilitytesting were performed by Vitek II system (BioMérieux,Marcy l’Étoile, France). Confirmatory and additional tests forβ-lactams (amoxicillin-clavulanic acid, mecillinam, cefoxitin,extended-spectrum cephalosporins, aztreonam, carbapenems),aminoglycosides (amikacin, gentamicin, netilmicin,tobramycin), fluoroquinolones (ciprofloxacin), folate pathwayinhibitors (trimethoprim, trimethoprim-sulfamethoxazole),chloramphenicol, fosfomycin, and colistin were assessed bystandard disc diffusion (Oxoid Ltd., Basingstoke, United



170


TABLE1|Epidemiologicaldata

ofK.pneumoniaeisolatescarryingblaKPC-3-T

n4401didentifiedin

non-hospitalizedpatients

inPortugal(O

ctober2014–November2015).

ST(no.)

PFGE-type

(no.)

wzi/K-

typea

Date

of

isolation

(month/year)

Source

(no.)b

Localofprevious

hospitalizations(no)c

Age

(range)

Gender

Sample

(no.)

Plasmids

associated

withblaKPC-3

(size;Inc

groups)(no.)

Other

β-

lactamasesd

ST147

(10)

Kp1(1)

wzi64/K14.64

Feb–Nov/2015

LTCFA(2)

Hosp

italB

(1),

unknown(1)

88–89

FUrine

∼130kb;

FIA+FII+

ColE

(6)

(OXA-9),

SHV-11or

SHV-28,

(TEM-1)

LTCFB(1)

unknown

89

FUrine

LTCFC(1)

Hosp

italD

76

FUrine

LTCFE(1)

Differenthosp

itals

86

FUrine

LTCFF(1)

Hosp

italE

81

MUrine

NHA(2)

Hosp

italA

78–82

F,M

Urine

∼50kb;N(4)

(OXA-9),

SHV-11

NHD(1)

Hosp

italC

e85

FUrine

Ambulatory(1)

Hosp

italA

77

FUrine

ST15(5)

Kp2(4)

wzi19/K19

Jul–Nov/2015

LTCFA(3)

Hosp

italB

(2)f

83–84

FUrine(2),

sputum

(1)

∼130–140kb;

FIA+FII+

ColE

(4)

OXA-9,

SHV-28,

(TEM-1)

LTCFD(1)

Hosp

italB

88

FUrine

Kp3(1)

wzi93/K60

Sep/2015

Ambulatory

(1)

Hosp

italF

72

MUrine

∼135kb;

FIA+FII+

ColE

(1)

CTX-M

-15,

OXA-9,

SHV-28

ST231(3)

Kp4(1)

wzi104/K-

Oct/2015

NHB(1)

Hosp

italA

88

FUrine

∼250kb;

FIA+FII+

ColE

(1)

SHV-1,

(TEM-1)

NHE(1)

Hosp

italG

89

FUrine

Ambulatory

(1)

Hosp

italC

e83

FUrine

ST348(1)

Kp5(1)

wzi94/K-

Oct/2014

NHC(1)

unknown

87

MUrine

∼105kb;

FIA+FII+

ColE

(1)

CTX-M

-15,

SHV-11,

OXA-1,TEM-1

ST109(1)

Kp6(1)

wzi22/K22.37

Sep/2015

LTCFB(1)

unknown

61

FUrine

∼130kb;

FIA+FII+

ColE

(1)

SHVnew

(N196S)g

aK-typeisreported

accordingwith

wziallele-serotypeassociations

reported

byBrisse

etal.(2013).

bLTCFs

A–D

andNHA–C

arelocatedintheNorthregionofP

ortugal,whileLTCFs

E–F

andNHD-E

arelocatedintheCentreregionofthe

country.

cHospitalsA,B,andDarelocatedintheNorthregionofP

ortugal,whileHospitalsC,E,F,andGarelocatedintheCentreregionofthe

country.

dVariabilityam

ong

isolatesisshowninparenthesis.

eOlderhospitalizationevents(4–9

months).

f Noprevious

hospitalizations

inone

ofthe

patients.

gGenBankaccessionnumberKX421191.

LTCF,long-termcarefacility;NH,nursinghome;F,female;M,male.



171


Kingdom), agar dilution (for fosfomycin; in the presence ofglucose-6-phosphate at 25 mg/L), broth microdilution (forcolistin) or E-test (for carbapenems) (Liofilchem, Italy) methodsaccording to EUCAST (www.eucast.org).

Production of carbapenemases was assessed by the Blue-Carbatest (Pires et al., 2013), and identification of carbapenemases(blaNDM, blaVIM, blaIMP, blaKPC,

blaOXA−48),or other bla genes(blaCTX−M, blaSHV, blaTEM, blaOXA) was performed by PCR andsequencing (Curiao et al., 2010; Bogaerts et al., 2013; Rodrigueset al., 2014).

Population Structure AnalysisPopulation structure characterization included XbaI-Pulsed-Field Gel Electrophoresis (PFGE) (electrophoresis conditions:5–20 s for 4 h and 25–50 s for 18 h, 14◦C, 6 V/cm2),and multi-locus sequence typing (MLST) (http://bigsdb.web.pasteur.fr/klebsiella/primers_used.html) in representativeisolates, as described (Rodrigues et al., 2014). Molecular capsuletyping was performed by PCR and sequencing ofwzi gene (Brisseet al., 2013).

Characterization of the GeneticEnvironment and Location of blaKPC GenesThe genetic context of blaKPC−3 was investigated by PCR andsequencing targeting Tn4401 conserved sequences (Chen et al.,2014a). Location of bla (blaKPC−3, blaCTX−M−15) genes andplasmid characterization were assessed by S1- and I-CeuI-PFGEand identification of replication genes by PCR, sequencing andhybridization (Carattoli et al., 2005; García-Fernández et al.,2009; Villa et al., 2010; Chen et al., 2014a; Rodrigues et al., 2014).

RESULTS

Carbapanemase Production and VariableAntibiotic Resistance PhenotypesAll isolates produced KPC-3 and demonstrated resistanceor intermediate phenotypes to ertapenem (MIC = 1 to 16mg/L), and susceptible, intermediate or resistance phenotypesto imipenem (MIC = 2 to 16 mg/L) and meropenem(MIC = 1 to 8 mg/L), with colonies growing within theinhibition zone of all carbapenems tested, a hetero-resistancephenotype usually observed for KPC producers (Nordmannet al., 2009; Table 2). Although for some isolates the MICvalues for imipenem and meropenem were interpretedas susceptible by the clinical breakpoints defined byEUCAST, in all cases they were above the epidemiologicalcut-off values (ECOFFs) defined for K. pneumoniae(http://www.eucast.org/mic_distributions_and_ecoffs/; Table 2).

All isolates were defined as multidrug resistant (MDR) inaccordance with the definition of MDR for Enterobacteriaceae(non-susceptible to ≥1 agent in ≥3 antimicrobial categories;Magiorakos et al., 2012), although some of them exhibited a lessextensive resistance profile to non-β-lactams, being resistant onlyto ciprofloxacin (Table 2). All isolates were susceptible to colistin(MIC= 0.25–2 mg/L; Table 2).

KPC-3 was Identified Among LocallyCirculating K. pneumoniae ClonesKPC-3-producing K. pneumoniae isolates were assigned to sixdifferent PFGE-types (arbitrarily designated as Kp1 to Kp6),each one of them linked to a specific capsular type (Table 1).Most isolates belonged to ST147 carrying wzi64 (K14.64; n =

10 Kp1, 50%; detected between February to November 2015)and produced additionally SHV-11 (n = 9) or SHV-28 (n =

1), OXA-9 (n = 6) and/or TEM-1 (n = 1; Table 1). Some(n = 4) of these patients had recently been hospitalized inhospitals (A and D) where KPC-3-producing ST147 isolatesexhibiting the same PFGE-type were detected (data not shown,Silva et al., unpublished data). ST15 was also frequent (n =

5, 25%), with two different clones being identified (n = 4Kp2 carrying wzi19/K19; and n = 1 Kp3 carrying wzi93/K60)between July and November 2015. These isolates co-producedOXA-9 and SHV-28. All ST15-Kp2 were identified in patientsfrom the same LTCF or region, and in one case there wasno previous hospitalization (Table 2). ST231 isolates carryingwzi104 (n = 3 Kp4; October 2015) co-produced SHV-1 andTEM-1 (n = 1), and were identified in patients for which noepidemiological link could be established. Sporadic clones suchas ST348 carrying wzi94 (n = 1 Kp5; October 2014) or ST109carrying wzi22 (K22.37; n = 1 Kp6; September 2015) producingother β-lactamases were also detected (Table 1).

IncFIA and IncN Plasmids Involved in theDissemination of blaKPC-3-Tn4401dIn all isolates, the blaKPC−3 was identified between ISKpn7(upstream) and ISKpn6 (downstream), in a structure previouslydescribed as Tn4401 isoform d, known to have a 68bp deletionbetween ISKpn7 and blaKPC gene (Chen et al., 2014b). Isolatesshowed a variable number of plasmids (2–5 plasmids) withdifferent sizes (40–500 kb), frequently from IncFIIK and IncRfamilies. In most of the cases (n = 16/20 isolates from differentclones), blaKPC−3 was located within cointegrated plasmids (105to 250 kb) carrying repAFIA (100% identity with that of pBK30661plasmid, GenBank accession number KF954759), repAFII (100%identity with that of pBK30683 plasmid, GenBank accessionnumber KF954760), and oriColE1 (100% identity with ori p15 genepKBuS13 plasmid, GenBank accession number KM076933). Inthe remaining isolates (n = 4/20 isolates belonging to ST147),blaKPC−3 was identified in a ca. 50 kb IncN plasmid [repNshowing 100% identity with that of pKPC_FCF/3SP plasmid(defined by pMLST as repN allele 7; ST15), GenBank accessionnumber CP004367] (Table 1). The two isolates for which aless extended resistance profile was observed (Table 2) carriedblaKPC−3 within IncN plasmids and no additional IncF plasmidswere observed. The blaCTX−M−15 (when present) was variablylocated in a ca. 200 kb-IncFIIK7 (ST348) or in a ca. 60 kb-IncR(ST15-Kp3) plasmid type.

DISCUSSION

In this study, we highlight a polyclonal structure of KPC-3 producing K. pneumoniae isolates among patients outside



172


TABLE 2 | Antimicrobial resistance patterns of KPC-3-producing K. pneumoniae clones.

Antimicrobial % of Resistance (MIC range, mg/L)a,b

All ST147 ST15-Kp2 ST15-Kp3 ST231 ST348 ST109

(n = 20) (n = 10) (n = 4) (n = 1) (n = 3) (n = 1) (n = 1)

Amoxicillin/clavulanic acid 100 100 100 100 100 100 100

Mecillinam 100 100 100 100 100 100 100

Ceftazidime 100 100 100 100 100 100 100

Cefotaxime 100 100 100 100 100 100 100

Cefepime 100 100 100 100 100 100 100

Cefotaxime 85 80 100 100 100 100 0

Aztreonam 100 100 100 100 100 100 100

Ertapenem 100 (1–16) 100 (1–4) 100 (4–16) 100 (8) 100 (4–8) 100 (8) 100 (1)

Imipenem 75 (2–16) 100 (4–8) 25 (2–8) 100 (8) 33 (2–8) 100 (16) 100 (4)

Meropenem 40 (1–8) 20 (1–4) 25 (2–4) 100 (8) 67 (2–8) 100 (4) 100 (4)

Amikacin 50 60 75 0 33 0 0

Gentamicin 70 70 100 0 67 100 0

Netilmicin 75 70 100 100 67 100 0

Tobramycin 80 70 100 100 100 100 0

Ciprofloxacin 95 100 100 100 100 100 0

Sulfamethoxazole/trimethoprim 90 80 100 100 100 100 100

Trimethoprim 90 80 100 100 100 100 100

Chloramphenicol 35 0 75 100 100 0 0

Fosfomycin 15 10 0 100 0 100 0

Colistin 0 0 0 0 0 0 0

aAll intermediate isolates were considered as resistant.bClinical Breakpoints (Ertapenem - S≤0.5 mg/L; Imipenem and Meropenem - S≤2 mg/L) and ECOFF values (Ertapenem - WT≤0.064 mg/L; Imipenem - WT≤1 mg/L; Meropenem -WT≤0.125 mg/L) for MIC defined by EUCAST for K. pneumoniae.

hospital boundaries in Portugal consistent with nosocomialacquisition, and unveil novel or uncovered plasmid backbonescarrying blaKPC−3 in Europe.

The first clinical cases of KPC-3 producers in Portugal weredetected in 2009 in a pediatric unit of a hospital from theLisbon and Tagus Valley region and involved 2 K. pneumoniaebelonging to ST11 (Machado et al., 2010). Months later anduntil 2011, an outbreak involving 41 KPC-3-producing isolates,most of them (n = 29) assigned to ST14, was reported (Calistoet al., 2012). More recently, a nationwide study reported 22K. pneumoniae producing KPC-3 (mainly ST11, ST14, ST15,and ST147 clones) in several hospitals between 2010 and2013 but plasmid backgrounds had been poorly characterized(Manageiro et al., 2015). However, the situation concerningcarbapenemase-producing Enterobacteriaceae in Portugal wasonly recently recognized in the EuSCAPE survey, where ourcountry appeared in level 2b (sporadic hospital outbreaks)mainly due to the expansion of KPC producers (Albiger et al.,2015). The data presented in this study strengthens an ongoingdissemination of KPC-3 producers in Portugal, where theidentification of these bacteria among such a high diversityof healthcare institutions other than hospitals might potentiatetheir impact for both hospital and community settings. It isthus advisable a reinforcement of infection control measures,surveillance, and tracking of isolates resistant to carbapenems

in clinical institutions, and a coordinated action betweenclinicians, epidemiologists and national reference laboratoriesfor guidance and harmonization of protocols (Albiger et al.,2015).

We observed a polyclonal structure of KPC-3-producingK. pneumoniae isolates, where most of the clones identified(ST15, ST147, ST348) exhibited the same PFGE-pattern asCTX-M-15 (ST348, ST15-Kp2, and -Kp3) or SHV-12 (ST147)producers previously involved in hospital- and community-acquired infections at least in the North region of Portugal(2010–2012; Rodrigues et al., unpublished data; Rodrigueset al., 2014). These “high-risk clones” have been linked tothe worldwide expansion of different ESBL (CTX-M-15 anddifferent SHV-types) and carbapenemases (KPC, VIM, NDM,OXA-48-like), including in Portugal (Rodrigues et al., 2014).This scenario suggests recent acquisition of blaKPC−3 by MDRK. pneumoniae genetic lineages that were already circulatingin Portugal (ST15, ST147, ST231, ST348), a situation observedless frequently than the amplification of CG258 lineages(Baraniak et al., 2015; Bonura et al., 2015; Ocampo et al.,2015).

The ST147 clone [clonal group (CG) 147] exhibiting capsulartype K14.K64 was identified in patients from diverse LTCFs andNHs for a long period of time and seems to be the predominantlineage among KPC-3-producing K. pneumoniae in different



173


healthcare settings (Silva et al., unpublished data; Manageiroet al., 2015). In fact, identical KPC-3-producing ST147 isolateswere recently involved in outbreaks in hospitals where someof the patients had been previously hospitalized. Although,nosocomial acquisition is the most probable origin for mostKPC-3 producers identified in patients included in this study,it is of notice that in three cases no obvious hospitalizationlink could be established. Indeed, considering the frequentdisplacement of these patients between institutions (integratednetwork of LTCFs in Portugal) and hospitals (we had only accessto the last hospitalization event) and that intestinal colonizationmight be persistent in time (Feldman et al., 2013), we cannotcompletely discard cross transmission events in the unitsanalyzed.

The identification of two distinct ST15 (CG15) lineagesin this study (ST15-K19 and ST15-K60) is in line withrecent studies based on wzi-capsule typing unveiling thecirculation of distinct lineages within this CG, that might havedifferences in their relative occurrence, geographical, or nichedistribution and/or host susceptibility (Bialek-Davenet et al.,2014; Holt et al., 2015; Rodrigues et al., 2015; Zhou et al., 2015,http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). TheST231 K. pneumoniae clone (CG231) had already been linked toGES-5 plus SHV-12 production in Portugal (Manageiro et al.,2015) and its association with community invasive infections(sepsis, lethal pneumonia, or meningitis), and high content invirulence and antimicrobial resistance genes have recently beenhighlighted (Holt et al., 2015). The ST109 clone is rarely reported(http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html) and it isdescribed for the first time in Portugal. However, it belongs to theCG17, associated with the expansion of CTX-M-15 and differentcarbapenemases worldwide (Rodrigues et al., 2014; Holt et al.,2015).

The blaKPC−3 was linked to Tn4401d isoform in allcharacterized KPC-3-producing Enterobacteriaceae fromPortugal (this study; Manageiro et al., 2015). In this study, weshow that in most cases (80%) Tn4401d-blaKPC−3 was locatedwithin cointegrated FIA, FII, and ColE1 plasmids (105–250kb; Table 1) corroborating the strong association betweenTn4401d-blaKPC−3 with IncFIA plasmids pointed out previouslyin large collections from the USA (Chen et al., 2014a; Deleoet al., 2014; Bowers et al., 2015; Chavda et al., 2015). We detectedFIA and FII replicons identical to those of pBK30683 plasmid(GenBank accession number KF954760) plus an additionaloriColE1 gene identical to that of the ColE1 plasmid pKBuS13(GenBank accession number KM076933), supporting the roleof these mobilizable plasmids in the assembly of MDR plasmids(Chen et al., 2014a; Garbari et al., 2015). These and other (IncFIAplus IncA/C2 or IncFIA plus IncX3) cointegration forms seemto play an important role in the intra- and inter-species spreadof carbapenem resistance genes (Chen et al., 2014a,b; Chavdaet al., 2015). To the best of our knowledge, we unveil for the first

time a cointegrate IncFIA platform carrying Tn4401d-blaKPC−3in Europe, characterized and highly represented by far only inisolates from the USA (mainly among non-ST258 and non-K.pneumoniae isolates; Chen et al., 2014a). In the remainingisolates (20%, 4 ST147), blaKPC−3-Tn4401d was located on ca.50 kb IncN plasmids, an association primarily described in thisstudy.

In conclusion, this study highlights a polyclonal structureamong KPC-3 producers identified in geographically dispersednon-hospitalized patients in Portugal, not always linkedto nosocomial acquisition, a situation that deserves closemonitoring due to its high clinical or epidemiological impact. Inall cases, a common platform (blaKPC−3-Tn4401d) was identifiedin plasmids still unnoticed in Europe (blaKPC−3-Tn4401d-IncFIA) or firstly reported here (blaKPC−3-Tn4401d-IncN).Their identification in previously circulating MDR K.pneumoniae lineages in our area (ST147, ST15, ST231, ST348)underlines the recent penetration of successful mobile geneticelements in locally circulating clonal backgrounds, rather thanthe importation of the most common global lineages fromCG258.

AUTHOR CONTRIBUTIONS

CR and JB performed the experiments and contributed with theacquisition of molecular data. CR and ÂN wrote the article andperformed analysis and interpretation of molecular data. EMand JA contributed with epidemiological data and revision ofthe manuscript. ÂN and LP contributed with the design of thestudy and final revision of the manuscript. All authors read andapproved the final version of the manuscript.

FUNDING

This work received financial support from the EuropeanUnion (FEDER funds) through Programa OperacionalFactores de Competitividade – COMPETE and NationalFunds (FCT, Fundação para a Ciência e Tecnologia)(UID/Multi/04378/2013). ÂN and CR were supported byfellowships (grant number SFRH/BPD/104927/2014 andSFRH/BD/84341/2012, respectively) from FCT throughPrograma Operacional Capital Humano (POCH).

ACKNOWLEDGMENTS

We thank Dr. Carlos Mendes, Dr. Alexandra Gomes, andDr. Bibiana Marques (Laboratório de Análises Clínicas DoutorCarlos Torres), Dr. Rui Campainha and Dr. SofiaMoniz (BotelhoMoniz Análises Clínicas - BMAC), and Dr. Ricardo Silva(Santa Casa da Misericórdia, Vila do Conde) for providing thestrains and the clinical/epidemiological data included in thisstudy.



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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2016 Rodrigues, Bavlovic, Machado, Amorim, Peixe and Novais. Thisis an open-access article distributed under the terms of the Creative CommonsAttribution License (CC BY). The use, distribution or reproduction in other forumsis permitted, provided the original author(s) or licensor are credited and that theoriginal publication in this journal is cited, in accordance with accepted academicpractice. No use, distribution or reproduction is permitted which does not complywith these terms.



177

Detection of VIM-34, a novel VIM-1 variant identified in the

intercontinental ST15 Klebsiella pneumoniae clone

Carla Rodrigues1, Ângela Novais1, Elisabete Machado1,2, Luísa Peixe1*

1UCIBIO-REQUIMTE. Laboratório de Microbiologia, Faculdade de Farmácia,

Universidade do Porto, Porto, Portugal; 2FP-ENAS/CEBIMED. Faculdade de Ciências da

Saúde, Universidade Fernando Pessoa, Porto, Portugal.

Journal of Antimicrobial Chemotherapy 2014; 69 (1): 274-275

Oxford University Press has authorized the reproduction of the final published PDF version of this

paper in this thesis through the License Agreement Number 4091970885366 on April 18, 2017.


178

J Antimicrob Chemother 2014doi:10.1093/jac/dkt314Advance Access publication 9 August 2013

Detection of VIM-34, a novel VIM-1variant identified in theintercontinental ST15Klebsiella pneumoniae clone

Carla Rodrigues1, Angela Novais1, Elisabete Machado1,2

and Luısa Peixe1*

1REQUIMTE, Laboratorio de Microbiologia, Faculdade de Farmacia,Universidade do Porto, Portugal; 2CEBIMED, Faculdade de Ciencias daSaude, Universidade Fernando Pessoa, Porto, Portugal

*Corresponding author. Universidade do Porto, Faculdade de Farmacia,REQUIMTE Research Laboratory, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Tel: +351-22-042-85-80; Fax: +351-22-042-85-90;E-mail: [email protected]

Keywords: carbapenem resistance, MBL, ESBL, pandemic, class 1integron

Sir,Enterobacteriaceae producing metallo-b-lactamases (MBLs), andparticularly VIM-type MBLs, have frequently been implicated in hos-pital outbreaks across Europe,1 blaVIM genes having been linked toTn402 derivatives, epidemic plasmids (IncN, IncI1, IncHI2) and occa-sionally with particular Enterobacteriaceae clones.1–4 VIM enzymeshave been classified in three clusters (VIM-1, VIM-2 and VIM-7)according to their amino acid sequences (http://www.lahey.org/studies), VIM-1 and VIM-2 being the most widespread variants.5

In this study, we report the molecular epidemiology and the anti-biotic susceptibility profiles of Klebsiella pneumoniae clinical isolatesproducing VIM-34, a novel VIM-1 variant identified in Portugal.

In October 2011 and October 2012, two K. pneumoniae isolates(strains K43 and K47, respectively) showing reduced susceptibilityto carbapenems (MICs 0.38–1.0 mg/L) were recovered fromurine samples of hospitalized patients in a general hospital innorthern Portugal (Hospital Pedro Hispano). They are the onlycarbapenemase-producing Enterobacteriaceae isolates identifiedin this hospital since the beginning of 2011, when reference proto-cols for carbapenemase detection were adopted.

Antimicrobial susceptibility tests were performed using theEtest for b-lactams and disc diffusion for all other antimicrobialagents. These showed that all isolates were resistant to diversecephalosporins, aztreonam,b-lactam/b-lactamase inhibitor com-binations (Table 1), nalidixic acid, ciprofloxacin, chloramphenicol,gentamicin, kanamycin, netilmicin, streptomycin, tobramycinand sulphonamides, but susceptible to trimethoprim andamikacin (http://www.eucast.org/).6 Standard disc diffusion pheno-typic tests using different b-lactams and b-lactamase inhibitors(cefotaxime, ceftazidime, imipenem; 0.2 mM EDTA, clavulanic acid),6

isoelectric focusing, PCR and sequencing7 demonstrated the pro-duction of VIM-34 (pI¼5.4) (GenBank accession numberJX013656), a novel VIM-type enzyme differing from VIM-1 by oneamino acid change (V113I, according to MBL standard numbering

scheme) and co-production of SHV-1 (pI¼7.6) and SHV-12(pI¼8.2) extended-spectrum b-lactamase. We could not identifythe origin of these isolates but as both patients had multiple previ-ous hospitalizations (including in other hospitals) and carried thesame novel blaVIM type, a common nosocomial source seemsmore plausible than community acquisition.

The blaVIM-34 from the K47 isolate was cloned in the pBGS18(kanamycinresistance)plasmidusingprimersVIM-EcoRI(5′-GGGAATTCGCAGTCGCCCTAAAACAAAG-3′) and VIM-PstI (5′-AACTGCAGCCGCTCCAACGATTTGTTAT-3′) (restrictionsitesareunderlined),andtheexpressionvector (pBGS18/VIM-34) was further introduced into Escherichiacoli DH5a, as previously reported.8 MICs of different b-lactamswere determined using the Etest (in triplicate) and compared withthose corresponding to a blaVIM-1-carrying clone obtained in thesame conditions (Table 1). The VIM-34-producing E. coli recombin-ant yielded b-lactam MIC values similar to those observed inthe VIM-1-encoding transformant (with the exception of cefoxitin;Table 1). Because our experiments were performed in an isogeniccontext and identical standard experimental conditions, we are ableto hypothesize that the substitution V113I has a low influence onthe MICs of carbapenems, although further studies of enzymaticactivity are required to confirm this observation.

The isolates exhibited identical XbaI-PFGE profiles and clonalidentification by multilocus sequence typing(http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) revealed thatthey belong to the intercontinental ST15 K. pneumoniae clone,widely disseminated in different European countries and associatedwith the spread of extended-spectrum b-lactamases (CTX-M-15;diverse SHV types) and/or MBLs (VIM-1, NDM-1).2,3,7,9,10Conjugationassays performed by broth and/or filter mating methods using E. coliHB101 (azide and kanamycin resistant, Lac-, plasmid free) as

Table 1. MICs of different b-lactam antibiotics for VIM-34-producingwild-type isolates and recombinant strains encoding VIM-34 or VIM-1

Antibiotic

MIC (mg/L)

Klebsiellapneumoniae

K43 (VIM-34)a

E. coli DH5a

pBGS18pBGS18/

VIM-1pBGS18/VIM-34

Amoxicillin/clavulanate 24 2 24 12Ticarcillin/clavulanate .256 2 .256 .256Piperacillin/tazobactam .256 0.75 6 6Cefalotin .256 2 64 32Ceftazidime .256 0.19 12 8Cefotaxime 32 0.125 2 2Cefepime 12 0.016 0.75 0.5Cefpirome 32 0.032 1.5 2Cefoxitin 32 4 12 4Aztreonam 32 0.023 0.023 0.016Ertapenem 0.38b 0.006 0.008 0.008Imipenem 1.0b 0.125 0.38 0.38Meropenem 0.5b 0.016 0.032 0.023

aK47 isolate exhibited identical antibiotic susceptibility profiles.bMIC values interpreted as susceptible by both EUCASTand CLSI guidelines,but above the epidemiological cut-off values defined for K. pneumoniae(http://www.eucast.org).6

Research letters

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recipient at 228C and 378C (selection of transconjugants in MacCon-key agar with 2 mg/L of ceftazidime and 130 mg/L of azide)7 failedto yield transconjugants either for blaVIM-34 or blaSHV-12. The locationof bla (blaVIM-34, blaSHV-12) genes and plasmid characterization wereaccomplished by S1- and I-CeuI-PFGE, and identification of incom-patibility groups.7 In both isolates, blaVIM-34, blaSHV-12 and repHI2probes hybridized in the same chromosomal band (I-CeuI-PFGE)whereas no signals were observed in the S1 gel, suggesting the ac-quisition of both bla genes by an IncHI2 plasmid and subsequentplasmid (whole or in part) integration. A chromosomal locationfor bla genes, including blaVIM, has been occasionally observed indifferent Enterobacteriaceae species.1

The linkage of blaVIM-34 to class 1 integrons and Tn402 deriva-tives was investigated by PCR (intI1, 5′CS-3′CS region, orf5, orf6,IS1326, IS1353, IS6100) and sequencing.4,11 blaVIM-34 waslocated within an "6 kb class 1 integron named In817 by INTE-GRALL (http://integrall.bio.ua.pt/) (GenBank accession numberJX185132), with an original array of gene cassettes comprisingblaVIM-34, aacA4′, aphA15, aadA1b and catB2 (Figure S1; availableas Supplementary data at JAC Online). The absence of tni402

sequences and the high similarity detected with In70 and In113,identified in VIM-1-producing Achromobacter xylosoxidans, K.pneumoniae and E. coli isolates, suggests that the In817 integronmight have arisen by both recombination and in vivo evolutionevents (Figure S1; available as Supplementary data at JAC Online).4

In summary, we present the first report of VIM-34, a VIM-1-likevariant embedded in the novel integron type In817 on the chromo-some of the intercontinental ST15 K. pneumoniae clone, asso-ciated with carbapenem susceptibility profiles similar to thoseobserved for VIM-1. This study highlights the risk of further dissem-ination of the multidrug-resistant ST15 K. pneumoniae clone andgenetic backgrounds containing metallo-b-lactamase genes inour country, which deserves future monitoring.

AcknowledgementsWe thank Valquıria Alves and Antonia Read (Hospital Pedro Hispano,Matosinhos, Portugal) for the gift of strains.

FundingThis study was supported by Fundacao para a Ciencia e Tecnologia, which ispart of the Ministry of Science, Technology and Innovation of Portugal(through grants no. PEst-C/EQB/LA0006/2011, PTDC/AAC-AMB/103386/2008, EXPL/DTP-EPI/0196/2012 and FCOMP-01-0124-FEDER-027745),Fundacao Ensino e Cultura Fernando Pessoa, and an ESCMID ResearchGrant 2012 awarded to Angela Novais. Carla Rodrigues was supported byFundacao para a Ciencia e Tecnologia through grant no. SFRH/BD/84341/2012. Angela Novais was supported by a Marie Curie Intra European Fellow-ship within the 7th European Community Framework Programme(PIEF-GA-2009-255512).

Transparency declarationsNone to declare.

Supplementary dataFigure S1 is available as Supplementary data at JAC Online (http://jac.ox-fordjournals.org/).

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3 Sanchez-Romero I, Asensio A, Oteo J et al. Nosocomial outbreak ofVIM-1-producing Klebsiella pneumoniae isolates of multilocus sequencetype 15: molecular basis, clinical risk factors, and outcome. AntimicrobAgents Chemother 2012; 56: 420–7.

4 Tato M, Coque TM, Baquero F et al. Dispersal of carbapenemase blaVIM-1

gene associated with different Tn402 variants, mercury transposons, andconjugative plasmids in Enterobacteriaceae and Pseudomonas aeruginosa.Antimicrob Agents Chemother 2010; 54: 320–7.

5 Cornaglia G, Giamarellou H, Rossolini GM. Metallo-b-lactamases: a lastfrontier for b-lactams? Lancet Infect Dis 2011; 11: 381–93.

6 Clinical and Laboratory Standards Institute. Performance Standards forAntimicrobial Susceptibility Testing: Twenty-Third Informational SupplementM100-S23. CLSI, Wayne, PA, USA, 2013.

7 Novais A, Rodrigues C, Branquinho R et al. Spread of an OmpK36-modifiedST15 Klebsiella pneumoniae variant during an outbreak involving multiplecarbapenem resistant Enterobacteriaceae species and clones. Eur J ClinMicrobiol Infect Dis 2012; 31: 3057–63.

8 Novais A, ComasI,Baquero Fetal. Evolutionarytrajectories ofb-lactamaseCTX-M-1 cluster enzymes: predicting antibiotic resistance. PLoS Pathog2010; 6: e1000735.

9 Damjanova I, Toth A, Paszti J et al. Expansion and countrywidedissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type b-lactamase-producing Klebsiella pneumoniae epidemic clonesin Hungary in 2005-the new ‘MRSAs’? J Antimicrob Chemother 2008; 62:978–85.

10 Poirel L, Benouda A, Hays C et al. Emergence of NDM-1-producingKlebsiellapneumoniae inMorocco.JAntimicrobChemother2011;66:2781–3.

11 Novais A, Baquero F, Machado E et al. International spread andpersistence of TEM-24 is caused by the confluence of highly penetratingEnterobacteriaceae clones and an IncA/C2 plasmid containing Tn1696::Tn1and IS5075-Tn21. Antimicrob Agents Chemother 2010; 54: 825–34.

J Antimicrob Chemother 2014doi:10.1093/jac/dkt315Advance Access publication 29 August 2013

Mutant prevention concentrations ofcolistin for Acinetobacter baumannii,Pseudomonas aeruginosa and Klebsiellapneumoniae clinical isolates

Myung-Jin Choi and Kwan Soo Ko*

Department of Molecular Cell Biology, Samsung BiomedicalResearch Institute, Sungkyunkwan University School of Medicine,Suwon 440-746, Korea

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Figure S1 is available as Supplementary data at JAC online

Supplementary data

Figure S1. Comparison of the novel class 1 integron (In817) containing the new blaVIM-34 gene described in this study with highly similar

integron structures (In70, In113) previously described.4 Open reading frames (ORFs) are indicated by arrows. Black boxes represent class 1

integron conserved (CS) regions and light grey boxes represent the gene cassettes. The P1/Pant promoter type, which directs the expression of the

gene cassettes, is indicated by fine black arrows. The 59-base elements (attC recombination site) of the corresponding gene cassettes are

indicated by white circles.


181

High rates of long-term care facilities (LTCFs) residents colonized with multidrug resistant Klebsiella pneumoniae lineages frequently causing

infections in Portuguese clinical institutions

Carla Rodrigues1, Ana C Mendes1,2, Filip Sima1, Jan Bavlovič3,4,

Elisabete Machado1,5, Ângela Novais1, Luísa Peixe1*

1UCIBIO/REQUIMTE. Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2Serviço de Microbiologia, Centro Hospitalar do Porto, Portugal; 3Faculty of Pharmacy in

Hradec Králové, Charles University, Prague, Czech Republic; 4Faculty of Military Health

Sciences, University of Defense, Brno, Czech Republic; 5FP-ENAS/CEBIMED. Faculdade

de Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal.

Running Tittle: Faecal carriage of MDR Enterobacteriaceae among LTCFs residents Keywords: intestinal colonization, ESBL, AmpC, MLST, capsule subtyping

Corresponding author:

*Luísa Peixe.

UCIBIO/REQUIMTE,

Laboratório de Microbiologia, Faculdade de Farmácia,

Universidade do Porto,

Rua Jorge de Viterbo Ferreira, n. 228,

4050-313 Porto, Portugal

Tel.: +351 220 428 580




182

Sir,

The recent increase of extended-spectrum β-lactamase (ESBL)-producing Klebsiella

pneumoniae (Kp) and the emergence of carbapenemase-producing Kp in Portuguese hospitals

parallels epidemiological trends described in other countries (1,2). Moreover, the recent

demonstration that Kp isolates causing hospital infection often correspond to the patient's own

colonizing strains, stresses the need to survey multidrug-resistant (MDR) bacteria among the

intestinal flora of patients from different clinical settings (3). Long-term care facilities (LTCFs) are

fundamental institutions in contemporary healthcare services, mainly in the assistance of elderly

people who due to frequent hospitalizations, recurrent antibiotic consumption and communal living

are at a high risk of infection by MDR bacteria (4,5). Different studies among European LTCFs

residents have pointed out the high rates of colonization by CTX-M-15-producing Escherichia coli

belonging to the pandemic ST131 clones, and a low occurrence of carbapenemase-producing

bacteria. However, little is known regarding the prevalence and diversity of other MDR non-E. coli

Enterobacteriaceae species (and particularly Kp) colonizing LTCFs residents, including in

Portugal (4,6,7). The aim of this study was to assess the faecal carriage rate and epidemiological

features of non-E. coli Enterobacteriaceae isolates resistant to extended-spectrum β-lactams among

Portuguese LTCFs residents.

Faecal samples from residents (n=47; 32 females/15 males) at LTCF I (25 beds/n=25

samples) and LTCF II (40 beds/n=22 samples) located in the North of Portugal were collected on

July 2015 and January 2016, respectively. A total of 81% of the residents were ≥70 years old, 44%

were previously hospitalized or shared a room with other residents, and 58% received antibiotic

treatment during the three months preceding sampling (mainly amoxicillin-clavulanate, but also

fosfomycin or levofloxacin). Faecal samples were suspended in 2 mL of saline solution and 0.2 mL

were seeded on chromID® ESBL for screening of isolates producing ESBL and/or acquired AmpC.

For carbapenemase detection, the 0.2 mL were seeded directly on the chromID® Carba SMART

agar plate (37ºC/24-48h) or after a preenrichment step (0.1 mL inoculum in 10 mL of trypticase soy

broth containing a 10-µg meropenem disk, and incubation at 37ºC/18h) (8,9). All presumptive non-

E. coli Enterobacteriaceae (representative morphotypes/plate) were selected for further


183

characterization. Bacterial identification was assessed by species-specific PCR and/or MALDI-

TOF MS (VITEK MS Biomérieux, Marcy l'Etoile, France). Production of ESBL/acquired AmpC

or carbapenemases was detected by phenotypic and Blue-Carba tests, respectively, PCR and

sequencing (8). Susceptibility testing to non-β-lactams antibiotics was performed by the disk

diffusion method (http://www.eucast.org/clinical_breakpoints/). Clonal relatedness among Kp

isolates was evaluated by Fourier transform infrared with attenuated total reflectance (FTIR-ATR)

spectroscopy, multilocus sequence typing (MLST) and wzi capsular typing

(http://bigsdb.pasteur.fr/klebsiella/primers_used.html), as described previously (10,11).

Microbiological air quality was also assessed using an air sampler (MAS100, Merck

Millipore, Germany) with a flow rate of 100 L/min. Air samples of 250 L were collected from

different common indoor (n=3, LTCF I; n=1, LTCF II) and outdoor (n=1 for both institutions)

spaces using different culture medium petri dishes [plate count agar (PCA), chromID® ESBL and

chromID® Carba SMART], which were further incubated at 25ºC/72h (PCA, for fungi

quantification) or at 37ºC/48h (PCA and other culture medium, for bacteria quantification). The air

bioburden values were expressed in CFUs per m3 (CFU/m3), and air quality was categorized

according to the Portuguese law (Ordinance No. 353-A/2013 4 December)

(https://www.academiaadene.pt/download/pt/portaria-n-353-a2013-recs-ventilacao-e-qai.pdf).

A high proportion of faecal samples (n=19/47; 40.4%) was positive for non-E. coli

Enterobacteriaceae producing ESBL (29.8%) or acquired AmpC (10.6%). It is of interest to

highlight that in 53% of the samples we also identified E. coli producing ESBL, leading to an

overall rate of faecal carriage with ESBL-producers of 81% (data not shown). Quality of air

samples was within the established standards at LTCF I, but fungi and bacteria counts were above

the limit in LTCF II (https://www.academiaadene.pt/download/pt/portaria-n-353-a2013-recs-

ventilacao-e-qai.pdf), whereas no growth was detected on selective plates (chromID® ESBL and

chromID® Carba SMART). Similar colonization rates (44% in LTCF I and 36% in LTCF II) were

observed in both institutions, despite some epidemiological differences (Table 1). The colonization

rates by ESBL-producing non-E. coli Enterobacteriaceae (29.8%) was significantly higher than

that encountered some years ago among LTCFs and nursing home residents in our country (~6%,


184

2008-2012), or LTCFs from other European countries (~8%, 2012-13) (6,12). Despite the low

sample size, this extraordinary increase (ca. 5-fold) depicts a worrying scenario taking into

consideration this at-risk population, probably influenced by the recent global expansion of MDR

Kp isolates in clinical institutions from our country

(http://ecdc.europa.eu/en/publications/Publications/antimicrobial-resistance-europe-2015.pdf).

Carbapenemases-producing Enterobacteriaceae were not detected despite they are increasingly

identified (mainly KPC-3 and OXA-48) among clinical isolates from either hospitalized and non-

hospitalized patients from LTCFs and nursing homes in Portugal (2,13).

CTX-M-15 (n=16 isolates, 14 samples) and DHA-1 (n=6 isolates, 5 samples) were,

respectively, the only ESBL and acquired AmpC variants identified (Table 1). In LTCF I, CTX-M-

15 was predominant (n=12 isolates in 91% of the samples), especially among Kp ST15 lineages (7

wzi19-K19, 3 wzi24-K24). Some of these residents shared rooms and had been previously

hospitalized or institutionalized in variable hospitals or LTCFs, respectively. A DHA-1-producing

ST11 (wzi75) Kp isolate was sporadically detected (Table 1). Both ST15 Kp clonal lineages (K19,

K24) or ST11-wzi75 have been frequently identified among isolates producing CTX-M-15, KPC-3

or DHA-1 in Portuguese clinical institutions (1,2,13). Conversely, in LTCF II, CTX-M-15 (n=4

isolates in 50% of the samples) was detected in 2 Kp clones (2 ST348-wzi94 and 1 ST15-wzi24)

and 1 Enterobacter aerogenes from residents with previous admissions at different LTCFs,

whereas DHA-1 (n=5 isolates in 50% of the samples) was identified in diverse species and lineages

(Kp, Proteus mirabilis, Enterobacter spp.) from residents with previous hospital admissions (Table

1). It is of note the co-colonization by isolates from the same or different species harbouring the

same enzyme (which suggests in vivo transfer of bla gene), as well as the strain variability in

residents with previous admission in the same institution.

Our study reveals increased and alarming colonization rates by MDR Enterobacteriaceae

among LTCFs residents that represent a challenge for these institutions in terms of stewardship and

infection prevention and control, and also highlight the risk of further dissemination of MDR

bacteria to healthcare professionals, residents’ families and the community in general. Most of the

isolates identified belonged to the same Kp lineages [ST11 (wzi75), ST15 (wzi19, wzi24) and


185

ST348 (wzi94)] of those causing infections in clinical institutions from the same geographic area,

evidencing a higher risk of infection by this vulnerable population. It is of note the variability

observed on the acquired antibiotic resistance genes of such lineages (ESBL, AmpC,

carbapenemases) among colonization/infection isolates in our settings, which might reflect the

open genome of such lineages for plasmid exchange. Finally, the antibiotic susceptibility data

obtained in this study could support an optimized empirical therapeutic approach in these

institutions.

Acknowledgments: We are grateful to the residents, the healthcare personnel and the management

board of the long-term care facilities that participated in this study. We thank to bioMérieux for

kindly providing chromID® ESBL and chromID® CARBA agar plates for this study.




UID/MULTI/04378/2013. CR and ÂN were supported by fellowships from FCT

(SFRH/BD/84341/2012 and SFRH/BPD/104927/2014, respectively).

Transparency declarations: Nothing to declare.

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188

Tabl

e 1.

Epi

dem

iolo

gica

l dat

a of E

SBL-

or a

cqui

red

Am

pC-p

rodu

cing

non

-E. c

oli i

sola

tes i

dent

ified

in re

siden

ts fro

m P

ortu

gues

e LTC

Fs (2

015

and

2016

).

NA, n

ot ap

plica

ble;

NI, n

ot id

entif

ied; S

, sha

red

room

; I, i

ndiv

idua

l roo

m.

a F, F

emale

, M, M

ale; b ST

, Seq

uenc

e Typ

e; c

Varia

bilit

y am

ong

isolat

es is

show

n in

par

enth

esis;

d AMK,

amik

acin

; CIP

, cip

roflo

xacin

; CLO

, chl

oram

phen

icol;

GEN,

gen

tamici

n; K

AN, k

anam

ycin

; NAL

, nali

dixi

c acid

; NET

, neti

lmici

n; S

TR,

strep

tom

ycin

; SUL

, sul

phon

amid

es; S

XT, s

ulfa

meth

oxaz

ole;

TET,

tetra

cycli

ne; T

MP,

trim

ethop

rim; T

OB, t

obra

myc

in; e

Sing

le Lo

cus V

arian

t (SL

V) o

f ST4

05.

LTC

F (S

ampl

ing

date

)

Res

iden

t(s)

Gen

der/

Age

a Dat

e of

adm

issio

n in

the

LTC

F Lo

cal o

f pre

viou

s in

tern

men

t St

rain

R

oom

type

Spe

cies

(no.

) ES

BL o

r A

mpC

ST

b FT

IR

clus

ters

wzi

Res

istan

ce to

non

-β-la

ctam

sc,d

LTCF

I (J

uly

2015

) LT

1-LT

6 F(

4)/7

4-85

M

(2)/6

0-71

14

.10.

2014

- 24

.6.2

015

H1,

H2,

H3,

LT

CF1,

LTC

F2,

LTCF

3

LT1.

1-LT

6.1

S (3

), I (

3)

K. p

neum

onia

e (6)

CT

X-M

-15

ST15

A

wz

i19

(AM

K), C

IP, C

LO, (

GEN)

, KAN

, NAL

, N

ET, S

TR, S

UL,

SX

T, (T

ET),

TMP,

TOB

LT7

F/78

02

.06.

2015

LT

CF3

LT7.

1 I

K. p

neum

onia

e CT

X-M

-15

ST15

A

wz

i19

CIP,

CLO

, GEN

, KA

N, N

AL,

NET

, STR

, SU

L, S

XT,

TM

P, T

OB

LT

7.2

K.

pne

umon

iae

CTX

-M-1

5 ST

15

B wz

i24

CIP,

GEN

, KA

N, N

AL,

STR

, SU

L, S

XT,

TE

T, T

MP

LT8

F/90

08

.07.

2915

H

4 LT

8.1

S K.

pne

umon

iae

CTX

-M-1

5 ST

15

B1

wzi2

4-lik

e A

MK

, CIP

, CLO

, GEN

, KAN

, NAL

, N

ET, S

TR, S

UL,

SX

T, T

MP,

TO

B LT

9 M

/75

15.0

6.20

15

LTCF

3 LT

9.1

I K.

pne

umon

iae

CTX

-M-1

5 ST

15

B wz

i24

CIP,

GEN

, KA

N, N

AL,

NET

, STR

, SU

L,

SXT,

TET

, TM

P, T

OB

LT

9.2

E.

aer

ogen

es

CTX

-M-1

5 N

A

NA

N

A

AM

K, C

IP, G

EN, K

AN

, NA

L, N

ET,

STR,

SU

L, S

XT,

TET

, TM

P, T

OB

LT10

M

/61

06.0

2.20

15

H3

LT10

.1

I K.

pne

umon

iae

CTX

-M-1

5 ST

2620

e C

wzi1

43

CIP,

GEN

, KA

N, N

AL,

NET

, STR

, SU

L,

SXT,

TET

, TM

P, T

OB

LT11

F/

76

27.0

5.20

15

H5

LT11

.1

I K.

pne

umon

iae

DH

A-1

ST

11

D

wzi7

5 CI

P, C

LO, K

AN

, NA

L, S

TR, S

UL,

TO

B

LTCF

II

(Jan

uary

20

16)

LT21

-22

M(2

)/53-

79

05.0

5.20

15

11.0

4.20

13

LTCF

4, H

2 LT

21.1

-LT

22.1

S

(1),

I (1)

K.

pne

umon

iae

(2)

CTX

-M-1

5 ST

348

H

wzi9

4 CI

P, (C

LO),

(GEN

), (K

AN

), N

AL,

(N

ET),

STR,

SUL

, SXT

, TM

P, (T

ET),

(TOB

) LT

23

M/8

0 12

.07.

2013

H

(NI)

LT23

.1

S K.

pne

umon

iae

CTX

-M-1

5 ST

15

B wz

i24

CIP,

KA

N, N

AL,

NET

, SU

L, S

XT,

STR

, TM

P, T

OB

LT24

M

/93

07.0

8.20

15

H6

LT24

.1

I E.

aer

ogen

es

CTX

-M-1

5 N

A

NA

N

A

AM

K, G

EN, K

AN

, NET

, TET

, TO

B

LT25

F/

84

08.0

7.20

15

LTCF

5 LT

25.1

S

K. p

neum

onia

e D

HA

-1

NI

E wz

i193

-like

CIP

, KA

N, N

AL,

SU

L, S

TX, T

MP,

TO

B

LT26

F/

84

21.0

9.20

15

LTCF

4 LT

26.1

S

K. p

neum

onia

e D

HA

-1

ST25

2 F

wzi8

1 CI

P, K

AN

, NA

L, S

UL,

TO

B

LT27

F/

81

15.1

0.20

15

LTCF

6 LT

27.1

S

K. p

neum

onia

e D

HA

-1

ST66

1 G

wz

i373

CI

P, K

AN

, NA

L, S

UK

, TET

, TO

B

LT28

F/

48

21.0

8.20

12

LTCF

7 LT

28.1

I

Ente

roba

cter

spp.

D

HA

-1

NA

N

A

NA

CI

P, K

AN

, SU

L, S

TR

LT28

.2

P.

mira

bilis

D

HA

-1

NA

N

A

NA

CI

P, K

AN

, NA

L, S

UL,

TO

B


189

3.3. Detailing multidrug resistant (MDR) Klebsiella pneumoniae by omic approaches: from classical to high-throughput modern typing

3.3.1. Congruence between capsular genotypic and phenotypic features of

multidrug-resistant (MDR) Klebsiella pneumoniae clones: a step-forward

on K- typing by Fourier Transform Infrared (FTIR) Spectroscopy.

3.3.2. Fourier Transform Infrared (FTIR) spectroscopy based-typing for “real-

time” analysis of an outbreak by carbapenem-resistant Klebsiella

pneumoniae isolates.

3.3.3. Elucidating constraints for differentiation of major human Klebsiella

pneumoniae clones by MALDI-TOF MS.

3.3.4. High-resolution analysis of the globally disseminated multidrug-resistant

Klebsiella pneumoniae Clonal Groups 14 and 15.


191

Congruence between capsular genotypic and phenotypic features of multidrug-resistant (MDR) Klebsiella pneumoniae clones: a step-

forward on K-typing by Fourier Transform Infrared (FTIR) Spectroscopy

Carla Rodrigues1, Clara Sousa2, João Almeida Lopes3, Ângela Novais1, Luísa Peixe1*

1UCIBIO/REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia,

Universidade do Porto, Porto, Portugal. 2LAQV/REQUIMTE, Departamento de Ciências

Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal. 3Research

Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa,

Lisboa, Portugal.

Running Title: FTIR-based typing of MDR K. pneumoniae clones

Keywords: high-risk clones, capsular typing, LPS, virulence, yersiniabactin

Corresponding Author: * Luísa Peixe. UCIBIO/REQUIMTE,

Laboratório de Microbiologia,

Faculdade de Farmácia, Universidade do Porto,

Rua de Jorge de Viterbo Ferreira, n. 228,

4050-313 Porto, Portugal

Tel.: +351 220 428 580




192

ABSTRACT

Multidrug resistant (MDR) Klebsiella pneumoniae (Kp) strains are overrepresented by

particular clonal groups (CGs), such as CG14, CG15, CG101, CG147 and CG258. Capsular

diversity within these clones has been uncovered by recent molecular methods for capsule typing,

but it has been essentially explored for CG258. Since capsule is a major virulence factor, the

correlation between genotypic and phenotypic features is necessary to clarify possible host-

pathogen interactions. The main goal of this study is to assess the potential of Fourier transform

infrared (FTIR) spectroscopy, a phenotype-based method yielding characteristic bacterial surface

fingerprinting, for strain level discrimination in an international collection of clinically relevant

MDR Kp clones.

One hundred and fifty-eight previously characterized MDR K. pneumoniae isolates

representative of different worldwide spread clones or CG (ST14, ST15, ST39, ST54, ST101,

ST147, ST336, CG258) producing diverse ESBLs and/or carbapenemases were studied. Capsular

types, (wzi sequencing, and association with cps locus using KAPTIVE database), O-types and

virulence genes were characterized by genotypic methods. Phenotypic characterization of K-types

was performed by FTIR-ATR, and data analysed by chemometric tools.

Despite the high capsular diversity found (prevailing rhamnose capsule types), specific

associations between capsular types and particular ST or lineages could be depicted in different

geographic regions over time, that were correctly (90,9-99,5%) discriminated by FTIR-ATR.

Additionally, variants within Kp ST11 and ST15 clones were inferred from genotypic methods and

further corroborated by FTIR-ATR. Conversely, a low diversity was observed among O-types, O1

and O2 being the most prevalent. Moreover, MDR Kp clones analysed were enriched in type 1 and

3 fimbriae and iron uptake systems (yersiniabactin and Kfu system).

We demonstrate for the first time the potential of FTIR-ATR to differentiate K.

pneumoniae capsular types, highlighting the potential of this methodology as a fast and accurate

alternative K-typing tool. The high prevalence of rhamnose capsule derivatives among MDR Kp is

also of relevance, and needs to be further explored in terms of host-pathogen interactions and the

design of therapeutic or preventive perspectives. Our study also provides a comprehensive


193

characterization of international MDR K. pneumoniae clones, unveiling their enrichment in specific

virulence factors, which is worrisome due to the possible clinical impact.

INTRODUCTION

Klebsiella pneumoniae (Kp), a Gram negative encapsulated bacterium from the

Enterobacteriaceae family, is a frequent cause of hospital- (mainly pneumonia, urinary tract or

wound infections and sepsis) and community-acquired (pyogenic liver abscess, meningitis,

pneumonia or urinary tract infections) infections (1,2). In the last years, the infection rates caused

by Kp and generally associated with resistance to multiple antibiotics increased alarmingly, rising a

serious global public health concern (1,3)

(http://ecdc.europa.eu/en/publications/publications/antimicrobial-resistance-europe-2014.pdf).

The distinct clinical manifestations combined with the output of the different typing

methods [capsular typing, multilocus sequence typing (MLST) and more recently whole-genome

sequencing (WGS)] settled the basis for the definition of two distinct Kp populations comprising

hypervirulent (HV) or multidrug resistant (MDR) strains (4,5). Capsule has been recognized in Kp

strains as a major virulence factor that protects bacteria from phagocytosis and from the

bactericidal effect of the human serum (6). In fact, the expression of specific capsular types or K-

types (mainly K1 and K2) linked to enhanced capsular production (due to the expression of mucoid

phenotype regulators - rmpA and rmpA2), together with particular virulence factors, such as

siderophore systems (yersiniabactin and aerobactin locus), seem to play a major role in the ability

of HV Kp to cause invasive infections (4,5,7). On the other hand, MDR Kp isolates usually have a

lower content in Kp recognized virulence factors (mainly type 1 and 3 fimbriae) and a greater

diversity of capsular types, although the significance in terms of infectivity potential is unclear

(4,7). At a global level, MDR strains causing human infection and commonly linked to the

production of diverse extended-spectrum β-lactamases (ESBL), acquired AmpCs and/or

carbapenemases belong to a few clonal groups (CGs) (CG14, CG15, CG101, CG147, CG258).

Among these, capsular diversity has been explored mainly for CG258 isolates, where different

lineages have been associated with specific capsular types (8–10).


194

Kp strains express on the cell surface K-antigens from the capsular polysaccharide (CPS)

and O-antigens from the lipopolysaccharide (LPS), being antigenically recognized 77 K-types and

9 O-types (2,6). Despite being still considered the reference technique for K- and O-type

determination, serotyping is restricted to reference centers (and thus has limited applicability) and

has not 100% coverage (2,6). More recently, the identification of particular discriminatory cps gene

markers or the massive use of WGS facilitated both O- and K-typing, which particularly boosted

the knowledge on capsular variation among Kp MDR strains (11–14). Nevertheless, the

information obtained by these methods is limited to a genotypic (wzi/wzc-based typing, targeting

conserved cps genes) or genomic (the KAPTIVE platform, targeting the whole cps cluster)

fingerprint of the cps locus (12–14). Considering that the expression of capsular types with variable

polysaccharide composition will condition host-pathogen interactions, it is mandatory to correlate

genotypic and phenotypic features. Within the techniques that allow a phenotypic characterization

of bacteria at the surface level, Fourier transform infrared (FTIR) spectroscopy-based

fingerprinting represents an attractive alternative. Some studies highlight its potential for

discrimination of capsular types, but it has never been explored for Kp (15–18). This rapid and

cost-effective methodology is based on the interaction of infrared radiation with the bacterial

isolate, which leads to the vibration of certain functional groups, providing a specific fingerprint

that reflects the structure and composition of the whole cell (nucleic acids, polysaccharides,

proteins, fatty acids) (15,19). The main purpose of this study is to assess the potential of FTIR for

strain level discrimination of a representative and international collection of MDR Kp strains and to

correlate phenotype-based FTIR-assignments with genotypic features.

MATERIAL AND METHODS

Bacterial Strains

One-hundred fifty-eight well-characterized MDR Kp clinical isolates were analysed in this

study. They represent a snapshot of major MDR clones responsible for outbreaks or endemic

situations in multiple countries (Brazil, Greece, Poland, Portugal, Romania, Spain) in a large period

of time (2003-15). Most of the isolates were producers of ESBLs, acquired AmpCs and/or


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carbapenemases and belonged to different pulsed-field gel electrophoresis (PFGE)-types (n=36),

sequence types (ST) (n=13) or CG (n=7). Details about the bacterial isolates included in this study

are summarized in Table 1.

Gene virulence Content

The presence of 9 putative virulence genes including siderophores [enterobactin (entB),

aerobactin (iutA), yersiniabactin (ybtS)], ferric ABC transporters (kfu), adhesins [type 1 (fimH) and

type 3 (mrkD) fimbriae], urease operon (ureA), allantoinase cluster (allS) and regulators of mucoid

phenotype (rmpA) was screened by PCR (20).

Genotypic characterization of CPS and LPS

Capsular genotyping was performed by PCR and sequencing of wzi (12) and occasionally

wzy genes (13). Specific LPS antigens (O1, O2, O3 and O5) were identified by PCR (11).

FTIR with attenuated total reflectance (ATR) spectra acquisition

Isolates were grown on Mueller-Hinton agar at 37ºC for 18h and colonies were directly

transferred from the agar plates to the ATR crystal and air-dried in a thin film. Spectra were

acquired using a Perkin Elmer Spectrum BX FTIR System spectrophotometer in the ATR mode

with a PIKE Technologies Gladi ATR accessory from 4000–600 cm-1 and a resolution of 4 cm-1

and 32 scan co-additions. For each isolate, three instrumental replicates (obtained from the same

agar plate in the same day) and three biological replicates (obtained in three independent days)

were acquired and analyzed, corresponding to a total of nine spectra per strain (21,22).

Spectra preprocessing and modeling

Original FTIR-ATR spectra were processed with standard normal variate (SNV) followed

by the application of a Savitzky-Golay filter (9 smoothing points, 2nd order polynomial and 2nd

derivative) and mean-centring (23,24). Due to the high amount of data generated, a mean spectrum

of each isolate (resulting from nine congruent replicates) was considered in the analysis performed.


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Spectra were further analysed by supervised (partial least squares discriminant analysis,

PLSDA) and unsupervised (principal component analysis, PCA) chemometric methods. The

selected spectral region was the one corresponding to the phospholipids/DNA/RNA and

polysaccharides vibrations (1500-900 cm-1). All chemometric analysis was performed using Matlab

version 6.5 release 13 (MathWorks, Natick, MA) and PLS Toolbox version 3.5 for Matlab

(Eigenvector Research, Manson, WA, USA). The PLSDA model is based on the PLS regression

method, and requires a previous knowledge about all the samples used. In PLSDA models, to each

isolate spectrum (xi) was assigned a vector of zeros with the value one at the position

corresponding to its ST or K-type (yi), in such a way that categorical variable values (ST or K-

types) can be predicted for samples of unknown origin. Model loadings and the corresponding

scores were obtained by sequentially extracting the components or latent variables (LVs) from

matrices X (spectrum) and Y (matrix codifying the ST or K-types). In PLSDA, a probability value

for each assignment is estimated for each sample. The number of latent variables (LVs) was

optimized using the leave-one-sample-out cross-validation procedure in order to prevent over-

fitting considering 70% of the available data (randomly selected). After optimization of the number

of LVs, the model was tested on the remaining 30% data in order to assess the proportion (%) of

correct predictions for each ST/K-type (21,22,25). PCA is an unsupervised method that offers the

possibility of projecting multivariate data without a priori knowledge of the sample. The direction

of maximum variability in the samples are obtained and used as a new set of axes, known as

principal components (PCs). They are created with the idea that they must be uncorrelated with one

another, with the first one describing as much variability in the data as possible, whereas the

second one explains the maximum amount of the remaining variability, and so forth (25).

RESULTS AND DISCUSSION

Genomic diversity of CPS and LPS among international MDR Kp clones: finding specific

associations

A high diversity of wzi alleles (n=22) and the corresponding capsular types (n=21, wzi24

and wzi101 codifying both for K24) was identified among the 158 MDR Kp isolates included in


197

this study. Four new wzi types (wzi89, wzi200-202) were detected and submitted to BIGSbd-Kp

Pasteur database (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html), most of them recognized

meanwhile in other studies (14). The 21 capsular types were inferred by the wzi allele or using the

KAPTIVE platform according to previously described typing tools (12,14). Among them, 13

correspond to serologically defined K-types (K2, K14, K16, K17, K19, K23, K24, K27, K41, K48,

K60, K62, K64), 6 are presumptive new K-types (KL105, KL107, KL110, KL112, KL127, KL151)

and 2 wzi alleles (wzi200 and wzi150) remain unclear (Table 1).

Interestingly, specific associations between capsular types and STs and/or CGs from

different geographic regions and large periods of time were detected (Table 1), most of them

consistently observed in other Kp collections (9,10,12,26–28). Moreover, diverse capsular types

were associated with particular clones such as ST15 or ST11 (5 different capsular types), ST14,

ST17 or ST258 (2 distinct capsular types), suggesting the circulation of distinct lineages with

defined capsular types and a suboptimal resolution of MLST typing. Furthermore, these results

reflect the high potential for CPS diversification probably driven by host-pathogen interactions, but

also the importance of CPS typing in population structure studies as a discriminatory and

evolutionary analysis tool (Table 1) (8–10). In contrast to previous studies, capsular switching was

infrequent and observed for isolates from ST11, ST15, ST17 and ST147 or eventually between

species (K. pneumoniae and K. varicola) (Table 1) (7,14).

A schematic representation of the different capsular types identified in this work was

performed in silico using Geneious software v10.0 (Biomatters Ltd, Auckland, New Zealand) and

the sequences from the reference strains available at the NCBI database (Figure 1). Notably, 58%

(11/19, considering also KL151) of the capsular locus identified are associated with genes involved

in synthesis and processing of rhamnose (rhamnosyltransferases and operon rmlABCD), 43% with

mannose synthesis while only 5% with fucose synthesis. Although the presence of rhamnose in Kp

capsules has been recognized in MDR isolates involved in outbreaks (10,29), we firstly clarify the

abundance of rhamnose, and to a lesser extent mannose, among capsular types of a representative

collection of MDR strains. We hypothesize that the presence of rhamnose in the capsule of MDR

strains might be linked to host immune evasion responses, and that rhamnose biosynthesis pathway


198

biosynthesis pathway could constitute a potential target for the development of antimicrobial

agents or vaccines against MDR Kp strains due to the absence of rhamnose in humans (30).

Regarding the genotypic characterization of O-types, O1 (45.6%, 72/158) and O2 (38.6%,

61/158) were the most prevalent among our collection, as expected, followed by O3 (1.8%, 3/158)

and O5 (0.6%, 1/158), while 13.2% (21/158) of the isolates did not belong to any of these O-types

(11,31,32). O1 was associated with 6 STs and 13 distinct K-types, while O2 was detected among 4

STs and 6 K-types (Table 1). O3 was only detected among isolates from ST54 harbouring

wzi14/K14 and O5 in one ST17-wzi93/KL112 isolate. It is of interest to highlight that no specific

associations were observed between K-types and O-types, but instead that multiple O-types (O1,

O2 and O5-wzi93/KL112; O1 and O2- wzi64/K64) were in some cases observed in isolates

exhibiting the same capsular type, corroborating previous observations (31).

Virulence genotypes

Multiple virulence genes have been recognized among Kp isolates based on murine models

of infections, such as siderophores (aerobactin, yersiniabactin, salmochelin), iron uptake systems

(kfuABC operon), citotoxins (colibactin), regulators of overexpression of capsule polysaccharides

(rmpA and rmpA2), allantoin and urea metabolism, and type 1 and 3 fimbrial adhesins (fim and mrk

operons) (33,34). In this work, we explored the occurrence of recognized Kp virulence genes in a

diversity of MDR strains and also their association with specific clones, K-types or O-types. Type

1 and 3 fimbriae and the urease cluster were present in almost all MDR isolates (Table 1), contrary

to what has been reported for HV Kp strains where these virulence genes are rare (31). Type 1

fimbriae are important in the adhesion to uroepithelial cells, while type 3 fimbriae have the ability

to link to different human cell types, and both have been associated with the promotion of biofilm

formation (33,35). Our data do not support a higher prevalence of type 1 fimbriae among O1

strains, as reported (31). As expected, allantoinase cluster, aerobactin and rmpA were not detected

among MDR strains, since these virulence factors are particularly associated with HV Kp strains

(5,33). Thus, our data support that specific virulence factors seem to condition the different clinical

manifestations observed for MDR or HV Kp strains. On the other hand, yersiniabactin and the


199

ABC iron transporter (Kfu system) were frequently detected (64% and 39%, respectively). These

data are worrisome, since yersiniabactin is a siderophore strongly associated with invasive

infection and it seems to be a first step for subsequent acquisition of other siderophores, and Kfu

plays an important role in intestinal colonization where free ferric iron is available (7,36,37).

Yersiniabactin was detected among ST14, ST101, ST336, ST405 or ST348 and certain ST11

(wzi75/KL105, wzi27/K27 and wzi64/K64) or ST15 (wzi93/KL112, wzi24/K24 and wzi19/K19)

lineages, and sporadically among ST258 (n=2) and ST147 (n=3). Kfu was mainly detected among

ST14 and ST15 and some isolates from ST147, ST101 and ST405 (Table 1). These results,

together with previous studies, reinforce that some widespread MDR ST (in our study ST14, ST15,

ST101 and ST147) are becoming enriched in virulence factors that augment the ability of Kp to

cause invasive infections (7), which associated with their MDR phenotypes, might be catastrophic

in terms of clinical practice and patient outcomes. Also, we hypothesize that the virulence

armamentarium of K. pneumoniae is far from being completely understood and that additional non-

recognized factors might be contributing to the success of particular lineages.

Phenotypic characterization of MDR Kp clones by FTIR spectroscopy

Spectra overview

FTIR-ATR spectra of all Kp isolates tested contained bands associated with lipids (3000–

2800 cm-1), proteins/amides I and II (1700–1500 cm-1), phospholipids/DNA/RNA (1500–1185 cm-

1), polysaccharides (1185–900 cm-1) and the fingerprint region (900–600 cm-1). The main spectral

differences were detected in the phospholipids/DNA/RNA and the polysaccharides regions (1500–

900 cm-1), which were selected for further analysis and discriminatory purposes.

Analysis workflow and FTIR-based discrimination of Kp capsular types

Spectral diversity was captured by PLSDA models considering either ST or K-types (based

on wzi typing) as sample classes (Figure S1 and Figure 2, respectively). In both cases, several well

defined clusters were observed, although the distribution of the isolates in the score plots, and


200

hence their relatedness was congruent with the capsular types of each isolate (Figure 2 – model 1).

Both models were superimposable when one ST harboured only one capsular type distinct from all

the others identified (e.g. ST336 - wzi150). The FTIR-based assignments obtained evidenced a very

good correlation between wzi alleles and the predicted K-types providing for the first time evidence

of a correlation between genotypic and phenotypic features of the cps locus. Moreover, subtle

differences on bacterial surface components were depicted within particular K-types.

In model 1 (Figure 2), we observed some independent and homogeneous clusters including

isolates exhibiting specific capsular types, and a large cluster comprising isolates exhibiting diverse

capsular types (KL112, K24, KL105, K27, K16, K17, K60, K41, K62), probably reflecting some

degree of similarity on the composition of these capsular types. The resolution of isolates grouped

in the large cluster was accomplished by three independent PLSDA models subsequently

generated: i) model 2, that included all the isolates with K-types other than those identified among

ST11 and ST15; ii) model 3 that comprised all ST15 isolates exhibiting diverse K-types, and iii)

model 4 that included all ST11 isolates exhibiting diverse capsular types. Isolates included in

model 1 harbouring the same capsular types than those detected among ST15 and ST11 isolates

were projected in models 3 and 4, respectively, to test the specificity of these models.

Capsular discrimination within PLSDA model 2

In PLSDA model 2, eleven well-defined clusters comprising isolates exhibiting the same

capsular type were depicted (Figure 3). The few exceptions identified were 2 ST258 carrying wzi29

(K41), 1 ST14 (wzi16) isolate and 3 ST101 (wzi137) isolates (Figure 3, signed with a red circle).

Since these isolates carried the same O-types (O1, O2) than those carrying the same capsular

type/wzi allele that would explain differences in the composition of the bacterial surface, we

hypothesize possible recombination events within the cps that need to be confirmed by sequencing

of the whole cps region (10,31). According to this model, 97.5% of the K-types were correctly

predicted (Table 2).



201

In PLSDA model 3, ST15 isolates were grouped in four clusters corresponding to the four

capsular types (K24, KL112, K19, KL110) characterized by genotypic methods (Figure 3). It is of

interest to highlight that the cluster containing isolates exhibiting K24 type contained isolates

carrying either wzi24 or wzi101 alleles, reinforcing that all of them shared similar phenotypic

properties, detected by FTIR (Table 1). The confusion matrix for this model revealed a high

proportion of correct predictions (90.9%) for these K-types (Table 3). The projection of the four

isolates sharing the same capsular type than ST15 isolates revealed only two correct predictions (2

ST17-wzi93/KL112-O2), although they did not fit in the central cluster of wzi93/KL112 (Figure

S2). These results are not surprising since ST17-wzi93/KL112 isolates are O-type O2 or O5 instead

of O1 (the rest of the wzi93/KL112 isolates). In fact, O1 and O2 are more similar in terms of

composition than O5, which might justify the slightly distinct phenotypic behaviour of the ST17-

wzi93/KL112-O5 (Figure S2) (11,31). In fact, our results corroborate that FTIR-based assignments

are mostly based on the composition of the capsule, probably due to its surface location and

density, which make its structure more accessible. Phenotypic variations in isolates with similar wzi

or cps locus might occur when the O-polysaccharide of the LPS is more accessible at the cell

surface resulting in variations in FTIR-based assignments (6).


In this PLSDA model, six well-defined clusters of ST11 isolates were observed (Figure 5).

Three of these clusters included exclusively isolates from wzi27/K27, wzi64/K64 and

wzi202/KL127 isolates, respectively, whereas the other three clusters differentiated isolates

carrying wzi75/KL105 (arbitrarily designed as KL105-1, KL105-2 and KL105-3). All of them

displayed O2-type and variably produced OXA-48 or KPC-3 (KL105-1, all isolates from Spain),

DHA-1 or DHA-6 (KL105-2 and KL105-2, all isolates from Portugal). According to this PLSDA

model, 99.5% of the K-types were correctly predicted (Table 4), and in fact, projection of ST147-

wzi64/K64 isolates revealed that they were all correctly predicted as wzi64/K64 (Figure S3).

The representation of the mean spectra of wzi75 isolates assigned to the three different

clusters revealed differences in the 1030-1080 cm-1 region (region involved in alcoholic C–OH


202

vibration of polysaccharide fraction) (Figure 6) (38), and in an additional PCA model including

only wzi75 isolates the same three well-defined clusters were observed (Figure 7). The whole cps

region should be sequenced in order to characterize the genetic alterations involved in the

expression of these different subtypes of wzi75-KL105. Thus, our results unveiled phenotypic

differences on capsule assembly or composition, in spite the apparent similarity depicted by

genotypic wzi typing, whose significance needs to be further explored. This finding reinforces the

need to compare genotypic and phenotypic methods in the characterization of capsular types and

Kp populations. In the study performed by Wyres et al., two different cps clusters were identified

among wzi75 isolates, distinguished by the presence of an insertion sequence (IS5) disrupting CDS

in the non-conserved region of the cps, which might alter the composition of the capsule (10).

CONCLUSIONS

We demonstrate for the first time the potential of FTIR-ATR coupled with chemometrics

analysis to differentiate K. pneumoniae capsular types. Besides, we unveil stable associations

between capsular types and specific MDR K. pneumoniae lineages in different geographic regions

and over time, which are correctly depicted by FTIR-based assignments, highlighting the potential

of this methodology as a fast and accurate alternative typing tool. Furthermore, the phenotypic

features provided by FTIR corroborate the differences already recognized at the genotype level in

K. pneumoniae ST11 and ST15 clones, and supports the existence of different lineages circulating

within these clones, which needs additional characterization by other methodologies. The high

prevalence of rhamnose capsule derivatives among MDR K. pneumoniae is also of relevance, and

needs to be further explored in terms of host-pathogen interaction and the design of therapeutic or

preventive measures. Beyond capsular characterization, our study also provides a deeper

characterization of international MDR K. pneumoniae clones, revealing their enrichment in specific

virulence factors such as type 1 and 3 fimbriae and iron uptake systems (yersiniabactin siderophore

and Kfu ABC transport system), which is worrisome due to the possible clinical impact.


203

Acknowledgments: We thank Marek Gniadkowski (National Medicines Institute, Poland), Vivi

Miriagou (Department of Bacteriology, Hellenic Pasteur Institute, Athens, Greece), Grigore

Mihăescu (University of Bucharest, Faculty of Biology, Deptartment of Microbiology, Bucharest,

Romania), Rafael Cantón and Teresa Maria Coque (Laboratorio de Microbiologia, Hospital

Universitario Ramón y Cajal, Madrid, Spain), Leonardo Neves de Andrade and Ana Lucia Darini

(Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto) for the

strains analysed in this study.




UID/MULTI/04378/2013). Carla Rodrigues and Ângela Novais were supported by fellowships

from FCT through Programa Operacional Capital Humano (POCH) (grants number

SFRH/BD/84341/2012 and SFRH/BPD/104927/2014, respectively). Clara Sousa was funded

through the NORTE-01-0145-FEDER-000024 – “New Technologies for three Health Challenges

of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases”.

Conflict of interest: The authors declare that they have no conflict of interest.

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Table 1. Epidemiological data and genotypic characterization of international MDR K. pneumoniae clinical isolates 1

analysed in this study. 2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Iron

uptake

systems

Regulator

of mucoid

phenotype

fimH mrkD entB ybtS iutA kfuBC ureA allS rmpA H1144 Portugal 2010 Kp1 CTX-M-15 93/KL112 O1

H1102 Portugal 2010 Kp1 CTX-M-15 93/KL112 O1


K8 Portugal 2010 Kp1 CTX-M-15 93/KL112 O1





C1709 Portugal 2012 Kp1 CTX-M-15 93/KL112 O1


C1699 Portugal 2012 Kp1 CTX-M-15 93/KL112 O1

H1120 Portugal 2010 Kp2 SHV-283 24/K24 O1



Kp55 Portugal 2014 Kp2 OXA-48 24/K24 O1

44 Portugal 2013 Kp2 OXA-48, CTX-M-15 24/K24 O1

C1686 Portugal 2012 Kp2 CTX-M-15 24/K24 O1





SC44 Brazil 2012 Kp2 CTX-M-15 24/K24 O1

B19H Brazil 2012 Kp2 CTX-M-8, SHV-2 24/K24 O1

H693 Portugal 2006 Kp2 CTX-M-15 24/K24 O1


K21 Portugal 2010 Kp2 CTX-M-15 101/K24 O1




K88 Portugal 2015 Kp3 KPC-3 19/K19 O1




K43 Portugal 2011 Kp4 VIM-34, SHV-12, OXA-17 89/KL110 O1

K47 Portugal 2012 Kp4 VIM-34, SHV-12, OXA-17 89/KL110 O1








H49 Portugal 2003 Kp6 TEM-24 2/K2 O1



13I15 Spain 2012 Kp7 OXA-48 75/KL105 O2

12E76 Spain 2012 Kp7 OXA-48 75/KL105 O2

12F14 Spain 2012 Kp7 OXA-48 75/KL105 O2

12F48 Spain 2012 Kp7 OXA-48 75/KL105 O2

12F64 Spain 2012 Kp7 OXA-48 75/KL105 O2

12F72 Spain 2012 Kp7 OXA-48 75/KL105 O2

12F73 Spain 2012 Kp7 OXA-48 75/KL105 O2

12F55 Spain 2012 Kp7 OXA-48 75/KL105 O2

12G17 Spain 2012 Kp7 OXA-48 75/KL105 O2

12H72 Spain 2012 Kp7 OXA-48 75/KL105 O2

H642 Portugal 2006 Kp8 DHA-1 75/KL105 O2



C1721 Portugal 2012 Kp8 DHA-1 75/KL105 O2


C1951 Portugal 2013 Kp8 DHA-6 75/KL105 O2


10E34 Spain 2010 Kp10 KPC-3 75/KL105 O2

H646 Portugal 2006 Kp11 DHA-1 24/K24 O1

RP29 Brazil 2012 Kp12 KPC-2, CTX-M-2 27/K27 O2


RP75 Brazil 2012 Kp12 CTX-M-2 27/K27 O2






HCC23 Brazil 2009 Kp15 KPC-2 202/KL127

SC51 Brazil 2012 Kp15 KPC-2 202/KL127


B40U Brazil 2012 Kp16 CTX-M-2 64/K64 O2

B31U Brazil 2012 Kp16 KPC-2, CTX-M-2 64/K64 O2

CRE01 KP Brazil 2008 Kp17 KPC-2 29/K41 O2

HCC52 Brazil 2007 Kp17 KPC-2 29/K41 O2


CRE38 Brazil 2006 Kp17 KPC-2 29/K41 O2



126/09 Poland 2008 Kp18 KPC-2, SHV-12 29/K41 O2

5023/09 Poland 2009 Kp18 KPC-2, CTX-M-3 29/K41 O2

5586/09 Poland 2009 Kp18 KPC-2, CTX-M-3, SHV-12 29/K41 O2

6595/09 Poland 2009 Kp18 KPC-2, SHV-12 29/K41 O2

Kp4077 Greece 2007 Kp19 KPC-2 29/K41 O2




4930/09 Poland 2009 Kp18 KPC-3 154/KL107 O2

2934/08 Poland 2008 Kp18 KPC-3, CTX-M-3 154/KL107 O2








K70 Portugal 2015 Kp20 KPC-3, SHV-12 64/K64 O2







10D79 Spain 2010 Kp21 VIM-1 64/K64 O1

18 Romenia 2012 Kp22 OXA-48, CTX-M-15 137/K17 O1



E45 Romenia 2012 Kp22 OXA-48, CTX-M-15 137/K17 O1

E7 Romenia 2012 Kp22 OXA-48, CTX-M-15 137/K17 O1

E16 Romenia 2012 Kp22 OXA-181, NDM-1, CTX-M-15137/K17 O1





SC26 Brazil 2012 Kp25 CTX-M-15 137/K17 O1

B10U Brazil 2012 Kp26

H1170 Portugal 2010 Kp27 CTX-M-15 150/-












F12 Brazil 2012 Kp28 SHV-2 93/KL112 O2

F29 Brazil 2012 Kp28 SHV-2 93/KL112 O2

C1748 Portugal 2012 Kp30 DHA-1, SHV-12 93/KL112 O5

09B53 Spain 2012 Kp29 VIM-1 200/-

08Z37 Spain 2008 Kp31 VIM-1 83/K23 O1

Kpn20 Spain 2008 Kp31 VIM-1 83/K23 O1

09C77 Spain 2009 Kp31 VIM-1 83/K23 O1

09A69 Spain 2009 Kp31 VIM-1 83/K23 O1

10D60 Spain 2010 Kp31 VIM-1 83/K23 O1

10F53 Spain 2010 Kp31 VIM-1 83/K23 O1

160 Spain 2010 Kp31 VIM-1 83/K23 O1

12G9 Spain 2012 Kp32 OXA-48 143/KL151

12G10 Spain 2012 Kp32 OXA-48 143/KL151

12H41 Spain 2012 Kp32 OXA-48 143/KL151

13I19 Spain 2013 Kp32 OXA-48 143/KL151

C1702 Portugal 2012 Kp33 CTX-M-15 143/KL151




Kp56 Portugal 2014 Kp34 KPC-3, CTX-M-15 94/K62 O1

09B51 Spain 2009 Kp35 VIM-1 14/K14 O3

10H66 Spain 2010 Kp35 VIM-1 14/K14 O3

161 Spain 2010 Kp35 VIM-1 14/K14 O3

09B76 Spain 2009 Kp36 VIM-1 201/K60 O1

09C12 Spain 2009 Kp36 VIM-1 201/K60 O1

10F74 Spain 2010 Kp36 VIM-1 201/K60

ST4054/-

ST348/-

ST54/-

ST253/-

Virulence Genes

Strain OriginYear of

isolation ST/CGPFGE

Cluster

ST258/CG258

ST147/CG147

ST101/CG101

ST336/CG17

ST17/CG17

ST39/CG39

Adhesins Siderophores

Nitrogen

source

utilization

ST15/CG15

ST14/CG14

ST11/CG258

-lactamases conferring

resistance to extended-

spectrum -lactams

wzi/K1O-types

2

137/K17 O1CTX-M-15


209

Figure 1. cps locus of Klebsiella pneumoniae K-types identified in this study inferred according to wzi allele. 31 On the left side the identification of wzi allele, the predicted K-types and also the Genbank accession number of the reference strain. In the right 32 side the Sequence types (ST) where the different K-types were identified in this study. 33 34

35

36

37

wzi93 – KL112 (LT174581)

wzi24/101 – K24 (AB924562)

ST15, ST17

ST15, ST11

wzi151– K48 (AB924585)

ST15

wzi19 – K19 (AB924559) ST15

wzi89 - KL110 (LT174579)

ST15

wzi16- K16 (AB742228)

wzi2- K2 (KJ541664)

wzi75- KL105 (KR007676)

wzi27- K27 (AB924565)

wzi202- KL127 (LT603704)

wzi64- K64 (AB924600)

wzi29- K41 (AB924578)

wzi154- KL107 (CP006918)

ST14

ST14

ST11

wzi137- K17 (AB924557)

wzi83- K23 (AB924561)

wzi94- K62 (AB371295)

wzi14- K14 (AB371294)

wzi201- K60 (AB924597)

ST11

ST11

ST11, ST147

ST258

ST258

ST101

ST39

ST348

ST54

ST253

core genes from the cps

wzx flippase and wzy polymerase

initial glycosyltransferases

mannose synthesis and processing

rhamnose synthesis and processing

fucose synthesis

HP, hypothetical protein sugar synthesis and processing


210

Figu

re 2

. Sco

re p

lot o

f the

PL

SDA

regr

essi

on m

odel

1 a

ccor

ding

to K

-typ

es, c

orre

spon

ding

to th

e fi

rst f

ive

late

nt v

aria

bles

(LV

s).

Hig

h di

vers

ity o

f K-t

ypes

K24

/wzi

101

K17

/wzi

137

K14

/wzi

14

KL

151/wzi

143

-/wzi

150

KL

107/wzi

154

K16

/wzi

16

K19

/wzi

19

K2/wzi

2 K

60/wzi

201

KL

107/wzi

202

K24

/wzi

24/1

01

K27

/wzi

27

K41

/wzi

29

K64

/wzi

64

KL

105/wzi

75

K23

/wzi

83

KL

110/wzi

89

KL

112/wzi

93

K62

/wzi

94

K-t

ype/wzi


211

Figu

re 3

. Sco

re p

lot o

f the

PL

SDA

regr

essi

on m

odel

2 a

ccor

ding

to K

-typ

es, c

orre

spon

ding

to th

e fi

rst t

hree

late

nt v

aria

bles

(LV

s).

Tabl

e 2.

Con

fusi

on m

atri

x fo

r K. p

neum

onia

e PL

SDA

mod

el 2

acc

ordi

ng to

K-t

ypes

def

ined

by

wzi

seq

uenc

ing.

K

-typ

es d

efin

ed b

y w

zi s

eque

ncin

g (K

-typ

e/w

zi a

llele

) (T

otal

of c

orre

ct p

redi

ctio

ns=9

7.5%

)

K23

/ wzi

83

K14

/ wzi

14

K60

/ wzi

201

K41

/ wzi

29

KL

151/

wzi

143

K17

/ wzi

137

KL

107/

wzi

154

K62

/ wzi

94

K16

/ wzi

16

K2/

wzi

2 -/w

zi15

0

K-types predicted by FTIR-ATR a,b

K23

/ wzi

83

100,

0%%

0,

0%%

0,

00%

0,

00%

0,

00%

0,

30%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

K14

/ wzi

14

0,0%

%

100,

0%%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

K60

/ wzi

201

0,0%

%

0,0%

%

100,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

K41

/ wzi

29

0,0%

%

0,0%

%

0,00

%

97,6

0%

0,00

%

0,80

%

0,00

%

0,00

%

15,7

0%

0,00

%

0,00

%

KL

151/

wzi

143

0,0%

%

0,0%

%

0,00

%

0,00

%

100,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

K17

/ wzi

137

0,0%

%

0,0%

%

0,00

%

2,40

%

0,00

%

98,3

0%

0,00

%

0,00

%

0,70

%

0,00

%

0,00

%

KL

107/

wzi

154

0,0%

%

0,0%

%

0,00

%

0,00

%

0,00

%

0,00

%

100,

00%

0,

00%

0,

00%

0,

00%

0,

00%

K62

/ wzi

94

0,0%

%

0,0%

%

0,00

%

0,00

%

0,00

%

0,00

%

0,00

%

100%

0,

00%

0,

00%

0,

00%

K16

/ wzi

16

0,0%

%

0,0%

%

0,00

%

0,00

%

0,00

%

0,80

%

0,00

%

0,00

%

83,7

0%

0,00

%

0,00

%

K2/

wzi

2 0,

0%%

0,

0%%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

0,

00%

10

0,00

%

0,00

%

-/wzi

150

0,0%

%

0,0%

%

0,00

%

0,00

%

0,00

%

0,00

%

0,00

%

0,00

%

0,00

%

0,00

%

100,

00%

a

Gre

y sh

aded

repr

esen

t the

per

cent

age

of is

olat

es c

orre

ctly

pre

dict

ed u

sing

FT

IR-A

TR

for e

ach

K-t

ype.

b V

alue

s ob

tain

ed c

onsi

deri

ng 1

4LV

s in

PL

SDA

mod

el.

K17

/wzi

137

K14

/wzi

14

KL

151/

wzi

143

-/w

zi15

0 K

L10

7/w

zi15

4 K

16/w

zi16

K

2/w

zi2

K60

/wzi

201

K41

/wzi

29

K23

/wzi

83

K62

/wzi

94

Exc

eptio

n is

olat

es

K-t

ype/

wzi


212

Fi

gure

4. S

core

plo

t of t

he P

LSD

A re

gres

sion

mod

el 3

acc

ordi

ng to

K-t

ypes

, cor

resp

ondi

ng to

the

firs

t fou

r lat

ent v

aria

bles

(LV

s).

Tabl

e 2.

Con

fusi

on m

atri

x fo

r K. p

neum

onia

e PL

SDA

mod

el 3

acc

ordi

ng to

K-t

ypes

def

ined

by

wzi

seq

uenc

ing.

K-t

ypes

def

ined

by

wzi

seq

uenc

ing

(K-t

ype/

wzi

alle

le)

(Tot

al o

f cor

rect

pre

dict

ions

=90.

9%)

K24

/ wzi

24/1

01

K19

/ wzi

19

KL

112/

wzi

93

KL

110/

wzi

89

K-t

ypes

pr

edic

ted

by

FTIR

-AT

R a,

b K24

/ wzi

24/1

01

90,6

0%

2,30

%

3,30

%

0,00

%

K19

/ wzi

19

0,60

%

86,3

0%

4,30

%

0,00

%

KL

112/

wzi

93

5,60

%

10,7

0%

92,5

0%

0,00

%

KL

110/

wzi

89

3,20

%

0,70

%

0,00

%

100,

00%

a G

rey

shad

ed re

pres

ent t

he p

erce

ntag

e of

isol

ates

cor

rect

ly p

redi

cted

usi

ng F

TIR

-AT

R fo

r eac

h K

-typ

e.

b Val

ues

obta

ined

con

side

ring

14L

Vs

in P

LSD

A m

odel

.

K19

/wzi

19

K24

/wzi

24/1

01

KL

110/

wzi

89

KL

112/

wzi

93

K-t

ype/

wzi


213

Figu

re 5

. Sco

re p

lot o

f th

e PL

SDA

reg

ress

ion

mod

el 4

acc

ordi

ng to

K-t

ypes

, cor

resp

ondi

ng to

the

firs

t fou

r la

tent

var

iabl

es (

LVs)

. Ta

ble

3. C

onfu

sion

mat

rix

for

K. p

neum

onia

e PL

SDA

mod

el 4

acc

ordi

ng to

K-t

ypes

def

ined

by

wzi

seq

uenc

ing.

a G

rey

shad

ed r

epre

sent

the

perc

enta

ge o

f is

olat

es c

orre

ctly

pre

dict

ed u

sing

FT

IR-A

TR

for

eac

h K

-typ

e.

b Val

ues

obta

ined

con

side

ring

14L

Vs

in P

LSD

A m

odel

.

K-t

ypes

def

ined

by

wzi

seq

uenc

ing

(K-t

ype/

wzi

alle

le)

(T

otal

of

corr

ect p

redi

ctio

ns=9

9.5%

)

K

L10

5/w

zi75

K

64/ w

zi64

K

L12

7/w

zi20

2 K

27/ w

zi27

K-t

ypes

pr

edic

ted

by

FTIR

-AT

R a,

b

KL

105/

wzi

75

99,0

0%

0,00

%

0,00

%

0,00

%

K64

/ wzi

64

0,00

%

100,

00%

0,

00%

0,

00%

K

L12

7/w

zi20

2 0,

00%

0,

00%

10

0,00

%

0,00

%

K27

/ wzi

27

1,00

%

0,00

%

0,00

%

100,

00%

KL

127/

wzi

202

K27

/wzi

27

K64

/wzi

64

KL

105/

wzi

75

K-t

ype/

wzi

KL

105-

2

KL

105-

1

KL

105-

3


214

Fi

gure

6. K

. pne

umon

iae

wzi

75 F

TIR

-AT

R s

pect

ra p

roce

ssed

with

SN

V a

nd S

avitz

ky-G

olay

(9

poin

ts f

ilter

siz

e, 2

nd d

egre

e po

lyno

mia

l, 2nd

der

ivat

ive)

cor

resp

ondi

ng to

th

e m

ean

± on

e st

anda

rd d

evia

tions

in th

e re

gion

900

-150

0 cm

-1.

KL

105-

1 K

L10

5 su

btyp

e

KL

105-

2

KL

105-

3


215

Figu

re 7

. Sco

re p

lot o

f the

PC

A u

sing

KL

105

subt

ypes

(arb

itrar

y de

sign

ed a

s 1,

2 a

nd 3

) cor

resp

ondi

ng to

the

firs

t tw

o la

tent

var

iabl

es (L

Vs)

. KL

105-

1 K

L10

5 su

btyp

e

KL

105-

2

KL

105-

3


216

Figu

re S

1. S

core

plo

t of t

he P

LSD

A re

gres

sion

acc

ordi

ng to

STs

, cor

resp

ondi

ng to

the

firs

t tw

o la

tent

var

iabl

es (L

Vs)

.

ST10

1 ST

11

ST14

ST

147

ST15

ST

17

ST25

3 ST

258

ST33

6 ST

348

ST39

ST

405

ST54

Sequ

ence

Typ

e (S

T)


217

Figu

re S

2. S

core

plo

t cor

resp

ondi

ng to

the

first

four

LV

s of

the

PLSD

A re

gres

sion

mod

el 3

with

the

proj

ecte

d te

st is

olat

es.

ST17

-wzi

93/K

L112

-O2

ST17

-wzi

93/K

L112

-O5

ST11

-wzi

24/K

24-O

1

K19

/wzi

19

K24

/wzi24

/101

K

L110

/wzi

89

KL1

12/wzi

93

K-t

ype/wzi

test

isol

ates


218

Figu

re S

3. S

core

plo

t cor

resp

ondi

ng to

the

firs

t fou

r LV

s of

the

PLSD

A re

gres

sion

mod

el 4

with

the

proj

ecte

d te

st is

olat

es.

KL

127/wzi

202

K27

/wzi27

K

64/wzi

64

KL

105/wzi

75

K-t

ype/wzi

test

isol

ates


219

Fourier Transform Infrared (FTIR) spectroscopy based-typing for

“real-time” analysis of an outbreak by carbapenem-resistant Klebsiella pneumoniae isolates

Liliana Silva1,2,3, Carla Rodrigues1, Agostinho Lira4, Mariana Leão4, Margarida Mota4,

Paulo Lopes4, Angelina Lameirão4, Gabriela Abreu4, Ângela Novais1, Luísa Peixe1*

1UCIBIO/REQUIMTE. Departamento de Ciências Biológicas, Laboratório de

Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal. 2ESALD.

Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal. 3FEUP. Faculdade de

Engenharia da Universidade do Porto, Porto, Portugal. 4CHVNG/E. Serviço de Patologia

Clínica, Centro Hospitalar de Vila Nova de Gaia/Espinho, Vila Nova de Gaia, Portugal.

Keywords: bacterial typing, nosocomial infection, carbapenemases, KPC producers

Corresponding Author: * Luísa Peixe.

UCIBIO/REQUIMTE,

Laboratório de Microbiologia,

Faculdade de Farmácia, Universidade do Porto,

Rua de Jorge de Viterbo Ferreira, n. 228,

4050-313 Porto,

Portugal. Tel.: +351 220 428 580.




220

Sir,

Carbapenem resistant Klebsiella pneumoniae isolates are responsible for a significant

number of hospital outbreaks worldwide (1). Early and reliable identification of outbreaks as

well as identification of clones associated with higher virulence or antimicrobial resistance to

critical antibiotics is important to adopt efficient antimicrobial therapy and infection control

measures (2). Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST),

whole genome sequencing (WGS) and other genotype-based strain typing methods are widely

used but often fail to yield quick responses (2,3). We recently demonstrated a very good

correspondence between international multidrug resistant (MDR) K. pneumoniae clones and

capsule types that were reliably discriminated by Fourier transform infrared (FTIR)

spectroscopy with attenuated total reflectance (ATR) (4). This method provides a high

bacterial resolution at infraspecies level with excellent turnaround times while no sample

processing is required, being also cost-efficient (4-6). Thus, we aimed to assess the ability of

FTIR spectroscopy to assist infection control in “real-time”, in the context of a nosocomial

outbreak of carbapenem resistant K. pneumoniae.

Twenty-five carbapenem-resistant K. pneumoniae isolates collected between August

and November 2015 were received by our laboratory in November 2015 and analysed.

Isolates were identified among clinical (n=11) or fecal (n=14) samples recovered in

hospitalized patients between 8 and 97 years old (12 females, 13 males) of different wards

(n=8 surgery, n=4 medicine, n=3 urgency, n=10 others] from a Portuguese hospital. Isolates’

relatedness was investigated by FTIR-ATR spectroscopy coupled with multivariate data

analysis as reported [4], and further compared with reference methods for strain (XbaI-PFGE,

MLST) and capsule typing (wzi sequencing) (7). Briefly, isolates were grown on Mueller-

Hinton agar (37ºC, 18h) and directly applied on the ATR crystal. Spectra were pre-processed

and compared each other and with those included in our in-house K. pneumoniae database

(including representative strains of 13 MDR sequence types (ST) with 21 different capsular

types). Strain diversity was captured by models based on principal component analysis (PCA)

and partial least squares discriminant analysis (PLSDA). Data analysis were performed in


221

Matlab R2015a version 8.5.0 (MathWorks, Natick, MA) and the PLS Toolbox version 7.9.5

for Matlab (Eigenvector Research, Manson, WA).

Most of the isolates (n=23) were highly related and appeared in a well-delimited

cluster (outbreak cluster), whereas the remaining 2 isolates were unrelated (to each other and

with the latter, Figure 1a). Furthermore, when the outbreak isolates were projected in our K.

pneumoniae model (4), they clustered tightly with ST147 isolates exhibiting capsule type K64

(Figure 1b). This high-risk clone has been linked to the worldwide expansion of different

extended-spectrum β-lactamases (ESBLs) and carbapenemases, including in different

Portuguese clinical institutions (hospitals, long-term care facilities and nursing homes) (8,9).

This assignment was confirmed by PFGE (identical PFGE-patterns for isolates of outbreak

cluster (Supplementary Figure 1), MLST (ST147) and wzi-typing (wzi64/K64). Isolates

unrelated to those of the outbreak cluster corresponded to 1 K. pneumoniae ST336 (wzi150,

no K-type defined) and 1 K. pneumoniae ST348 (wzi94/K62). It is of interest to highlight that

the absolute congruence between all methods supports a good correlation between relevant

genotypic and phenotypic features for discrimination of K. pneumoniae and furthermore

demonstrates the potential of FTIR spectroscopy for typing isolates of this important

pathogen (4). Using this approach, we were able to assess isolates’ relatedness and

communicate the results obtained in less than 36 hours (including an overnight growth step),

which represents a very good alternative to existing methods (3,10). Also, our quick results

were of great support to infection control since measures were immediately adopted to control

the outbreak.

Additional characterization of the isolates included the evaluation of carbapenemase

production by Blue-Carba test (11), identification of blaKPC and its genetic context by PCR

and sequencing (12), and plasmid content (replicon typing, S1-PFGE and hybridization). The

results show that all ST147 and the ST336 isolate produced KPC-3 carbapenemase, whereas

the ST348 isolate was negative. Different genetic backgrounds were identified within ST147

(Tn4401d in an IncN plasmid) or ST336 (Tn4401b in an IncR plasmid) isolates, enlarging the

number of KPC-encoding platforms previously described in our setting (9). The susceptibility


222

profiles to different non-β-lactam antibiotics (aminoglycosides, tetracyclines, quinolones,

folate pathway inhibitors, chloramphenicol, nitrofurantoin, and colistin) were obtained by

disc diffusion/agar microdilution methods, according to EUCAST (www.eucast.org) or CLSI

guidelines (13). All isolates exhibited MDR phenotypes, retaining susceptibility against

amikacin and colistin (MIC values: 0.063 – 0.125 mg/L).

Our results suggest that this methodology is a suitable alternative for a reliable, quick

and low-cost identification and typing of MDR K. pneumoniae clones/capsular types, at least

in local/short-term epidemiological contexts. We envision that the proximity of this

methodology and analysis workflow to routine clinical microbiology practices to assist

epidemiological typing in real-time would have a significant impact to improve the

effectiveness of infection control measures and therapeutic decisions.




UID/MULTI/04378/2013 ). Liliana Silva and Carla Rodrigues were supported by FCT grants

(SFRH/BD/88028/2012 and SFRH/BD/84341/2012, respectively). Ângela Novais was

supported by a fellowship (SFRH/BPD/104927/2014) from FCT through Programa

Operacional Capital Humano (POCH).

Conflict of interest: The authors declare that they have no conflict of interest.

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Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases.

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3. Li W, Raoult D, Fournier PE. 2009. Bacterial strain typing in the genomic era. FEMS

Microbiol Rev. 2009;33(5):892-916.

4. Rodrigues C, Sousa C, Lopes JA, Novais Â, Peixe L. Congruence between capsular

genotypic and phenotypic features of multidrug-resistant (MDR) Klebsiella pneumoniae

clones: a step-forward on K-typing by Fourier Transform Infrared (FTIR) spectroscopy. 2017

(manuscript final draft).



2013;3:3278.

6. Sousa C, Silva L, Grosso F, Lopes J, Peixe L. Development of a FTIR-ATR based model

for typing clinically relevant Acinetobacter baumannii clones belonging to ST98, ST103,

ST208 and ST218. J Photochem Photobiol B. 2014;133:108-14.

7. Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, et al. wzi

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Clin Microbiol. 2013;51(12):4073-8.




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and Uncommon Genetic Platforms (Tn4401d-IncFIA and Tn4401d-IncN). Front Microbiol.

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10. Belkum AV, Tassios PT, Dijkshoorn L, Haeggman S, Cookson B, Fry NK, et al.

Guidelines for the validation and application of typing methods for use in bacterial

epidemiology. Clin Microbiol Infect. 2007;3(13):1-46.

11. Pires J, Novais Â, Peixe L. Blue-Carba, an Easy Biochemical Test for Detection of

Diverse Carbapenemase Producers Directly from Bacterial Cultures. J Clin Microbiol.

2013;51(12):4281-83.

12. Bogaerts P, Rezende de Castro R, Mendonça R, Huang TD, Denis O, Glupczynski Y.

Validation of carbapenemase and extended-spectrum β-lactamase multiplex endpoint PCR

assays according to ISO 15189. J Antimicrob Chemother. 2013;68(7):1576-82.

13. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for

Antimicrobial Susceptibility Testing. 26th ed. CLSI supplement M100-S. Wayne, PA: Clinical

and Laboratory Standards Institute; 2016.


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Elucidating constraints for differentiation of major human Klebsiella

pneumoniae clones by MALDI-TOF MS

Carla Rodrigues1, Ângela Novais1, Clara Sousa2, Helena Ramos3, Teresa Maria Coque4,5,

Rafael Cantón4, João Almeida Lopes6, Luísa Peixe1*

1UCIBIO-REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia,

Universidade do Porto, Porto, Portugal; 2LAQV/REQUIMTE, Departamento de Ciências

Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 3Serviço de

Microbiologia, Centro Hospitalar do Porto, Porto, Portugal; 4Servicio de Microbiologia,

Hospital Universitario Ramón y Cajal and Instituto Ramón y Cajal de Investigación

Sanitaria (IRYCIS), Madrid, Spain; 5CIBER Epidemiología y Salud Pública, Madrid, Spain; 6Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade

de Lisboa, Lisboa, Portugal.

European Journal of Clinical Microbiology and Infectious Diseases. 2017; 36(2):379-86

Springer has authorized the reproduction of the final published PDF version of this paper in this

thesis through the License Agreement Number 4091971048884 on April 18, 2017.


228

ORIGINAL ARTICLE

Elucidating constraints for differentiation of major humanKlebsiella pneumoniae clones using MALDI-TOF MS

C. Rodrigues1 & Â. Novais1 & C. Sousa2 & H. Ramos3 & T. M. Coque4,5 & R. Cantón4,6 &

J. A. Lopes7 & L. Peixe1

Received: 30 June 2016 /Accepted: 10 October 2016# Springer-Verlag Berlin Heidelberg 2016

Abstract The establishment of matrix-assisted laser desorp-tion time-of-flight mass spectrometry (MALDI-TOF MS) inroutine microbial identification boosted many developmentstowards high-throughput applications, including bacterial typ-ing. However, results are still controversial for different bac-terial species. We aim to evaluate the suitability of MALDI-TOF MS for typing clinically relevant multidrug resistant(MDR) Klebsiella pneumoniae subsp. pneumoniae clonesusing routine conditions and a previously validated chemo-metric analysis workflow. Mass spectra of 83K. pneumoniaeclinical isolates representing major human MDR clones [11sequence types (STs), 22 PFGE-types] recovered in Portugaland Spain during outbreaks and non-outbreak situations

(2003–2012) were obtained from cell extracts (CE) and intactcells (IC), and analysed with different chemometric tools. Weobserved a highly consistent peak pattern among isolates fromdifferent clones either with CE or IC, suggesting a high degreeof conservation of biomolecules analysed (a large part corre-sponding to ribosomal proteins). Moreover, the low degree ofagreement between MALDI-TOF MS and other methods(from 34.9 % to 43.4 % of correct assignments for CE andfrom 40.8 % to 70.1 % for IC) corroborates the low discrim-inatory potential of the technique at infraspecies level. Ourresults suggest a low discriminatory power of MALDI-TOFMS for clinically relevant MDR K. pneumoniae clones andhighlight the need of developing tools for high-resolution typ-ing in this species.

Introduction

Matrix-assisted laser desorption time-of-flight mass spectrome-try (MALDI-TOFMS) has revolutionized routine identificationof microorganisms in clinical, environmental and food microbi-ology [1–3]. In principle, it consists of a proteotyping high-throughput analytical technique based on the ionization and sep-aration of ions according to their mass-to-charge (m/z) ratio,originating a characteristic mass spectra fingerprint of a givenbacterial cell, which reflects mostly the content of peptides andsmall proteins (mainly ribosomal proteins) [4]. Recently, effortsare being performed to push the limits of the technique andanalysis tools towards different clinical microbiology applica-tions including typing at infraspecies level [1, 4–6].

Notwithstanding the potential of MALDI-TOF MS forinfraspecies typing, available data are still not consistent due tovariability of the sample analysed (size, representativeness), ex-perimental or technical design and analysis tools [1, 6]. In fact,available studies constitute sensu stricto proof of principle

Electronic supplementary material The online version of this article(doi:10.1007/s10096-016-2812-8) contains supplementary material,which is available to authorized users.

* L. [email protected]

1 UCIBIO-REQUIMTE, Laboratório de Microbiologia, Faculdade deFarmácia, Universidade do Porto, Rua Jorge de Viterbo Ferreira, n.228, 4050-313 Porto, Portugal

2 LAQV-REQUIMTE, Departamento de Ciências Químicas,Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

3 Serviço de Microbiologia, Centro Hospitalar do Porto,Porto, Portugal

4 Servicio deMicrobiologia, Hospital Universitario Ramón y Cajal andInstituto Ramón y Cajal de Investigación Sanitaria (IRYCIS),Madrid, Spain

5 CIBER Epidemiología y Salud Pública, Madrid, Spain6 Red Española de Investigación en Patología Infecciosa (REIPI),

Madrid, Spain7 Research Institute for Medicines (iMed.ULisboa), Faculdade de

Farmácia, Universidade de Lisboa, Lisboa, Portugal

Eur J Clin Microbiol Infect DisDOI 10.1007/s10096-016-2812-8


229

lacking long-term evaluation and inter-laboratory reproducibili-ty [1]. Moreover, different resolution potential was observed forthe few clinically relevant Gram-positive and Gram-negativespecies evaluated, even when the same workflow was used[7–12]. Understanding the usefulness and limitations ofMALDI-TOF MS-based typing for a wide range of bacterialspecies, and the intra- and interspecies proteotypic diversity,justifies further research on the topic [5, 6].

The number of community- and hospital-acquired infectionscaused by Klebsiella pneumoniae subsp. pneumoniae (K.pneumoniae sensu stricto) increased alarmingly in recent years[13], and highly virulent or multidrug resistant (MDR) subpop-ulations are overrepresented in all the available series [14, 15]. Aquick and reliable detection of clinically relevant clones is neededto support effective therapeutic options and the implementationof prompt and adequate infection control measures to combattheir spread. Significant advances have recently been achievedto accurately define K. pneumoniae populations based on gold-standard genomics-based methods such as pulsed-field gel elec-trophoresis (PFGE) or multilocus sequence typing (MLST), andmore recently capsule typing (K-typing) and whole-genome se-quencing (WGS) [14, 16–18]. Nevertheless, these methods aregenerally costly, time-consuming or technically demanding, andunsuitable for routine application. Other alternative methods forK. pneumoniae typing have been explored, including MALDI-TOFMS, but only for small or random collections of isolates andusing diverse analysis workflows, yielding suboptimal discrimi-natory or low reproducible results [19–22].

In this work, we aim to evaluate the potential of MALDI-TOF MS for typing major human clinically relevant MDRK. pneumoniae clones, using a robust chemometric analysispreviously validated for other bacterial species [10, 12].

Materials and methods

Bacterial isolates

A set of 83 previously well-characterized K. pneumoniaesubsp. pneumoniae clinical isolates was analysed in this study.They represent a snapshot of major MDR clones responsiblefor outbreaks or endemic situations in two neighbouring coun-tries (Portugal and Spain) over a long period of time (2003–2012) [18, 23–27] (Table 1). Most of the isolates were pro-ducers of ESBL, acquired AmpC and/or carbapenemases andbelonged to different PFGE-types (n = 22), sequence types(ST, n = 11) or clonal groups (CGs, n = 7) (Table 1).

MALDI-TOF MS experiments

Mass spectra obtained from cell extracts (CE) of all isolates(n = 83) and from intact cells (IC) of a highly representativesubset (n = 67/83, 81 %; 11 ST11, 14 ST15, 8 ST14, 9 ST147,

6 ST39, 6 ST336, 3 ST54, 3 ST416, 3 ST405, 2 ST1, 2 ST253)were analysed. CE were obtained according to the MALDIBiotyper protocol (extraction procedure, matrix, sample:matrixratio and solvents) suggested by the manufacturer (BrukerDaltonics, Bremen, Germany) and previously described [12].IC were obtained from an overnight culture in Mueller-Hintonagar (18 h/37°C) and a small amount of a single colony directlyapplied using the ‘direct transfer method’. Samples (1 μL of CEor the IC) were spotted onto MALDI ground steel target(AnchorChipTM), air dried and overlaid with 1 μL of a saturatedα-cyano-4-hydroxycinnamic acid (HCCA) matrix solution in50 % of acetonitrile and 2.5 % of trifluoroacetic acid.

Mass spectra were acquired with a Microflex III instrumentcontrolled by FlexControl version 3.3 data acquisition soft-ware (Bruker Daltonics, Bremen, Germany), as previouslydescribed [10, 12]. Two independent CE were tested in qua-druplicate using four distinct spots of the MALDI target (in-strumental replicates) in at least two different days (biologicalreplicates), while IC were acquired in quadruplicate in onesingle day.

Chemometric analyses

Mass spectra were analysed by a workflow previously de-scribed and validated for other bacterial species [10, 12]. Allspectra were preprocessed using the FlexAnalysis software(Bruker Daltonics, Bremen, Germany) applying theBsmoothing^ and Bbaseline subtraction^ procedures. Spectrawith zero-line and low signal-to-noise (S/N) ratio wererejected after visual examination. The raw spectral data intext-file format were imported and loaded in Matlab R2015a(MathWorks, Natick, MA, USA). The instrumental and bio-logical replicates were used to generate a mean spectrum foreach isolate, which was considered for further analysis.

The discriminatory potential of MALDI-TOF MS wascompared with that obtained by MLST or PFGE. Initially,all spectra were visually inspected in order to identify partic-ular discriminatory peaks [at a given m/z ratio] or regions ofthe spectra for the different ST/CGs and PFGE-types tested.Mass spectra were preprocessed with vector-normalization (2-norm) and analysed with the peakfind function of the PLSToolbox version 7.9.5 for Matlab (Eigenvector Research,Manson, WA, USA) in order to evaluate the intra- and inter-ST variability among the 11 STs tested [10]. The followingsettings were used: nine points for the Savitzky-Golay filter,six units for peak tolerance, and 19 points to estimate localmaxima. The method started by estimating the peak mass-to-charge ratios of each ST. For this task, spectra of all isolatesbelonging to the same STwere averaged. The result was sub-mitted to the peak identification method and the peak loca-tions were stored in a vector producing a Bpeak profileprototype^ for each ST (BST prototype^). Then, the same peakidentification method was run for each isolate individually.

Eur J Clin Microbiol Infect Dis


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Peak positions of each BST prototype^ were compared withpeak positions of each isolate. When a BST prototype^ peaklocation matched a peak location of an isolate, a value 1 wasassigned for that peak; if not, the value 0 was given. Thisprocedure creates a vector of 0 s and 1 s for each pair BSTprototype^/isolate. Peak locations were considered to match iftheywere located within a mass-to-charge ratio difference low-er than 7 m/z units [if for a certain peak location n, |m/z(prototype)n–m/z(sample)n| < 7, that peak is considered tomatch]. For each isolate, a percentage of matching peaks wasestimated for each ST. Isolates were associated with a given STyielding the highest percentage of peak matches [10].

Mass spectra were further analysed by supervised (partialleast squares discriminant analysis, PLSDA) and unsupervised(principal component analysis, PCA) chemometric methodsusing PLS Toolbox version 7.9.5 for Matlab (EigenvectorResearch, Manson, WA, USA). Spectra were preprocessed withvector-normalization (2-norm) and before building a PCA orPLSDA model, the spectral dataset was subjected to mean-centring. Mass spectra were analysed by PLSDA, a methodbased on the partial least squares (PLS) regression method. InPLSDA models, a relationship between the proteomic data xi(mass spectra) and the categorical variable yi (ST) was devel-oped in such a way that categorical variable values can be pre-dicted for samples of unknown origin [10, 12, 28]. Model

loadings and the corresponding scores were obtained by sequen-tially extracting the components or latent variables (LVs) frommatrices X (the mass spectra) and Y (the matrix codifying theSTs). In PLSDA, a probability value for each assignment isestimated for each sample [10, 12, 28]. The number of LVwas optimized using the leave-one-sample-out cross-validationprocedure in order to prevent over-fitting considering 70 % ofthe available data (randomly selected). After optimization of thenumber of LV, the model was tested on the remaining 30 % ofthe data in order to assess the proportion (%) of correctpredictions for each ST (Table 2) [10]. As expected, a higherdiscriminatory power was observed with PFGE than withMLST, since different PFGE-types were identified amongST11 (5 PFGE-types), ST14 (5 PFGE-types), ST15 (3 PFGE-types) and ST147 (2 PFGE-types). For this reason, an additionalPCA analysis was performed considering PFGE-basedassignments.

Results

Spectral analysis

Each of the 11 STs tested revealed a highly consistent meanmass profile when isolates from the same or different ST or

Table 1 Epidemiological data ofK. pneumoniae clinical isolatesanalysed in this study

ST/CG (no.) PFGE-type (no.) Acquired β-lactamase (no.) Country Years

11/258 (16) Kp1 (10) OXA-48 Spain 2012

Kp2 (3) DHA-1 Portugal 2006–08

Kp3 (1) TEM-52 Portugal 2003

Kp4 (1) DHA-1 Portugal 2006

Kp5 (1) KPC-3 Spain 2010

15/15 (16) Kp6 (9) CTX-M-15 Portugal 2010

Kp7 (5) SHV-28 (2), SHV-2, SHV-12, CTX-M-15 Portugal 2010

Kp8 (2) VIM-43 + SHV-12 Portugal 2011–12

14/14 (10) Kp9 (6) SHV-106 (4), SHV-55 (2) Portugal 2010

Kp10 (1) CTX-M-15 Portugal 2010

Kp11 (1) GES-1 Portugal 2004

Kp12 (1) TEM-24 Portugal 2003

Kp13 (1) - a Portugal 2010

147/147 (10) Kp14 (9) SHV-12 Portugal 2006–10

Kp15 (1) VIM-1 Spain 2010

39/39 (7) Kp16 (7) VIM-1 Spain 2008–10

336/17 (7) Kp17 (7) CTX-M-15 Portugal 2010

54/- (4) Kp18 (4) VIM-1 Portugal 2009–10

416/- (4) Kp19 (4) DHA-1 + SHV-5/-12/-90 Portugal 2003–04

1/1 (3) Kp20 (3) VIM-1 Spain 2009–10

253/- (3) Kp21 (3) VIM-1 Spain 2009–10

405/- (3) Kp22 (3) OXA-48 Spain 2012

ST sequence type, CG clonal group, no. number; a Isolate with mutation in OmpK35 (resistance to carbapenems)involved in a Portuguese outbreak



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PFGE-type were compared either using CE or IC (Fig. 1,Fig. S1). However, differences were observed in mass spectraobtained from the different procedures, i.e. a higher number ofpeaks (4000-9000 Da) and a higher range (4000-18000 Da)was detected in CE spectra than in IC spectra (4000-12000 Da). Still, differences between the mean spectra of eachST inferred from visual inspection (shift of specific peaks inthe range of 4900 and 9900 Da) were more notorious using ICthan CE (Fig. S1, A1 and B1).

STs discrimination based on peaks identification

The peak positions found in the mass spectra of all isolateswith the peakfind function are represented for CE (a) and IC(b) in Fig. 1. We observed a highly consistent peak patternamong all isolates, corroborating the low intra- and inter-STspectra variability. Accordingly, it was possible to correctlypredict the ST for only 70.1 % of the isolates using IC andeven less (43.4 % of the isolates) using CE (Fig. 2), which arein both cases unsatisfactory for an accurate discrimination of

K. pneumoniae clones. In fact, although isolates belonging tosome of the ST analysed are always correctly predicted(100 %) using IC (ST1, ST54, ST147, ST253, ST405 andST416) or CE (ST253 and ST336), some of them show verydiscrepant prediction results using IC or CE (e.g. ST1, ST39,ST54 or ST336). Furthermore, a high proportion of isolatesfrom other STs are predicted as belonging to one of these STs,revealing a very low specificity. For example 10.8 % (9/83)and 21.7 % (18/83) of the isolates were erroneously predictedas ST253 and ST336 (using CE), while multiple isolates wereerroneously predicted as ST11 (11.9 %, 8/67), ST1 (6 %,4/67), ST15 (6 %, 4/67), ST405 (3.0 %, 2/67) or ST416(1.5 %, 1/67) (using IC) (Fig. 2).

STs discrimination based on PLSDA modeling

The PLSDA model constructed based on ST for CE and ICmass spectra is shown in Fig. 3. In both PLSDA models,isolates belonging to each STare widely dispersed in the scoreplot, and it is not possible to identify consistent clusters for

Table 2 Confusion matrix for K. pneumoniae PLSDA models according to MLST

STs obtained by MLST

Cell extracts (Total of correct predictions=34,9%) Intact cells (Total of correct predictions=40,8%)ST39 ST54 ST1 ST253 ST147 ST11 ST405 ST14 ST15 ST336 ST416 ST39 ST54 ST1 ST253 ST147 ST11 ST405 ST14 ST15 ST336 ST416

ST p

redi

cted

by

MA

LDI-

TOF

MS

a,b

ST39 28,0% 11,5% 15,0% 0,0% 1,3% 5,2% 27,0% 0,3% 8,0% 0,0% 1,5% 25,5% 44,0% 45,0% 25,0% 0,0% 6,0% 9,0% 4,3% 0,8% 0,0% 8,0%ST54 6,0% 8,5% 5,0% 0,0% 0,3% 0,0% 0,0% 0,0% 1,6% 0,0% 1,5% 17,5% 54,0% 0,0% 16,0% 0,0% 0,0% 0,0% 0,0% 1,6% 0,0% 0,0%ST1 4,0% 2,5% 0,0% 0,0% 0,0% 1,0% 0,0% 0,0% 0,0% 0,0% 0,5% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0%ST253 0,3% 0,5% 0,0% 16,0% 0,0% 0,6% 0,0% 0,0% 0,4% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0%ST147 11,3% 0,0% 0,0% 0,0% 39,7% 5,4% 14,0% 7,7% 10,0% 25,0% 11,0% 3,5% 0,0% 0,0% 57,0% 74,0% 15,3% 0,0% 2,3% 9,0% 19,0% 0,0%ST11 38,3% 56,0% 65,0% 72,0% 10,3% 56,8% 45,0% 17,3% 17,4% 8,0% 20,5% 23,0% 2,0% 1,0% 0,0% 21,7% 40,8% 1,0% 35,0% 7,4% 11,0% 14,0%ST405 0,3% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 9,0% 0,0% 0,0% 0,0% 0,0% 0,7% 0,2% 0,0% 2,0%ST14 0,7% 0,0% 8,0% 0,0% 7,3% 5,0% 7,0% 48,3% 13,0% 13,7% 0,0% 5,5% 0,0% 27,0% 0,0% 0,0% 6,3% 40,0% 25,7% 2,4% 0,0% 5,0%ST15 9,0% 18,5% 7,0% 12,0% 35,3% 26,0% 6,0% 25,3% 44,8% 12,7% 64,0% 24,5% 0,0% 18,0% 2,0% 3,3% 29,3% 21,0% 31,3% 73,2% 46,5% 71,0%ST336 0,0% 0,0% 0,0% 0,0% 5,7% 0,0% 1,0% 1,0% 4,4% 40,7% 1,0% 0,5% 0,0% 0,0% 0,0% 1,0% 2,5% 29,0% 0,7% 5,4% 23,5% 0,0%ST416 2,0% 2,5% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,4% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0% 0,0%

a Grey shaded represent the percentage of isolates correctly predicted using MALDI-TOF MS for each ST.b Values obtained considering 5LVs in the PLSDA model.

Fig. 1 Peak positions (m/z) of the K. pneumoniae isolates obtained with peak identification function fromMATLAB for cell extracts (a) and intact cells(b). The 11 lines at the bottom of the figure represent the mean sequence type peak profiles (BST prototype^)



232

each class. Accordingly, only 34.9 % and 40.8 % of the STswere correctly predicted for CE or IC models, respectively(Table 2). It is of note that in the case of CE, the best scorewas observed for ST11 (with 56.8 % of corrected predictions),while in the case of IC, the best scores were observed forST147, ST15 or ST54 (74.0 %, 73.2 % and 54.0 %,respectively).

PFGE-types discrimination based on PCA modeling

The PCAmodels constructed based on PFGE-types for ST11,ST14, ST15 and ST147 isolates using CE or IC mass spectra

are shown in Fig. S2. In both PCA models, isolates belongingto each PFGE-type are broadly distributed in the score plot,hindering the identification of consistent clusters for eachPFGE-type and corroborating results obtained with PLSDAand peak identification analysis.

Discussion

Analysis of the mass spectra with three different chemometricapproaches (mean spectra overview, peak identification, super-vised and unsupervised regressionmethods) revealed the lack of

Fig. 2 Proportion (%) of correctsequence type (ST) assignmentsconsidering the spectral matchingmethod for cell extracts (CE) (a)and intact cells (IC) (b). Eachcolour represents a given STwhile the bars correspond to theST predicted by the peakidentification function. Therewere 43.4 % correct predictions(CP) with CE and 70.1 % with IC

Fig. 3 Score plot of the PLSDA regression model according to MLST, corresponding to the first three latent variables (LVs) using cell extracts (a) andintact cells (b)



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suitability of MALDI-TOF MS for typing major clinical MDRK. pneumoniae clones. In fact, the high similarity observedamong the mass profiles of K. pneumoniae lineages is behindthe poor discrimination observed, and the very low or inconsis-tent sensitivity and/or specificity. Even considering the differ-ences in the resolution potential of the two reference typingmethods used (the suboptimal resolution of MLST contrastswith the high discriminatory power of PFGE), no correlationcould be inferred from MALDI-TOF MS results and any ofthese methods. Interestingly, data from this and previous studiesusing the same experimental workflow for infraspecies typing inseveral bacterial species (K. pneumoniae, Escherichia coli andAcinetobacter baumannii) allowed us to recognize a species-dependent discriminatory potential of MALDI-TOF MSreflecting different degrees of variation in core molecules eval-uated by this technique and the need of optimized protocols foreach bacterial species [10, 12].

According to the literature, most of the proteins and/or pep-tides visualized in mass spectra have low mass (<12000 Da),with about half of the peaks corresponding to ribosomal proteins[4, 29, 30]. Although amino acid sequences of ribosomal pro-teins are generally highly conserved in the same species [31],some studies have demonstrated their potential to discriminateNeisseria meningitidis and Pseudomonas putida isolates atstrain level [32, 33]. However, in this study, protein alignmentresults revealed a high degree of conservation ofK. pneumoniaesubsp. pneumoniae ribosomal proteins (99–100 % for the 56proteins recognized to date), while differences were observedbetween species and subspecies of the K. pneumoniae complex(K. pneumoniae subsp. rhinoscleromatis,K. pneumoniae subsp.ozaenae, Klebsiella variicola, Klebsiella quasipneumoniaesubsp. quasipneumoniae and Klebsiella quasipneumoniaesubsp. similipneumoniae) (data not shown). These observationssupport the poor ability of MALDI-TOF MS to discriminateK. pneumoniae subsp. pneumoniae at strain level under the ex-perimental conditions used [20–22, this study].

On the other hand, specific non-ribosomal proteins werecrucial for typing purposes in other bacterial species. As ex-amples, a 9716 Da peak corresponding to a mature HdeAprotein was specific for the identification of isolates of theB2 phylogroup ofE. coli, whereas a 7650Da peak correspond-ing to a mature YhaO protein allowed identification of isolatesfrom the B2-ST131 lineage [12, 34, 35]. In addition, differen-tiation of H antigens of E. coli and Salmonella using massspectrometry (including MALDI-TOF MS) revealed verypromising results for rapid and cost-effective serotyping [5].

K. pneumoniae strains lack H antigen but typically expressthe O-antigen polysaccharide (O-typing) and K-antigen cap-sular polysaccharide (K-typing) on their surface [36]. WhileK. pneumoniae O-antigens show limited heterogeneity, andare not usually accessible at the cell surface [37], K-antigensare highly diverse with particular K-types being linked to spe-cific clones [14–17]. However, polysaccharides and other

potential capsule biomarkers are not favoured in the experi-mental conditions used. In fact, attempts to discriminate atserotype level by MALDI-TOF MS in Haemophilusinfluenzae and Streptococcus pneumoniae were based on dif-ferences on ribosomal proteins [38, 39]. Thus, it would be ofinterest to optimize procedures for selective enrichment ofpolysaccharides and/or particular discriminatory proteinsfrom the capsule polysaccharide locus (such as Wzi) in orderto increase the discriminatory potential of MALDI-TOF MSin K. pneumoniae.

Nowadays, MALDI-TOF MS-based typing is competingwith WGS as a fast and automatized typing method. MALDI-TOF MS is already widely used and data for further discrim-ination at infraspecies level is included for free in the condi-tions used for microbial identification, favouring a shorterturnaround time for routine incorporation. Nevertheless, thedelay in the identification of good biomarkers for differentia-tion at infraspecies level and the massive advances in geno-mics might constitute a caveat for further MALDI-TOF MStechnological advances for strain typing [1, 4, 40]. Knowledgearising from genomic and proteomic based typing might alsocontribute to unravel aspects of biological significance forrelevant bacterial pathogens.

In conclusion, we demonstrated, using a robust chemomet-ric analysis of data obtained fromMALDI-TOFMS with rou-tine experimental conditions, a limited potential of this meth-odology to discriminate clinically relevant MDRK. pneumoniae clones. Notwithstanding the limitation of thesample analysed (restricted to some STs represented by a lim-ited number of isolates recovered in specific countries fromSouth Europe), the findings of this study suggest a degree ofconservation of ribosomal proteins within K. pneumoniaesubsp. pneumoniae. Further studies with other experimentalconditions could be explored in order to provide new insightson MALDI-TOF MS high-resolution typing in this species.

Author’s contributions C.R., A.N. and L.P. contributed to the studydesign. C.R. performed the experimental work related to the acquisitionof mass spectra in MALDI-TOF MS, performed chemometric analysisand wrote the manuscript. A.N. participated in data analysis and wrote themanuscript. C.S. and J.A.L. provided expertise in chemometric analysis,participated in the analysis of data, and the revision of the manuscript.H.R. provided access to the MALDI-TOF MS (Bruker Daltonics,Bremen, Germany) equipment, reagents and software, and part of thePortuguese strain collection. T.M.C. and R.C. provided the Spanish straincollection used in this study, expertise and participated in the revision ofthe manuscript. L.P. contributed for the general conceptualization of thestudy and methodological approach, the analysis of data and revision ofthe manuscript. All authors read and approved the final version of thismanuscript.

Compliance with ethical standards

Funding This work received financial support from the EuropeanUnion (FEDER funds) through Programa Operacional Factores deCompetitividade—COMPETE and Portuguese National Funds (FCT,



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Fundação para a Ciência e Tecnologia) (UID/Multi/04378/2013). CarlaRodrigues and Ângela Novais were supported by fellowships from FCTthrough Programa Operacional Capital Humano (POCH) (grants numberSFRH/BD/84341/2012 and SFRH/BPD/104927/2014, respectively).Spanish isolates were recovered during execution of grants founded bythe European Commission (TROCAR-FP7-HEALTH-F3-2008-223031and, R-GNOSIS-FP7-HEALTH-F3-2011-282512) and the Instituto deSalud Carlos III of Spain (REIPI RD12/0015, Spanish Network forResearch in Infectious Diseases) co-financed by the EuropeanDevelopment Regional Fund, AWay to Achieve Europe.

Conflict of interest The authors declare that they have no conflict ofinterest.

Ethical approval This article does not contain any studies with humanparticipants or animals performed by any of the authors.

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FigureS1.RepresentationofthemeanspectraofthedifferentSTsstudiedbetween2000and20000m/zratioforCE(A)andIC(B).

A1andB1 representtheexpansionofthediscriminatoryregionsvisualizedidentiHiedinthespectraofCEandIC,respectively.

A)

A1)

B1)

B)

SequenceType(ST)


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FigureS2.ScoreplotofthePCAusingPFGE-types(ST11,ST14,ST15andST147)correspondingtotheCirstthreelatentvariables(LVs)

usingCE(A)andIC(B).

PFGE-types

ST11

ST15

ST14

ST147

A)

B)

PFGE-types

ST11

ST15

ST14

ST147


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High-resolution genomic analysis of the globally disseminated

multidrug resistance Klebsiella pneumoniae Clonal Groups 14 and 15

Carla Rodrigues1,2, Val F Lanza2,3,4, Luísa Peixe1,

Ângela Novais1, Teresa M Coque2,3,4*

1UCIBIO/REQUIMTE. Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2Servicio de Microbiología, Hospital Universitario Ramón y Cajal (IRYCIS), Madrid, Spain; 3CIBER en Epidemiología y Salud Pública (CIBER-ESP), Madrid, Spain. 4Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (RYC-CSIC), Madrid, Spain. Running Title: Delineation of different lineages among CG14 and CG15 K. pneumoniae *Corresponding author Teresa M. Coque Senior researcher Departamento de Microbiologia y Parasitologia, Hospital Universitario Ramón y Cajal Carretera de Colmenar Km 9.100 28024-Madrid, Spain E-mail: [email protected]



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ABSTRACT

The Klebsiella pneumoniae (Kp) Clonal Groups (CG) 14 and 15 are overrepresented

among MDR Kp strains worldwide, but characterization of the different CG14 and CG15

lineages at the genome level is still missing. WGS settled down as a high-resolution tool,

providing a detailed overview into the population structure, resistome, virulome and

mobilome of Kp strains. In this study, we performed a high-resolution genomic analysis of Kp

CG14 and CG15, in order to elucidate factors responsible for its expansion.

WGS of nine representative Portuguese ST15 (4 wzi24, 1 wzi93, 1 wzi19, 1 wzi89)

and ST14 (1 wzi2, 1 wzi16) isolates showing variable PFGE-types (2003-2013) were obtained

using Illumina MiSeq (2x300bp pair-ended runs; ~6Gb per genome, coverage:100x).

Genomic sequences of all CG15 (n=61; 2004-2015; America/Asia/Europe) and CG14 (n=29;

1986-2014; Africa/America/Asia/Europe/Oceania) isolates available on NCBI and BIGSdb

databases (as from August 2016) were included. Assembly was performed using SPAdes and

core genome (>80% similarity/coverage) was defined using home Perl scripts. Core genes

were concatenated, aligned, and SNPs extracted to generate a maximum likelihood

phylogenetic tree (100 bootstraps) in R (Phangorn package). The cps operon within and

between lineages was analyzed using EasyFig and BRIG. Plasmid content of representative

isolates determined by PLACNET (n=17) and from complete genomes (n=5) was analysed by

hierarchical clustering of plasmid proteomes (n=21 genomes/66 plasmids). Virulence,

wzi/wzc, antibiotic resistance (ABR) and metal tolerance genes were searched using BIGSbd-

Kp and/or ResFinder.

Phylogenetic analysis of the core genomes of CG15(~3.1Mb) and CG14(~3.7Mb)

supported the circulation of seven and two distinct lineages, respectively, each one of them

harbouring the same wzi type (wzi24/wzi93/wzi19/wzi89/wzi118/wzi178/wzi274 for CG15;

wzi2/wzi16 for CG14). The whole cps operon was highly conserved within each lineage (with

exception of one hybrid strain K24/K39). The virulence content (100% mrk or kfu) was

similar in both CGs being of note the high prevalence of yersiniabactin cluster (64%-

CG15/58%-CG14). Plasmid analysis revealed the circulation of a high diversity of plasmid


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groups shared within the different lineages and CGs, with some particular groups

[HI1B+FIB(pNDM-MAR)/FIB(pKpn3)±FIIK/R replicons] comprising a diversity of ABR

and/or specific metal tolerance genes [ter+mer-HIB+FIB(pNDM-MAR)/ars+sil+pco±mer -

FIB(Kpn3)/mer-R]. This study provides evidences that CG14 and CG15 predominant

Kp lineages carrying specific cps operons are circulating in different geographic regions for

several years, thus suggesting divergent evolution in different host backgrounds.

Comprehensive analysis of the accessory genome revealed turbulent flux of plasmids of

disparate families containing a wide number of adaptive traits (virulence/ABR), frequently

associated with specific plasmids. MOBF12/IncF plasmids were pervasive and greatly

contribute to the CG14/15 pangenome. CG14/15 has an open genome for rapid and efficient

exchange that might explain their fast adaptation under different selective pressures.


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INTRODUCTION

Klebsiella pneumoniae subsp. pneumoniae (Kp) strains is one of the main drivers of

antibiotic resistance (ABR) (http://ecdc.europa.eu/en/eaad/antibiotics-

news/Documents/antimicrobial-resistance-EARS-Net-summary-2015.pdf) (1). This species

has a recombinant population structure that makes inefficient the use of standard genotypic

methods such as MLST for typing purposes (2–5). Core genome MLST (cgMLST) schemes

(based on 694 core genes) (6) or single nucleotide polymorphisms (SNPs) phylogenetic

analysis across the whole genome (3,7,8) allowed the identification of clonal groups (CGs),

such as CG14, CG15, CG17, CG101, CG147 and CG258. They are overrepresented among

multidrug resistant (MDR) Kp strains and strongly associated with the worldwide expansion

of extended-spectrum β-lactamases (ESBLs) (mainly CTX-M-15 and SHV-ESBL variants)

and carbapenemases (KPC, OXA-48-like, VIM, NDM) (6,9,10). To date, most phylogenomic

analysis of Kp have explored the CG258, as reflected by the number of genomes available in

public databases (674/1576, 43% of Kp genomes in NCBI database at December 2016) and

the growing number of publications concerning the evolution and diversification of CG258

(2,3,11,12).

MDR Kp CG14 [central genotype sequence type (ST) 14] and CG15 (central

genotype ST15) are increasingly associated with nosocomial and community-acquired human

infections all over the world, and also with animal infections or human colonization (7,8,13–

22). Despite their apparent relevance, the number of genomes of Kp CG14 and CG15 are less

represented in public databases (106/1576; 7% of Kp genomes in NCBI database at December

2016). Moreover, characterization of a limited number of CG15 and CG14 isolates reflects a

remarkable variability within these CGs based on the PFGE-type and diversity of wzi alleles,

suggesting the circulation of distinct lineages (4,8,16–20,23,24).

The location of genes conferring resistance to extended-spectrum β-lactams and also

fluoroquinolones and aminoglycosides on plasmids carrying specific replicons (FIIK, R, FIB,

FIA, HI1B, A/C, L, ColE-like) led to the assignment of a relevant role of these mobile genetic

elements in the transmission of ABR (7,10,14–17,25). However, the diversity and role of the


243

plasmidome in the evolution and host adaptation of Kp CG14 and CG15 MDR lineages

remains largely underexplored, mainly due to the scarcity on available bioinformatics tools

for plasmid data extraction (PLACNET, PlasmidSPAdes) (26,27).

In this work, we aimed to perform a high-resolution analysis of all known Kp

genomes belonging to CG14 and CG15, including those sequenced in this study and those

available in public databases in order to elucidate factors (phylogeny, cps evolution,

plasmidome) that might be contributing to the global expansion of these CGs.

RESULTS

Core genome phylogeny of Kp CG14 and CG15

To estimate the phylogenetic relationships within Kp CG14 and CG15 strains, 9

isolates (7 ST15 and 2 ST14) representing distinct lineages identified among Portuguese

clinical settings (16,28,29) were fully sequenced and compared with the 90 Kp genomes

available in GenBank and BIGSdb databases. In silico MLST analysis identified 61

ST15/CG15, 28 ST14/CG14 and 1 ST2316/CG14 (single locus variant of ST14). A further

SNPs-based phylogenomic analysis revealed a small core genome of 2.7 Mbp and a SNP

distance between the CG14 and CG15 of 2.200 SNPs/Mbp, and therefore the convenience to

analyze each one of them independently. Larger core genomes were then obtained for CG14

(3.7 Mbp) and CG15 (3.1 Mbp), representing a genomic overlay of isolates from the same CG

of ~60-70% (considering a Kp genome size of ~5.4 Mbp).

Characterization of the different Kp CG14 and CG15 phylogenetic lineages

Within the CG14 (n=31; core genome of 3.7 Mbp) isolates, we identified two distinct

branches corresponding to different wzi types (CG14-wzi2 and CG14-wzi16) (Figure 1), with

an average distance between them of about 2300 SNPs (622 SNPs/Mbp). The first branch

comprised isolates harbouring the same cps locus (n=22, wzi2/K2) and a divergence of about

370 SNPs (100 SNPs/Mbp) in the core genome. The cluster of CG14-wzi2 comprises isolates

recovered from 1986 to 2015 in all continents, with an overrepresentation of strains causing


244

human infections (Table 1, Figure 2). CG14 isolates often carry genes conferring resistance to

extended spectrum β-lactams (13/22, 60%, mainly NDM-1/-5 plus CTX-M-15, KPC-4, CTX-

M-15 or TEM-24). Most of the isolates harboured the chromosomal blaSHV-28 gene (73%,

16/22) and at lesser extent, blaSHV-1 (14%, 3/22), blaSHV-7 and blaSHV-11 (Figure 2). The second

cluster included isolates with wzi16/K16 (n=9), which differed up to 350 SNPs (95

SNPs/Mbp) (Figure 1). They included isolates recovered between 2002 and 2014 in distinct

continents (4 America, 3 Europe, 2 Oceania). Five out of 9 isolates (56%) from this lineage

produced ESBLs, mostly SHV-types (2 SHV-2, 1 SHV-5, 1 SHV-106). Four isolates

harboured the chromosomal blaSHV-1 and 3 blaSHV-28, while none of these blaSHV was detected

in two isolates (Figure 2).

The CG15 (n=68, core genome of 3.1 Mbp) split in seven clusters (Figure 3) each of

them linked to a specific wzi type (wzi24, wzi93, wzi19, wzi118, wzi89, wzi274, and wzi178).

From these, CG15-wzi24, CG15-wzi93 and CG15-wzi19 represent 93% (n=63/68) of the

CG15 sample (Table 1, Figure 2). The distance between isolates from each lineage ranged

from ~400 SNPs (130 SNP/Mbp) to 1400 SNPs (452 SNP/Mbp). Detailed analysis of each

CG15 lineage is performed below.

The CG15-wzi24/K24 (n=40) comprised isolates from human infections (n=17) or

colonization (n=6) or non-identified sources (n=17), from Europe (n=27), America (n=10)

and Asia (n=3) recovered between 2006 and 2015 with less than 120 SNPs among them (40

SNPs/Mbp) (Table 1, Figure 2 and Figure 3). Most isolates were CTX-M-15 producers

(n=32), and involved (66%) in outbreaks in The Netherlands (n=14, 2012-13, Groningen) and

North America (n=7, 2013-14, Boston). The remaining isolates produced CTX-M-14, KPC-3,

VIM-4 or SHV-2. Most CG15-wzi24 harbored blaSHV-28, with the exception of the isolates

involved in the American outbreak (carrying ESBL-blaSHV-5) and one Portuguese isolate

(carrying ESBL-blaSHV-2) (Figure 2).

CG15-wzi93/KL112 (n=19) lineage included isolates from different countries (10

Europe, 6 Asia, 3 America; 2004-2015) (Table 1, Figure 2). The core genome of CG15

isolates differed in less than 50 SNPs (16 SNPs/Mbp) indicating their close phylogenetic


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relationship (Figure 3). Most isolates produced CTX-M-15 (89%, 17/19) (3 isolates co-

produced NDM-1 or 1 KPC-2) and all of them harboured the chromosomal blaSHV-28 (Figure

2).

CG15-wzi19/K19 (n=4) was identified in European (n=2) and Asian (n=2) countries

between 2005 and 2013 in isolates from human infections and colonization, and in a food

product (Table 1). Three of the isolates were closely related phylogenetically with less than

30 SNPs (10 SNPs/Mbp) and linked to the production of diverse extended-spectrum acquired

β-lactamases (NDM-1, GES-11, CTX-M-15) (Figure 2 and Figure 3). Three out of four

isolates harboured chromosomal blaSHV-28. The remaining lineages were sporadic and only

two of them were linked to the production of ESBLs (SHV-2 in CG15-wzi118) and ESBLs

plus carbapenemases (VIM-34+SHV-12+OXA-17 in CG15-wzi89) (Figure 2).

The content of ABR genes was highly variable within and between the different

lineages identified, while the content in virulence genes was highly consistent, being common

the detection of type 3 fimbrial gene cluster (mrk, 100%), iron ABC transporters (kfu, 100%),

and yersiniabactin siderophore cluster (ybt, irp1, irp2, fyuA,62%).

Phylogenetic distribution and characterization of cps locus in Kp CG14 and CG15

lineages

We identified highly conserved cps locus (arbitrarily designated from A to J) among

the 99 genomes of Kp CG14 and CG15 using Kaptive (Figure 4). Comparison of the whole

cps locus of 31 representative isolates with those of the serotype reference strains (Table S2)

revealed concordance for cps clusters A-wzi2/K2, B-wzi16/K16, C-wzi24/K24, D-wzi24,

wzc39:21/K39, G-wzi274/K30, and I-wzi19/K19 by all methods tested (wzi, wzc and Kaptive).

The exception was cps type D, which was predicted as K24 by wzi and as K39 by

wzc/Kaptive and cps structure. The remaining cps locus detected within CG15 were classified

as new capsular types (E-wzi93/KL112; F-wzi89/KL110, H-wzi118/KL146 and J-wzi178),

three of them previously characterized by the Kaptive platform (E, F, H), and one firstly


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described in this study (J). It is of note that cps locus E was predicted by wzi as K60 but its

structure was different from the K60 reference strain, being assigned by Kaptive as KL112.

The 10 types/subtypes of the cps locus of CG14 and CG15 described to date ranged

from 18 to 27 Kb and from 13 to 22 CDSs [excluding insertion sequences (IS)] (Figure 4).

They comprise a common structure at the beginning (5’) and at the end (3’) of the locus and a

highly variable group of genes between wzc and gnd, and between gnd and ugd (30). The

conserved region included eight genes at 5’ end: galF (UTP–glucose-1-phosphate

uridylyltransferase), orf2 (putative acid phosphatase), wzi (responsible for capsule assembly),

wza, wzb and wzc (involved in the translocation of mature capsular polysaccharide to cell

surface) and two genes at 3´end: gnd (6-phosphogluconate dehydrogenase) and ugd (UDP-

glucose 6-dehydrogenase). Regarding the variable region of the cps, 89 unique CDSs were

identified across the ten cps locus identified (Figure 4, Figure S1). Eighty-three CDSs were

found in the wzc-gnd region encoding mainly for initial (wbaP and wcaJ present in 6 and 4 of

the cps locus types identified, respectively) and non-initial glycosyltransferases, modifying

enzymes (acetyltransferases, pyruvyl transferases, glycosyl hidrolases), hypothetical proteins

and the core assembly components Wzx (flippase) and Wzy (polymerase; not present in cps

locus B and F in the range analysed) involved in capsular type-specific repeat unit synthesis

and polymerization. The remaining six CDSs were found in the gnd-ugd region codifying for

proteins implicated in mannose (manB and manC; cps types A-H) and/or rhamnose (rmlA,

rmlB, rmlC and rmlD, cps types I and J) synthesis (Figure 4). In three cps locus, IS5 (present

in two cps locus-Bi, Ci and Cii) and IS1 (present in one cps locus-Ei) were detected (Figure

4). The ISs occurred upstream wzi (cps Bi, Cii, Ei) and/or in the central region of the cps (cps

Ci and Cii, in the case of cps Ci disrupting CDS) (Figure 4). It is of note the differences

observed in guanine+cytosine (GC) content between the conserved (>55%, similar to the CG

content observed for the rest of Kp chromosome) and nonconserved (<40%, the region

between wzc-gnd) regions of the cps locus, suggesting a distinct origin.


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Plasmid replicon analysis among Kp CG14 and CG15

Plasmid replicons from the Enterobactericeae family were searched through

PlasmidFinder platform (https://cge.cbs.dtu.dk/services/PlasmidFinder/) to get a snapshot of

the plasmid diversity among the 99 genomes from Kp CG14 and CG15. A high diversity of

plasmid replicons was observed among the strains of these CGs, with replicons belonging to

F (89%, 88/99) and R (43%, 42/99) family being the most frequent and represented in all

branches, independently of the lineage studied (Figure 2). Within the F plasmid family, we

detected a remarkable diversity of replicons, more than one rep gene per strain being frequent

– 59 FIIK (RNAI-FII), 69 FIB(KPN3) (repFIB from pKPN3), 12 FIB(MAR) (repFIB from

pNDM-MAR), 12 FIB(pKPHS1) (repFIB from pKPHS1 phage-related), 4 FIB(KpQIL)

(repFIB from KpQIL), 24 FIA(HI1) (repFIA from H1), 3 FII (RNAI-FII from pC15-1a).

Other plasmid families detected were HI1B, A/C2, L, M, X3, X4, HI2, N, Q2 and ColE-like

[colKP3, Col(pVC)] (Figure 2) (31).

Plasmid reconstruction from de novo sequenced Kp CG14 and CG15 genomes

The plasmid content of the 9 Portuguese isolates sequenced in this study was

reconstructed from whole-genome data using two different platforms, PLACNET and

PlasmidSPAdes (26,27), and the results obtained were confirmed with the plasmid content

assessed by S1-PFGE. For the set of plasmid genomes analysed, PLACNET showed a better

resolution than PlasmidSPAdes (Table S1), once a higher concordance was obtained with

plasmid sizes previously determined by S1-PFGE and because FIIK and R plasmids were

identified as separate entities. Moreover, the results obtained using PlasmidSPAdes were

completely concordant with those achieved using PLACNET and S1-PFGE for a single

isolate (K47) (Table S1).

A more comprehensive view of the plasmidome was obtained when we analyzed the

plasmids extracted from our genomes with those of all CG14 and CG15 genomes available at

public databases (Table S2). They included the plasmids of 5 complete genomes, 3 CG14 K2

(Kp617_ASM131717, PittNMD01_ASM73325, NUHL24835_ASM152189) and 2 CG15-


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KL112 (BR_ASM166319, PMK1_ASM76461). We also reconstructed the plasmids of seven

[3 CG14 (2 K2, 1 K16) and 4 CG15 (3 K24, 1 K112)] genomes available at public databases,

which represent different lineages according to the phylogenetic tree on Figure 1.

In total, we identified 66 plasmids in 21 genomes, with the number of plasmids per

isolate ranging from 1 to 9 (average of 3 plasmids per genome) (Figure 5). The most common

groups of plasmids belong to MOBF12/IncF (18 plasmids), MOBH11/IncHI1B-IncFIB (6

plasmids), MOBP (1 IncX3, 1 IncX4, 2 IncL, 18 ColE) and MOBH (1 IncHI2, 2 IncA/C2).

Seventeen plasmids lacked MOB or had an unclassified relaxase [including 4 IncFIB

(pKPHS1/pKPN3) and 7 IncR plasmids] (Figure 5).

The relationship between plasmids is shown in the dendrograms of the 66 plasmids

identified in CG14 and CG15 genomes (Figure 6), while a dendrogram including these 66

plasmids plus reference plasmids from different families is shown on Figure 7 (Table S2).

Plasmids clustered into 12 main groups that correspond to the different Inc/MOB families

identified. Plasmids classified as IncF/MOBF12, IncR and IncHIB-IncFIB/MOBP11 plasmid

families represented 52% of all plasmids detected (Figure 5). Detailed analysis according to

main plasmid families is provided below.

IncF plasmids

Twenty-one IncF/MOBF12 plasmids were found in each of the CG14 and CG15

genomes for which plasmid analysis was performed, with the exception of three isolates (K2 -

Kp617_ASM131717, KL112-BR_ASM166319 and KL110-K47) in which MOB and tra

regions (n=4) were absent but contained backbone genes (including RIP proteins) of IncF

plasmids (Figure 5). In the dendrogram represented in Figure 7, IncF plasmids grouped in

four main branches, and each branch was individually analysed.

The first group includes IncFIIA/MOBF12 (~65 kb) plasmids identified in 2 CG15

(K24, KL112). These plasmids are closely related to the widely disseminated plasmid pC15-

1a (blaCTXM-15) from the pandemic ST131 E. coli clone, although lacking a region of


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approximately 30 kb that contain the ABR genes (blaCTX-M-15, blaTEM-1, blaOXA-1, aac(6’)-Ib-cr,

tetA and tetR) (Figure S2).

The second group is represented by an IncFIB/MOBF12 plasmid (96 kb) from one

CG15-K24 isolate that did not contain any gene of resistance to ABR. This plasmid showed

high similarity with IncFIB plasmids (repFIB allele 36) circulating among different species of

Enterobacteriaceae despite the original sequences were detected in Citrobacter freundii (100

kb-pCAV1741-101, 135 kb-pCAV1321-135, 171 kb-SL151 p2) (Figure S3). They are

designated as pKPC_UVA01 group as they are good drivers of the spread of KPC genes in

hospitals (32).

The third group was the most heterogeneous and was subdivided in three sub-groups:

i) the first included IncFIB (pKPN3)/MOBF12 plasmids of ~100 kb identified in 2 CG14-wzi2

with no ABR and metal tolerance genes; ii) the second sub-branch contained IncFIB

(pKPN3)/MOBF12 or IncFIB(pKPN3)-IncFIIK/MOBF12 plasmids with sizes ranging from 100

kb to 275 kb detected in a large part of the genomes analyzed (67%, 14/21; CG15-6 K24, 3

KL112 and 1 K19, CG14- 2 K2 and 2 K16); in this subgroup most of the plasmids were MDR

(71%) but the ABR content was variable, and all plasmids harboured genes conferring metal

tolerance to copper (pco and sil cluster), silver (sil cluster, one exception) and arsenic (ars

operon); iii) the third sub-group comprised one IncFIIK2/MOBF12 MDR plasmid of ~100 Kb

detected in one CG14-K2 isolate with no FIB replicon and with no metal tolerance genes

(Figure S4).

The fourth group encompassed phage-related IncFIB plasmids (~110 kb), detected in

1 CG14-K2 and 2 CG15-KL112 isolates, closely related to pKPHS1 plasmid though lacking

MOB and tra regions and the approximately 10 kb region where blaCTX-M-14 is harboured. No

ABR and/or metal tolerances genes were detected in CG14 and CG15 harbouring

IncFIB(pHPHS1) phage-like plasmids and, interestingly, both isolates lacking the gene retA

codifying for a reverse transcriptase. The third and fourth groups are represented by IncF

plasmids almost exclusively found in Kp isolates (Figure S5).


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IncR plasmids

IncR plasmids (n=7) were highly heterogeneous and all of them were MDR. They

were located in one main branch of the dendrogram that was subdivided in two sub-branches.

The first group included IncR plasmids eventually harbouring an FIA replicon (45-70 kb) and

were found in 2 CG14-K2 and 2 CG15-1 K19 and 1 KL112. Most of them carried blaCTX-M-15

and mer operon. The second group included mosaics of IncR and IncN plasmid backbones

(35-76 kb) and were detected in CG15 isolates (2 K24, 1 KL112) with variable content in

ABR genes and low prevalence of mer operon. Relaxases for IncR plasmids were not

detected. H1122_p3 plasmid contained IncR backbone regions (except the replicon) and the

FIA replicon (Figure S6).

IncHI1B-IncFIB mosaic plasmids

A main branch comprises large plasmids (>250 kb) harbouring HI1B and FIB

replicons and MOBP11 relaxases (IncHIB-IncFIB/ MOBP11) from either CG14 (3 K2) or CG15

(2 KL112, 1 K24) isolates (Figure S7). These plasmids are closely related to IncHI1 plasmids

of Kp, which differ from other IncHI1 plasmids predominant in Salmonella or other

Enterobacteriaceae species. The IncHIB-IncFIB/MOBP11 plasmids circulating in CG14/CG15

collection were MDR (6/7) highly similar with those previously described, and four of them

carried blaNDM-1 in a Tn3-like transposon (tnpA- blaNDM-1-bleMBL-groES-groEL-tnpA) (33). All

plasmids harboured genes conferring metal tolerance to tellurium (ter operon), while presence

of mer operon was variable (Figure S7).

Other plasmid families

Plasmids of other six families were identified, namely IncX/MOBP3, IncL/MOBP13,

IncA/C2/MOBH121 or IncHI2/MOBPH11, ColE and a phage-like plasmid.

IncX/MOBP3 plasmids were divided in two subgroups, IncX3 and IncX4. The IncX3

(46 kb) from the CG14-K2 isolate was identical to pNDM-5-X3 (blaNDM-5) detected in 2015

in one clinical Kp isolate (Figure S8) (34). IncX4 (similar to pCROD2 reference plasmid) was


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detected in 1 CG15-K24 isolate and without ABR genes (Figure S9). IncL/MOBP13 plasmids

(62 kb) were detected among 1 CG14-K2 (OXA-162) and 1 CG15-K24 (OXA-48) and were

identical to the pOXA-48 plasmid which has been linked to the worldwide expansion of

OXA-48-like enzymes (Figure S10) (35). Concerning IncA/C2/MOBH121 plasmids they were

detected in 1 CG14-K2 (167 kb) and 1 CG15-K24 (188 kb) and were both MDR plasmids.

H49_p2 was identical to pEA1509_A plasmid, a widespread plasmid responsible for the

dissemination of TEM-24 in different Enterobacteriaceae species manly between 1999 and

2004 (36), MGH111_p5 harbouring OXA-10 was also closely related with the reference

plasmids pEA1509_A and pR55, but lacks the TEM-24 platform (Figure S11). The

IncHI2/MOBPH11 plasmid detected among 1 CG15-KL110 was unique (compared to those on

public databases) and different from the R478 reference plasmid (only 49% identity). The

phage-like plasmid (55 kb) detected among 1 CG15-K24 was similar to others detected

among Kp (pKPN-852) and also K. variicola (pKV2), and didn’t harbor any ABR genes

(Figure S12).

A great diversity was observed within ColE-like plasmids, with some typical families

being recognized, ColKP3 (7 kb) found in 3 CG14-K2 recently linked to the dissemination of

OXA-232 (37), and Col(pVC) (2.5-3 kb) found in 4 CG15 (3 K24, 1 KL110), found also

among other Enterobacteriaceae species (Figure 5).

Some unclassified plasmids (either by REP or MOB) were also detected, such as

PMK1-C and C1686_p1, which carried a repA belonging to the IncFII_superfamily.

Interestingly, these plasmids didn’t harbour ABR genes (except strAB in PMK1-C) but some

known virulence factors (mrk operon by PMK1-C or type VI secretion system and the fec

iron-enterobactin operon by C1686_p2).

DISCUSSION

The comprehensive genomic analysis of available Kp CG14 and CG15 isolates

performed in this study (core and accessory genome, cps) allows clearly define two distinct

lineages that evolved separately in different settings. Till very recently, isolates classified as


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ST14 and ST15 were considered to be highly related since they differed only in a single point

mutation in allele infB according to the MLST scheme defined

(http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Analysis of large Kp MDR collections by

whole genome sequencing, cgMLST and SNPs-based phylogeny approaches defined them as

individual CGs (4,6). Nevertheless, the precise definition of the different CG14 and CG15

lineages, and their relative predominance and biological significance has never been explored

by high-resolution genomic analysis.

The SNP-based phylogeny of Kp CG14 and CG15 performed in this work allowed

the identification of distinct phylogenomic subgroups of CG14 (two) and CG15 (seven), each

one of them harbouring a specific cps locus. Some of these subgroups, such as CG14-K2 and

CG14-K16 or CG15-K24, CG15-K19 and CG15-KL112 seem to be well-established in

different geographic regions for several years, similarly to what happened with Kp CG258

(3,11). Those that were detected sporadically could be the result of single capsular switching

(CG15-K30, KL146 and wzi178) but they could also be underrepresented in the available

databases. Besides, the differences in GC content observed within the different regions of the

cps suggest different events of horizontal gene transfer within the variable region (5,11). Even

recognizing the potential for capsule diversification, probably related with it being the first

element of recognition in the host-pathogen interaction, it is of interest to highlight the

identification of apparently stable lineages carrying the same capsular type in large

geographical areas and/or periods of time, which suggests local evolution under different

selection pressures. The results of this work, along with those resulting from the studies on

CG258, highlight the interest of cps subtyping as marker to distinguish epidemiological

relevant Kp subgroups that can be exploited by a number of genotypic and phenotypic rapid

diagnostic tests (5,11,23,38).

The in-depth genomic analysis of Kp CG14 and CG15, reveals that the core genome

of these CG represents only approximately 60-70% of a typical Kp genome, pointing out the

impact of the accessory genome in the diversification and adaptation of these strains. Mobile

genetic elements (phages, plasmids, transposons among others), are major drivers of


253

acquisition and dissemination of accessory genes (39). The study of the mobilome, and

especially of the plasmidome, has been scarcely explored (4,10), mainly due to the few

efficient bioinformatics tools available for extraction of plasmid information from whole

genome sequencing data (26,27,40,41). Most of the studies address the plasmid diversity only

by inferring the incompatibility groups based on replicon typing using platforms such as

PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) (31), which fail to establish

whole plasmid entities. The combined use of two bioinformatics approaches, PLACNET and

PlasmidSPAdes, allowed us characterizing the plasmidome and further discriminating IncFIIK

and IncR plasmids with high level of resolution. Moreover, the comprehensive exercise of

comparative genomics of all CG14 and CG15 available genomes contributed to define these

plasmid families among MDR K. pneumoniae, which definitely seem to have different

evolutionary histories. To the best of our knowledge, this is the first work using these

approaches to reconstruct Kp plasmids from whole-genome data, and PLACNET seems to

work as well as for E. coli plasmids (26,42).

As expected, we describe a stormy flux of plasmids of different families among Kp

CG14 and CG15 populations, some of them also able to be transferred to other lineages of the

same or different species of Enterobacteriaceae eventually reflecting the wide open genome

of these MDR Kp strains. Furthermore, we identified 53 plasmids (reconstructed using

PLACNET) circulating in representative CG14 and CG15, significantly enlarging the number

of Kp plasmids deposited in public databases. Plasmids were overrepresented among these

CGs, such as MOBF12/IncF, MOBH11/HI1B-FIB and IncR comprising a diversity of ABR and

enriched in some cases with specific metal tolerance genes. Interestingly, we unveiled a

higher diversity of FIB replicons than that previously recognized, detected among different

MOB families (F12 and H11) and in phage-related plasmids, whose significance needs to be

further explored in larger Kp populations. The predominance of F plasmids in all species of

Enterobacteriaceae has not been explored despite their association with adaptive traits

(virulence, antibiotic and heavy metal resistance, ability to biofilm formation, amongst other

metabolic features). It is also of note the high number of heterogeneous small plasmids


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(ColE1-type) observed, harbouring or not ABR genes, and further studies are needed to

understand the role of these elements in the pangenome of MDR Kp strains (26,43).

Interestingly, we identified a plasmid harbouring the mrk operon (type 3 fimbriae, usually

located on the chromosome of Kp), whose plasmid location has recently been associated with

increased expression and biofilm formation (44).

Finally, Kp has an ubiquitous distribution, infecting and colonizing diverse animals

and sites of the human body and being disperse in the environment (water, soil and plants).

For this reason, a flexible armamentarium might facilitate adaptation to shifting conditions

and exploitation of new niches, as well as the exposure to a large pool of accessory DNA

might enhance the ability of these bacteria for horizontal gene transfer, favoring selection,

diversification and adaptation of Kp populations (39,45,46).

CONCLUSIONS

This study provides evidences that CG14 and CG15 predominant Kp lineages

carrying specific cps operons are circulating in different geographic regions for several years,

thus suggesting divergent evolution in different host backgrounds. Comprehensive analysis of

the plasmidome revealed turbulent flux of plasmids of disparate families containing a wide

number of adaptive traits (ABR/virulence/metal tolerance genes), frequently associated with

specific plasmids. MOBF12/IncF, MOBH11/IncHIB-FIB and IncR plasmids were pervasive and

greatly contribute to the CG14 and CG15 pangenome. These CGs present an open genome for

rapid and efficient exchange that might explain the fast adaptation of Kp under different

selective pressures.

MATERIAL AND METHODS

Bacterial Isolates and Sequencing

Seven MDR ST15 (4 wzi24, 1 wzi93, 1 wzi19, 1 wzi89) and two ST14 (1 wzi2, 1

wzi16) isolates recovered in Portugal between 2003 and 2013 were selected for whole-


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genome sequencing (WGS) (16,28,29). Epidemiological characteristics of the strains are

shown in Table 1.

Total DNA was extracted using QIAmp DNA Mini Kit (Quiagen) and concentration

measured using Nanodrop 2000 (Thermo Scientific) and Qubit Fluorometer (Life

Technologies). DNA libraries were prepared using the Nextera XT kit (Illumina, San Diego,

CA, USA) and 300 bp paired-end sequence reads with mean coverage of 100x were generated

on Miseq plataform (Illumina, San Diego, CA, USA). De novo assembly was performed with

SPAdes v3.9.0 using k-mers of 101, 111, 121 and 127 (47), and the quality of the assembly

was evaluated using QUAST software (48). Assembly statistics of the nine sequence datasets

analysed are available on Table S3. Genome annotation was performed with Prokka 1.12

(49).

In addition, a comparison of the nine genomes described in this paper with the 90 K.

pneumoniae genomes of CG15 (n=61; 2004-2015; America, Asia, Europe) and CG14 (n=29;

1986-2014; Africa, America, Asia, Europe, Oceania) available on NCBI and BIGSdb

databases (last update August 2016) was performed. Detailed information of all the isolates

analysed in this study is listed in Table 1.

Phylogenetic Analysis of CG14 and CG15 K. pneumoniae core genome

The core genome was defined as the collection of genes present in all CG14 and

CG15 analyzed, respectively, with 80% of identity and 80% of coverage. CD-HIT-EST was

used to cluster genes, and the core genome set was defined using home Perl scripts (26,50).

Core genes were concatenated and aligned using MUSCLE (51) and SNPs extraction was

performed with HarvestTools (http://harvest.readthedocs.io/en/latest/content/harvest-

tools.html). The results outputted in a SNP matrix were used to generate a maximum

likelihood phylogenetic tree with 100 bootstrap replicates under the general time-reversible

model with Gamma correction (GTR+G) in R-3.2.2 (phangorn package) (52).

Identification and comparison of cps locus


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First, we perform the assignment of wzi/wzc alleles and cps locus in all genome

sequences using BIGSbd

(http://bigsdb.pasteur.fr/perl/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef_public&page=s

equenceQuery) and Kaptive, respectively (5). Thereafter, we proceeded to the in silico

extraction of the cps locus from whole genome data in representative isolates (n=31) using

BLAST to identify sequence regions with homology to the flanking genes galF and ugd of

the cps locus. In some genomes, galF and ugd were not found in the same contig, and in that

case the nucleotide sequences from, and including, galF or ugd until the end of their

respective contigs were extracted and put together. To confirm the correct contiguity of the

contigs, sequences were mapped against the cps of the reference strain (according to wzi

allele or K locus identified by Kaptive) using ProgressiveMauve (Table S4) (53). Even with

this approach, some contigs could not be joined, and the annotated cps of these isolates reveal

contig breaks. Representative cps locus were annotated with Prokka and subsequent

comparison within and between CG14 and CG15 lineages (using the K-type reference strains

when available) was performed using BRIG and EasyFig (Table S4) (54,55).

Plasmid identification and analysis

A preliminary analysis of the plasmid content of CG14 and CG15 was obtained by

searching the diversity of plasmid replicons on the genomes using PlasmidFinder. Next, the

plasmid content of the nine Portuguese isolates sequenced was achieved using both Plasmid

Constellation Networks (PLACNET) and PlasmidSPAdes (26,27,56). PLACNET combines

three types of information contained in the contigs to identify plasmids from whole genome

sequences, namely, i) scaffold links and coverage in the WGS assembly; ii) plasmids proteins

as replication initiator proteins (RIP) and relaxases (REL); and iii) comparison to reference

sequences. The combination of these data it produces a graphical representation (network)

that can be pruned by the user to adjust the sequencing results (26). On the other hand,

PlasmidSPAdes use the coverage information to improve the assembly graph, and assumes


257

that contigs belonging to each genomic element (e.g. chromosome, plasmids) have an

identical coverage (27).

To further strengthen the plasmidome analysis of the CG14 and CG15, we proceeded

to the plasmid reconstruction of seven additional genomes belonging to CG14 and CG15

located at different positions in the phylogenetic tree and carrying different ABR genes.

Plasmids reconstructed here using PLACNET (n=16) and plasmids from closed genomes of

CG14 and CG15 (n=5) available on NCBI database, as well as reference plasmids (Table S4),

were used to construct a hierarchical clustering dendrogram of plasmid proteomes. Plasmid

Neighbour-Joining dendrograms were constructed using CD-HIT to cluster references and

query plasmids (60% sequence identity and 80% coverage) (50). Based on the protein clusters

defined, a presence/absence table was created to represent the protein profile of each plasmid

(26,57). The Euclidean formula was used to calculate the distance matrix between plasmid

protein profiles using vegan package on R 3.2.2 (58), and subsequently bootstrap confidence

values were calculated and the Neighbour-Joining dendrogram was constructed with APE

package on R 3.2.2 (59). Representative query and reference plasmids appearing in the same

branch were compared using BRIG (54). Plasmids were classified according to MOB families

as described by Alvarado et al (60) and Inc (replicons) families using PlasmidFinder (31).

Gene Content Analysis

Genes presumptively associated with virulence such as siderophores (yersianabactin,

aerobactin, colibactin, salmochelin), iron uptake systems and regulators (kfu operon and

kvgAS operon, respectively), bacteriocins (microcin cluster), regulators of mucoid phenotype

(rmpA and rmpA2), allantoinase cluster and adhesins (mrk cluster) and metal tolerance genes

[copper (pco cluster), arsenic (ars operon), silver/copper (sil cluster) and tellurite (ter

operon)] were searched using BIGSbd database

(http://bigsdb.pasteur.fr/perl/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef_public&page=s

equenceQuery). Mer operon (conferring tolerance to mercury) was searched using BLAST.

Antimicrobial resistance (ABR) genes were searched using ResFinder database


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(https://cge.cbs.dtu.dk/services/ResFinder/). These data were used to construct the heat maps

on R 3.2.2.




UID/MULTI/04378/2013 Ângela Novais was supported by a fellowship from FCT through

Programa Operacional Capital Humano (POCH) (and SFRH/BPD/104927/2014,

respectively). Carla Rodrigues was supported by a fellowship from FCT through POCH

(grant number SFRH/BD/84341/2012) and a FEMS Research Grant (FEMS-RG-2014-0089).

Competing interests: The authors have declared that no competing interests exist.

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fect

ion

PRJN

A25

2925

A

SM15

9698

(KPO

II-7

) 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

H

uman

ur

ine

Infe

ctio

n PR

JNA

2529

25

ASM

1596

99 (K

POII

-10)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2013

H

uman

sp

utum

In

fect

ion

PRJN

A25

2925

A

SM15

9709

(KPO

II-1

) 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

H

uman

re

ctal

sw

ab

Col

oniz

atio

n PR

JNA

2529

25

ASM

1597

11 (K

POII

-6)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

H

uman

ur

ine

Infe

ctio

n PR

JNA

2529

25

ASM

1597

16 (K

POII

-9)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

H

uman

re

ctal

sw

ab

Col

oniz

atio

n PR

JNA

2529

25

ASM

1597

17 (K

PEII

-1)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

E

nvir

onm

ent

Bed

-

PRJN

A25

2925

A

SM15

9719

(KPE

II-2

) 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

E

nvir

onm

ent

Toi

let c

hair

-

PRJN

A25

2925

A

SM15

9722

(KPE

II-3

) 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2013

E

nvir

onm

ent

Toi

let c

hair

-

PRJN

A25

2925

C

LC

(KP-

10)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

H

uman

re

ctal

sw

ab

Col

oniz

atio

n PR

JNA

2529

25

ASM

1597

14 (K

POII

-8)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

ethe

rlan

ds

2012

H

uman

re

ctal

sw

ab

Col

oniz

atio

n PR

JNA

2529

25

1809

0_8_

59 (P

B46

6)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

T

haila

nd

2015

H

uman

ur

ine

NA

PR

JEB

1140

3 12

082_

2_22

(e16

02)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

nite

d K

ingd

om

2007

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

12

082_

5_17

(k17

86)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

nite

d K

ingd

om

2008

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

B

IDM

C_3

3B

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

SA

2012

H

uman

sp

utum

N

A

PRJN

A20

2045

12

045_

7_41

(k23

25)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

nite

d K

ingd

om

2010

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

19

PV

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

It

aly

NA

H

uman

N

A

hum

an

PRJE

B65

43

44*

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

Po

rtug

al

2013

H

uman

ur

ine

NA

C16

86*

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

Po

rtug

al

2012

H

uman

ur

ine

Infe

ctio

n (U

TI)

C16

93*

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

Po

rtug

al

2012

H

uman

ur

ine

Infe

ctio

n (U

TI)

MG

H63

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

SA

2013

H

uman

bi

liary

flui

d N

A

PRJN

A23

4262

M

GH

65

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

SA

2013

H

uman

bi

liary

flui

d N

A

PRJN

A23

4264

M

GH

75

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

SA

2013

H

uman

ab

dom

inal

w

ound

In

fect

ion

PRJN

A23

4115

MG

H11

1 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

A

2014

H

uman

N

A

NA

PR

JNA

2718

99

MG

H11

7 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

A

2014

H

uman

N

A

NA

PR

JNA

2718

99

MG

H11

5 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

A

2014

H

uman

N

A

NA

PR

JNA

2718

99

MG

H81

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

A

2014

H

uman

N

A

NA

PR

JNA

2718

99

U_1

2567

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

L

aos

2008

H

uman

ur

ine

Infe

ctio

n (U

TI)

E

RS0

1187

8

1198

3_8_

85 (k

1806

) 15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

nite

d K

ingd

om

2008

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

12

082_

4_95

(k14

36)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

nite

d K

ingd

om

2006

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

12

045_

7_44

(k23

29)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

N

A

2010

H

uman

N

A

NA

PR

JEB

5065

12

082_

2_21

(k23

25)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

nite

d K

ingd

om

2009

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

A

SM76

463

(BA

MC

07-

18)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

U

SA

2012

H

uman

w

ound

hu

man

PR

JNA

2472

86


267

* Is

olat

es s

eque

nced

in th

is s

tudy

; Sha

ded

grey

cor

resp

ond

to c

ompl

ete

geno

mes

; NA

, dat

a no

t ava

ilabl

e. 1 w

zi a

nd w

zc d

eter

min

ed in

sili

co a

ccor

ding

with

Bris

se e

t al.

and

Pan

et a

l., r

espe

ctiv

ely,

usi

ng B

IGSb

d da

taba

se (

http

://bi

gsdb

.pas

teur

.fr/p

erl/b

igsd

b/bi

gsdb

.pl?

db=p

ubm

lst_

kleb

siel

la_s

eqde

f_pu

blic

&pa

ge=s

eque

nceQ

uery

); 2

K lo

ci d

eter

min

ed a

ccor

ding

to

Wyr

es e

t al

. (5

) us

ing

Kap

tive

softw

are.

3 Sing

le L

ocus

V

aria

nt (S

LV) o

f ST1

4.

Stra

in

ST

wzi

/K-t

ype1

wzc

/K-t

ype1

Kap

tive2

Cou

ntry

C

olle

ctio

n da

te

Hos

t Sa

mpl

e

Infe

ctio

n st

atus

A

cces

sion

1208

2_5_

11 (k

1679

) 15

w

zi24

/K24

w

zc25

:8/K

24

KL2

4 U

nite

d K

ingd

om

2007

H

uman

bl

ood

hum

an

(infe

ctio

n)

PRJE

B50

65

1809

0_8_

75 (W

2-4-

ER

G)

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

Thai

land

20

15

Hum

an

NA

N

A

PRJE

B11

403

UC

I96

15

wzi

24/K

24

wzc

25:8

/K24

K

L24

NA

20

14

Hum

an

NA

N

A

PRJN

A27

1899

H

1119

15

w

zi24

/K24

w

zc39

:21/

K39

K

L39

Portu

gal

2010

H

uman

ur

ine

Infe

ctio

n (U

TI)

1204

5_6_

92 (k

981)

15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Uni

ted

Kin

gdom

20

04

Hum

an

bloo

d In

fect

ion

PRJE

B50

65

1208

2_4_

89 (k

1234

) 15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Uni

ted

Kin

gdom

20

05

Hum

an

bloo

d In

fect

ion

PRJE

B50

65

1208

2_4_

90 (k

1238

) 15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Uni

ted

Kin

gdom

20

05

Hum

an

bloo

d In

fect

ion

PRJE

B50

65

DU

3306

2_05

15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Sing

apor

e 20

05

Hum

an

urin

e C

olon

izat

ion

ERS0

1190

9 A

SM76

461

(PM

K1)

15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Nep

al

2011

H

uman

bl

ood

Infe

ctio

n PR

JNA

2533

00

ASM

1640

18

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

Le

bano

n 20

15

Hum

an

urin

e In

fect

ion

(UTI

) PR

JNA

3108

37

ST15

(ST

15 N

DM

-1)

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

N

epal

20

12

Hum

an

trach

eal

aspi

ratio

n In

fect

ion

PRJE

B80

09

ASM

1663

19 (B

R)

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

C

hina

20

15

Hum

an

NA

N

A

PRJN

A32

3746

A

SM15

9701

(KP-

33P)

15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Net

herla

nds

20

10

Hum

an

bloo

d In

fect

ion

PRJN

A25

2925

12

082_

2_3

(k13

10)

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

U

nite

d K

ingd

om

2006

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

12

082_

4_86

(k10

56)

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

U

nite

d K

ingd

om

2005

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

12

045_

7_22

(k21

41)

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

U

nite

d K

ingd

om

2009

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

C

1699

* 15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Portu

gal

2012

H

uman

ur

ine

Infe

ctio

n (U

TI)

LF_

7160

15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Laos

20

07

Hum

an

pleu

ral f

luid

In

fect

ion

ER

S011

869

18PV

15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

Italy

N

A

Hum

an

NA

N

A

PRJE

B65

43

99SG

R

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

Ita

ly

NA

H

uman

N

A

NA

PR

JEB

6543

A

SM78

800

(101

7129

) 15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

USA

20

07

Hum

an

wou

nd

Infe

ctio

n PR

JNA

2612

39

gkp3

2 (U

HK

PC17

9)

15

wzi

93/K

60

wzc

923:

66/K

- K

L112

U

SA

2009

H

uman

bl

ood

Infe

ctio

n PR

JNA

1873

10

gkp3

1 (U

HK

PC57

) 15

w

zi93

/K60

w

zc92

3:66

/K-

KL1

12

USA

20

08

Hum

an

cath

eter

In

fect

ion

PRJN

A18

7309

K

47*

15

wzi

89/K

- -

KL1

10

Portu

gal

2012

H

uman

ur

ine

Infe

ctio

n

2033

15

w

zi19

/K19

w

zc20

:74/

K19

K

L19

Indo

nesi

a 20

05

Hum

an

urin

e N

A

ERS0

1205

3 A

SM16

5369

(IN

Sali3

90)

15

wzi

19/K

19

wzc

20:7

4/K

19

KL1

9 Po

rtuga

l 20

13

Envi

ronm

ent

vege

tal

- PR

JNA

3119

32

C16

94*

15

wzi

19/K

19

wzc

20:7

4/K

19

KL1

9 Po

rtuga

l 20

12

Hum

an

urin

e In

fect

ion

(UTI

)

Kp3

03K

1 15

w

zi19

/K19

w

zc20

:74/

K19

K

L19

Taiw

an

2010

H

uman

re

ctal

sw

ab

Col

oniz

atio

n PR

JNA

2140

29

CH

S112

15

w

zi11

8/K

- -

KL1

46

USA

20

14

Hum

an

NA

N

A

PRJN

A27

1899

M

GH

101

15

wzi

118/

K-

- K

L146

U

SA

2014

H

uman

N

A

NA

PR

JNA

2718

99

1208

2_5_

23 (k

2135

) 15

w

zi17

8/K

- -

- U

nite

d K

ingd

om

2009

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065

12

045_

7_79

(k27

08)

15

wzi

274/

K-

wzc

903:

18/K

30

KL3

0 U

nite

d K

ingd

om

2011

H

uman

bl

ood

Infe

ctio

n PR

JEB

5065


268

Figure 1. The tree is based on a 3.7 Mbp core genome (3,902 core genes: 80% identity and 80% coverage) and 100

bootstrap replicates.

ASM130717

ASM73325

ASM131686

CHS175

ASM157450

ASM146316

12082 2 14

K102An 1

AJ094 1

AJ097 1

ASM170157

18090 8 51

ASM152189

ASM146347

CHS66

WGLW2

MTE1

MGH124

12045 7 16

BIDMC11

AFX 1

H49

CHS140

UCI4

ASM148305

AJ289 1

AJ290 1

MGH46

H1122

12045 5 67

12082 4 85

100

100

82

10094

100

100

84100

100

100

100

100

100

100

98

82

100

100

100

91

82

96

90

0.10

CG14 - wzi2/K2

CG14-wzi16/K16

~370 SNPs (100 SNPs/Mbp)




269

Strain ST wzi/K-type wzc/K-type Kaptive Continent Collection Data Host PlasmidFinder (Inc) a

ASM130717 (KP617) 14 wzi2/K2 wzc2:14/K2 KL2 Asia 2013 Human HI1B-FIB(pNDM-MAR), ColKP3

ASM73325 (PittNDM01) 14 wzi2/K2 wzc2:14/K2 KL2 America 2013 Human HI1B-FIB(pNDM-MAR),FIB(pKPN3), R-FIA(HI1), ColKP3

ASM131686 14 wzi2/K2 wzc2:14/K2 KL2 Africa NA Human HI1B-FIB(pNDM-MAR),FIB(pKPN3), R, ColKP3, ColE

CHS175 14 wzi2/K2 wzc2:14/K2 KL2 America 2014 Human HI1B, FIB(pNDM-MAR), FIIK5, FIB(pKPN3), R, FIA(HI1)

ASM157450 (U41) 14 wzi2/K2 wzc2:14/K2 KL2 Asia 2015 Human HI1B, FIB(pNDM-MAR), FIIK5, FIB(pKPN3), R, FIA(HI1)

ASM146316 (U25) 14 wzi2/K2 wzc2:14/K2 KL2 Asia 2010 Human -

12082_2_14 (k2671) 14 wzi2/K2 wzc2:14/K2 KL2 Europe 2011 Human FII(pC15-1a), R, FIA(HI1), FIB(pKPN3)

K102An 14 wzi2/K2 wzc2:14/K2 KL2 Asia 2004 Human FIIK8, FIB(pKPN3), R

AJ094 14 wzi2/K2 wzc2:14/K2 KL2 Oceania 2002 Human FIIK1, FIB(pKPN3)

AJ097 14 wzi2/K2 wzc2:14/K2 KL2 Oceania 2002 Human FIIK1, FIB(pKPN3)

ASM170157 2316 1 wzi2/K2 wzc2:14/K2 KL2 Asia 2012 Human HI1B, FIB(pNDM-MAR)

18090_8_51 (PB256) 14 wzi2/K2 wzc2:14/K2 KL2 Asia 2015 Human FIIK2, FIIK9, FIB(pKPN3)

ASM152189 (NUHL24835) 14 wzi2/K2 wzc2:14/K2 KL2 Asia 2014 Human FIIK2, X3

ASM146347 14 wzi2/K2 wzc2:14/K2 KL2 Europe 2014 Human FIIK5-FIB(pKPN3), FIB(pKPHS1), L

CHS66 14 wzi2/K2 wzc2:14/K2 KL2 America 2013 Human -

WGLW2 14 wzi2/K2 wzc2:14/K2 KL2 America NA Human FIB(pKPN3), FIB(pNDM-MAR)

MTE1 (JHCK1) 14 wzi2/K2 wzc2:14/K2 KL2 America 1986 Human HI1B, FIB(pNDM-MAR), FIIK9, FIB(pKPN3), FIB(pKpQIL)

MGH124 14 wzi2/K2 wzc2:14/K2 KL2 America 2014 Human FIB(pKPN3)

12045_7_16 (k1970) 14 wzi2/K2 wzc2:14/K2 KL2 Europe 2008 Human FIIK3, FIB(pKPN3), FIA

BIDMC11 14 wzi2/K2 wzc2:14/K2 KL2 America 2008 Human FIB(pKPN3), N(ST6)

AFX (SB2390) 14 wzi2/K2 wzc2:14/K2 KL2 America 2002 Human FIB(pKPN3), FIB(pKPHS1), R

H49* 14 wzi2/K2 wzc2:14/K2 KL2 Europe 2003 Human A/C2 (ST3), FIB(pKPN3)

CHS140 14 wzi16/K16 wzc16:85/K16 KL16 America 2014 Human FIB(pKPN3)

UCI4 14 wzi16/K16 wzc16:85/K16 KL16 America NA Human FIB(pKPN3)

ASM148305 (K1) 14 wzi16/K16 wzc16:85/K16 KL16 America 2011 Human FIB(pKPN3)

AJ289 14 wzi16/K16 wzc16:85/K16 KL16 Oceania 2002 Human A/C2 (ST1), FIIK5, R, FIB(pKPHS1)

AJ290 14 wzi16/K16 wzc16:85/K16 KL16 Oceania 2002 Human A/C2 (ST1), FIB(pKPHS1)

MHG46 14 wzi16/K16 wzc16:85/K16 KL16 America 2012 Human FIIK3, FIB(pKPN3), FIA

H1122* 14 wzi16/K16 wzc16:85/K16 KL16 Europe 2010 Human FIIK1-FIB(pKPN3), ColE (n=2)

12045_6_67 (k787) 14 wzi16/K16 wzc16:85/K16 KL16 Europe 2004 Human FIIK1, FIB(pKPN3), FIB(pKPHS1)

12082_4_85 (k1037) 14 wzi16/K16 wzc16:85/K16 KL16 Europe 2005 Human FIIK1, FIB(pKPN3), FIB(pKPHS1)

C1686* 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2012 Human HI1B-FIB(pNDM-MAR), FIIK5-FIB(pKPN3), IncFII_repA_superfamily

C1693* 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2012 Human FIIK5-FIB(pKPN3)

44* 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2013 Human FIIK5-FIB(pKPN3), L, X4

12045_7_41 (k2325) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2010 Human FIIK5, FIB(pKPN3), Col(pVC)

19PV 15 wzi24/K24 wzc25:8/K24 KL24 Europe NA Human FIIK5, FIB(pKPN3), FIIK7

BIDMC_33B 15 wzi24/K24 wzc25:8/K24 KL24 America 2012 Human FIIK5, FIB(pKPN3)

12082_2_22 (e1602) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2007 Human L, R, ColRNAI, Col(pVC)

12082_5_17 (k1786) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2008 Human R, Col(pVC)

U_12567 15 wzi24/K24 wzc25:8/K24 KL24 Asia 2008 Human FIIK1, FIB(pKPN3), R, Col(pVC), ColRNAI

MGH111 15 wzi24/K24 wzc25:8/K24 KL24 America 2014 Human HI1B(pNDM-MAR), A/C2 (ST3), FIIK1, R, Col(pVC), ColE

MGH63 15 wzi24/K24 wzc25:8/K24 KL24 America 2013 Human HI1B(pNDM-MAR), A/C2 (ST3)

MGH65 15 wzi24/K24 wzc25:8/K24 KL24 America 2013 Human HI1B(pNDM-MAR), A/C2 (ST3), FIIK1, R, Col(pVC)

MGH75 15 wzi24/K24 wzc25:8/K24 KL24 America 2013 Human HI1B(pNDM-MAR), A/C2 (ST3), FIIK1

MGH117 15 wzi24/K24 wzc25:8/K24 KL24 America 2014 Human HI1B(pNDM-MAR), A/C2 (ST3)

MGH115 15 wzi24/K24 wzc25:8/K24 KL24 America 2014 Human HI1B(pNDM-MAR), A/C2 (ST3), Col(pVC)

MGH81 15 wzi24/K24 wzc25:8/K24 KL24 America 2014 Human HI1B(pNDM-MAR), A/C2 (ST3), FIIK1, R

CLC (KP-10) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2012 Human FIIK1, FIB(pKPN3)

ASM159722 (KPEII-3) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2013 Environment FIIK1, FIB(pKPN3)


ASM159714 (KPOII-8) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2012 Human FIIK1, FIB(pKPN3)



ASM159690 (KPOII-4) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2012 Human FIB(pKPN3)




ASM159691 (KPOII-2) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2012 Human FIB(pKPN3)




18090_8_59 (PB466) 15 wzi24/K24 wzc25:8/K24 KL24 Asia 2015 Human FIIK2, FIB(pKPHS1), ColRNAI

11983_8_85 (k1806) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2008 Human FIIK9, FIB(pKPN3), M, R, FIA(HI1), Col(pVC)

12082_4_95 (k1436) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2006 Human FIIK9, FIB(pKPN3), R, Col(pVC)

12045_7_44 (k2329) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2010 Human FIIK5, FIB(pKPN3), FIB(pKpQIL ), R, FIA(HI1)

12082_2_21 (k2325) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2009 Human FIIK5, FIB(pKPN3), R, Col(pVC)

ASM76463 (BAMC 07-18) 15 wzi24/K24 wzc25:8/K24 KL24 America 2012 Human R

12082_5_11 (k1679) 15 wzi24/K24 wzc25:8/K24 KL24 Europe 2007 Human FIIK9, FIB(pKPN3)

18090_8_75 (W2-4-ERG) 15 wzi24/K24 wzc25:8/K24 KL24 Asia 2015 Human R

UCI96 15 wzi24/K24 wzc25:8/K24 KL24 America 2014 Human FIB(pKPN3)

H1119 15 wzi24/K24 wzc39:21/K39 KL39 Europe 2010 Human FIIK1-FIB(pKPN3), FII (pC15-a), R, Col(pVC), ColE

12082_4_89 (k1234) 15 wzi93/K60 wzc923:66/K- KL112 Europe 2005 Human R, FIA(HI1), Col(pVC)



DU33062_05 15 wzi93/K60 wzc923:66/K- KL112 Asia 2005 Human R, FIA(HI1), FIIK1, FIB(pKPN3), FIB(pKPHS1), Col(pVC)

ASM164018 15 wzi93/K60 wzc923:66/K- KL112 Asia 2015 Human HI1B, FIB(pNDM-MAR), FIIK1, FIB(pKPN3), FIB(pKPHS1)

ASM76461 (PMK1) 15 wzi93/K60 wzc923:66/K- KL112 Asia 2011 Human HI1B-FIB(pNDM-MAR), FIIK1-FIB(pKPN3), FIB(pKPHS1), IncFII_repA_superfamily

ST15 (ST15 NDM-1) 15 wzi93/K60 wzc923:66/K- KL112 Asia 2012 Human HI1B-FIB(pNDM-MAR), FIIK1-FIB(pKPN3), FIB(pKPHS1), FII(pC15-1a), ColE (n=5)

ASM166319 (BR) 15 wzi93/K60 wzc923:66/K- KL112 Asia 2015 Human R

ASM159701 (KP-33P) 15 wzi93/K60 wzc923:66/K- KL112 Europe 2010 Human R, FIA(HI1), Col(pVC)


12045_7_22 (k2141) 15 wzi93/K60 wzc923:66/K- KL112 Europe 2009 Human R, FIIK9, FIB(pKPN3)

C1699* 15 wzi93/K60 wzc923:66/K- KL112 Europe 2012 Human R-FIA(HI1), FIIK9-FIB(pKPN3)

LF_7160 15 wzi93/K60 wzc923:66/K- KL112 Asia 2007 Human R, FIA(HI1), FIB(pKPN3), Col(pVC), ColRNAI

12082_4_86 (k1056) 15 wzi93/K60 wzc923:66/K- KL112 Europe 2005 Human R, FIA (HI1), FIIK9, FIB(pKPN3), FIB(pKPHS1), Col(pVC)

18PV 15 wzi93/K60 wzc923:66/K- KL112 Europe NA Human R, FIA (HI1), FIIK9, FIB(pKPN3)

99SGR 15 wzi93/K60 wzc923:66/K- KL112 Europe NA Human R, FIA (HI1), FIIK9, FIB(pKPN3)

ASM78800 (1017129) 15 wzi93/K60 wzc923:66/K- KL112 America 2007 Human R, FIA (HI1), FIIK9, FIB(pKPN3), Col(pVC)

gkp31 (UHKPC57) 15 wzi93/K60 wzc923:66/K- KL112 America 2008 Human R, FIA(HI1), FIB(pKPN3), N, Col(pVC)

gkp32 (UHKPC179) 15 wzi93/K60 wzc923:66/K- KL112 America 2009 Human R, FIB(pKPN3), Col(pVC)

CHS112 15 wzi118/- - KL146 America 2014 Human R, FIA (HI1)

MGH101 15 wzi118/- - KL146 America 2014 Human R, FIA (HI1),FIIK9, FIB(pKpQIL)

12045_7_79 (k2708) 15 wzi274/- wzc903:18/K30 KL30 Europe 2011 Human R, FIIK5, FIB(pKPN3), Col(pVC)

12082_5_23 (k2135) 15 wzi178/- wzc931:73 - Europe 2009 Human R, FIA (HI1), Col(pVC)

2033 15 wzi19/K19 wzc20:74/K19 KL19 Asia 2005 Human FII(pC15-1a), Col(pVC)

ASM165369 (INSali390) 15 wzi19/K19 wzc20:74/K19 KL19 Europe 2013 Environment FIIK1, FIB(pKPN3), R, FIB(pKPHS1), Q2

C1694* 15 wzi19/K19 wzc20:74/K19 KL19 Europe 2012 Human FIIK5-FIB(pKPN3), R-FIA(HI1), ColE

Kp303K1 15 wzi19/K19 wzc20:74/K19 KL19 Asia 2010 Human FIIK7, FIB(pKPN3), R, FIA(HI1), FIB(pKpQIL)

K47* 15 wzi89/- - KL110 Europe 2012 Human HI2, Col(pVC)

Yers

inia

bact

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ster

Col

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TX-M

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aGE

S-1

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C-2

blaK

PC

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C-4

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A-9

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HV-

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sul2

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12045_6_9212082_4_8912082_4_90DU33062_05ASM76461ASM164018ST15ASM166319ASM15970112082_2_312082_4_8612045_7_22C1699LF_716018PV99SGRASM78800gkp32gkp31CHS112MGH101K472033ASM165369C1694Kp303K112082_5_2312045_7_79

Virulencegenes AcquiredResistancegenesMetal

ToleranceGenes

CoreResistancegene

s

Beta-lactams

* * *

FQs Aminoglycosides Folatepathwayinhibitors

Phenicols

Macrolid

es

Tetracyclin

es

Rifampicin

ST – Sequence type; NA– Not available data; FQs-Fluoroquinolones; *Isolates sequenced in this study; Shaded grey correspond to complete genomes and plasmids while greenish shade correspond to reconstructed plasmids using PLACNET. aReplicons detected in silico by PlasmidFinder platform (https://cge.cbs.dtu.dk/services/PlasmidFinder/). The allelic variant of FIIK plasmids and the ST of A/C2 and N plasmids (pMLST) was identified. 1Single Locus Variant of ST14.

* *

Figure 2. Heat map of Kp CG14 and CG15 genomes showing the content on virulence, ABR and metal tolerance genes.


270

CG15 – wzi24/K24

CG15 – wzi93/KL112

CG15 – wzi118/KL146

CG15 – wzi19/K19

CG15 – wzi89/KL110

CG15 – wzi274/KL30 CG15 – wzi178/-








~940 SNPs (303 SNPs/Mbp) ~1020 SNPs

(329 SNPs/Mbp)


*

Figure 3. The tree is based on a 3.1 Mbp core genome (3,332 core genes: 80% identity and 80% coverage) and 100

bootstrap replicates.


271

A) 

H11

22/C

G14

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16

2749

7 bp

B) H

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288

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14 - wzi

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488

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24

2492

5 bp

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86/C

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24

2573

3 bp

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H11

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G15

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24/K

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2705

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39:2

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2372

1 bp

E

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99/C

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L11

2 24

370

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Ei)

BR

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15 - wzi

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L11

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146

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PM

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CG

15 - wzi

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921

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927

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2627

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H

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G15

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KL

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2637

1 bp

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1694

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15 - wzi

19/K

19

2497

8 bp

J) 1

2082

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3/C

G15

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178

2712

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unca

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Figu

re 4

. Str

uctu

res

of c

ps lo

cus

iden

tifie

d in

Kp

CG

14 a

nd C

G15

.


272

H49 chromosome 14 K2 5,307,286 - NUHL24835chromosome 14 K2 5,387,996 - KP617 chromosome 14 K2 5,416,282 - PittNDM01 chromosome 14 K2 5,348,284 - ASM131686 chromosome 14 K2 3,375,157 - ASM146347 chromosome 14 K2 5,239,463 - H1122 chromosome 14 K16 5,310,697 - UCI4 chromosome 14 K16 5,306,876 - 44 chromosome 15 K24 5,452,413 - C1686 chromosome 15 K24 5,451,000 - C1693 chromosome 15 K24 5,301,118 - MGH111 chromosome 15 K24 5,337,343 - 12082_2_21 chromosome 15 K24 5,221,397 - UCI96 chromosome 15 K24 5,223,844 - H1119 chromosome 15 K24 5,262,486 - PMK1 chromosome 15 KL112 5,317,001 - BR chromosome 15 KL112 5,344,467 - ST15 chromosome 15 KL112 5,269,938 - C1699 chromosome 15 KL112 5,301,437 - K47 chromosome 15 KL112 5,242,008 - C1694 chromosome 15 KL112 5,169,564 - 44_p1 15 K24 31,877 X4/MOBP3 NUHL24835_p2 14 K2 46,161 X3/MOBP3 C1686_p2 15 K24 55,328 phage-like 44_p2 15 K24 62,852 L/MOBP13 ASM146347_p1 14 K2 64,518 L/MOBP13 ST15_p6 15 KL112 62,94 FII(pC15-a)/MOBF12 12082_2_21_p4 15 K24 64,671 FII(pC15-a)/MOBF12 pPMK1-C 15 KL112 69,947 IncFII_repA_superfamily/- MGH111_p3 15 K24 95,858 FIB/MOBF12 MGH111_p4 15 K24 185,508 FIIK1-InHI1B-R-N/- C1686_p4 15 K24 304,754 HI1B-FIB(pNDM-MAR)/MOBH11 PittNDM01_plasmid1 14 K2 283,371 HI1B-FIB(pNDM-MAR)/MOBH11 KP617-plasmid1 14 K2 273,628 HI1B-FIB(pNDM-MAR)/MOBH11 pPMK1-NDM 15 KL112 304,526 HI1B-FIB(pNDM-MAR)/MOBH11 ST15_p9 15 KL112 292,517 HI1B-FIB(pNDM-MAR)/MOBH11 ASM131686_p5 14 K2 261,264 HI1B-FIB(pNDM-MAR)/MOBH11 pK47_2 15 89/- 484,666 HI2/MOBH11 MGH111_p5 15 K24 188,858 A/C2/MOBH121 pH49_1 14 K2 166,342 A/C2/MOBH121 H49_p2 14 K2 119,803 FIB(Kpn3)/- pPMK1-A 15 KL112 187,571 FIIK1-FIB(Kpn3)/MOBF12 ST15_p8 15 KL112 224,041 FIIK1-FIB(Kpn3)/MOBF12 C1694_p3 15 K19 211,512 FIIK5-FIB(Kpn3)/MOBF12 H1122_p4 14 K16 226,269 FIIK1-FIB(Kpn3)/MOBF12 H1119_p3 15 K24 210,731 FIIK1-FIB(Kpn3)/MOBF12 12082_2_21_p5 15 K24 209,921 FIIK1-FIB(Kpn3)/MOBF12 UCI96_p1 15 K24 196,922 FIB(Kpn3)/MOBF12 UCI4_p1 14 K16 185,403 FIB(Kpn3)/MOBF12 C1699_p2 15 KL112 198,629 FIIK9-FIB(Kpn3)/MOBF12 ASM147347_p3 14 K2 274,757 FIIK5-FIB(Kpn3)/MOBF12 C1686_p3 15 K24 204,27 FIIK5-FIB(Kpn3)/MOBF12 44_p3 15 K24 201,996 FIIK5-FIB(Kpn3)/MOBF12 C1693_p1 15 K24 212,689 FIIK5-FIB(Kpn3)/MOBF12 NUHL24835_p1 14 K2 101,03 FIIK2/MOBF12 ASM131686_p4 14 K2 95,773 FIB (Kpn3)/MOBF12 PittNDM01_plasmid2 14 K2 103,694 FIB (Kpn3)/MOBF12 C1686_p1 15 K24 42,543 IncFII_repA_superfamily/- ASM147347_p2 14 K2 110,959 FIB(pKPHS1)/- [phage-like] ST15_p7 15 KL112 111,769 FIB(pKPHS1)/- [phage-like] pPMK1-B 15 KL112 111,693 FIB(pKPHS1)/- [phage-like] pH1122_3 14 K16 39,189 FIA(HI1)/- 12082_2_21_p3 15 K24 34,756 R-ΔN/- pH1119_p2 15 K24 45,342 R-ΔN/- pWSZBR 15 KL112 76,251 R-ΔN/- C1694_p2 15 K19 73,693 R-FIA(HI1)/- ASM131686_p3 14 K2 45,482 R/- PittNDM01_plasmid3 14 K2 70,814 R-FIA(HI1)/- C1699_p1 15 KL112 69,326 R-FIA-ΔN/- ASM131686_p2 14 K2 7,85 ColKP3 /MOBP5 PittNDM01_plasmid4 14 K2 6,141 ColKP3/MOBP5 KP617_p2 14 K2 6,141 ColKP3/MOBP5 ST15_p5 15 KL112 5,737 ColE1-like/MOBP5 ST15_p4 15 KL112 5,332 ColE1-like/MOBP5 pH1122_2 14 K16 3,671 ColE1-like/MOBP5 ST15_p2 15 KL112 3,753 ColE1-like/MOBP5 ST15_p1 15 KL112 3,703 ColE1-like/MOBP5 MGH111_p1 15 K24 2,155 ColE1-like/MOBP5 12082_2_21_p2 15 K24 2,732 ColE1-like/MOBP5 ASM131686_p1 14 K2 2,655 ColE1-like/MOBP5 C1694_p1 15 K19 2,811 ColE1-like/MOBP5 H1122_p1 14 K16 2,365 ColE1-like/MOBP5 K47_p1 15 KL110 2,564 Col(pVC)/MOBP5 H1119_p1 15 K24 3,357 Col(pVC)/MOBP5 MGH111_p2 15 K24 2,155 Col(pVC)/MOBP5 12082_2_21_p1 15 K24 2,275 Col(pVC)/MOBP5 ST15_p3 15 KL112 3,867 ColE1-like/MOBP5

Yers

inia

bact

in c

lust

erAe

roba

ctin

clu

ster

Col

ibac

tin c

lust

erSa

lmoc

helin

clu

ster

Mic

roci

n cl

uste

rkf

u op

eron

kvgA

Grm

pArm

pA2

Alla

ntoi

nase

clu

ster

mrk

clu

ster

fosA

oqxAB

bla SH

Vbl

a CTX

-M-1

5bl

a KPC

-2bl

a ND

M-1

bla N

DM

-5bl

a OXA

-1bl

a OXA

-9bl

a OXA

-10

bla O

XA-1

7bl

a OXA

-48

bla O

XA-1

62bl

a OXA

-232

bla SH

V-2

bla SH

V-5

bla SH

V-11

bla SH

V-12

bla SH

V-10

6bl

a VIM

-34

bla TE

M-1

bla TE

M-2

4qn

rA7

qnrB

1qn

rB4

arm

Aaa

c3-Ia

aac3

-IIa

aac3

-IId

aacA

4aac6’-I

b.cr

*aa

c6-II

caa

dA1

aadA

2aa

dA15

aadA

16ap

h3-Ia

aph3

-Icap

h3-V

Iaap

h3-X

Vst

rAst

rBsu

l1su

l2df

rA1

dfrA

8df

rA12

dfrA

14df

rA27

cmlA

1flo

Rca

tA1

catB

2ca

tB3

msr

Em

phA

mph

Eer

eAer

mB

tetA

tetD

ARR

-2AR

R-3

pco

clus

ter

sil c

lust

ermer

ope

ron

ars

oper

onte

r ope

ron

Virulencegenes AcquiredResistancegenes

Metal

Tolerance

Genes

CoreResistance

genes

Beta-lactams

Fluoroquinolones

Aminoglycosides

Folate

pathway

inhibitors

Tetracyclines

Phenicols

Macrolides

Rifampicin

* * *Strain ST K

locus Size (kb)

Plasmid Family [Inc(replicon)/MOB]

Figure 5. Heat map of Kp CG14 and CG15 genomes showing the content on virulence, ABR and metal tolerance genes in the chromosome and in the plasmids..


273

ST15

p8

C169

9 p1

1208

2 2

21 p

1

PittN

DM01

pla

smid

2

UCI4

p1

H49

p1

C169

4 p3

1208

2 2

21 p

2

H111

9 p1

UCI9

6 p1

KP61

7 p2

1208

2 2

21 p

5

pPM

K1−N

DM

MG

H111

p5

pPM

K1−A

C168

6 p3

ASM

1316

86 p

5

ST15

p3

pPM

K1−B

ASM

1463

47 p

3

H112

2 p1

1208

2 2

21 p

4

MG

H111

p1

H112

2 p2

PittN

DM01

pla

smid

3C168

6 p1

MG

H111

p2

MG

H111

p3

H111

9 p3

NUHL

2483

5 p2

pPM

K1−C

ASM

1316

86 p

2

ASM

1316

86 p

3

KP61

7 p1

H112

2 p3

pWSZ

BR

K47

p1

C168

6 p4

ST15

p7

H49

p2

ASM

1316

86 p

4

ASM

1463

47 p

1

C169

9 p2

K47

p2

C169

3 p1

44 p

3

ASM

1463

47 p

2

H111

9 p2

ST15

p9

ST15

p4

C168

6 p2

MG

H111

p4

C169

4 p2

PittN

DM01

pla

smid

1

ASM

1316

86 p

1

1208

2 2

21 p

3

44 p

2

ST15

p5

H112

2 p4

44 p

1

ST15

p2

C169

4 p1Pi

ttNDM

01 p

lasm

id4

NUHL

2483

5 p1

ST15

p6

ST15

p1

5

100

100

47 45

100 21 15 16 8 8 7 919 16

72 5694

6370

100 73

71

99 4994

6961

73

49

89 44

41 17100

100

36

38

55

92

59100

93

99 5594

100 75

96

60

8985

967560

100

53

60100

100

94

99

100

100

100

100

IncX

/MO

BP3

IncL

/MO

BP1

3 In

cFII

A(p

C15

-1a)

/MO

BF1

2

IncH

I1B

-FIB

(pN

DM

-MA

R)/M

OB

H11

IncH

I2B

/MO

BH

11

IncA

/C2/

MO

BH

121

IncF

/MO

BF1

2

IncF

IB (p

KPH

S1)/-

IncR

-(In

cFIA

[HI1

])/-

Col

E-lik

e/M

OB

P5

(col

KP3

)

[col

(pV

C)]

Figu

re 6

. Hie

rarc

hica

l clu

ster

ing

dend

rogr

am o

f Kp

CG

14 a

nd C

G15

pla

smid

s.

The

Nei

ghbo

r-Jo

inin

g de

ndro

gram

was

bas

ed o

n pr

otei

n cl

uste

r an

alys

is u

sing

60%

seq

uenc

e id

entit

y an

d 80

% c

over

age.

Diff

eren

t co

lour

bac

kgro

unds

are

sho

wn

to e

mph

asiz

e br

anch

es o

f re

late

d pl

asm

ids

and

the

Inc

and

MO

B ty

pes

are

indi

cate

d.

IncF

IB(p

KPN

3) -n

o tr

a sy

stem

IncF

IB(p

KPN

3) –

(Inc

FII K

)

IncF

IB(p

KPN

3) (n

o to

lera

nce

gene

s to

met

als)

In

cFII

K (n

o to

lera

nce

gene

s to

met

als)

IncF

IB(S

L151

_p2)

/MO

BF1

2

bact

erio

phag

e


274

MG

H111

p4

H112

2 p2

pUTI

89 C

P000

244

pEC1

4 35

JN9

3589

9

fosm

id H

Q41

9283

pKDO

1 JX

4244

23

pUUH

239

2 CP

0024

74

pCRO

D 2

FN54

3504

pKPN

−IT

JN23

3704

ST15

p4

C168

6 p4

pKP0

48 F

J628

167

ST15

p6

pKF3−9

4 FJ

8768

26

ColE

1 CE

1CG

13pInc

X−SH

V JN

2478

52

p1 C

P015

501

1208

2 2

21 p

5

ST15

p3

pKp1

3a C

P003

996

pSH1

46 J

X258

655

H112

2 p4

pEK5

16 E

U935

738

p995

6 AY

3625

54

C169

9 p1

ST15

p9

pASP

−a58

CP0

1477

5

1208

2 2

21 p

2

PittN

DM01

pla

smid

4

ST15

p5

pKPN

4 CP

0006

4

1208

2 2

21 p

3

H111

9 p3

R478

BX6

6401

5

ASM

1316

86 p

5

NUHL

2483

5 p2

pc15−a

AY4

5801

6

ASM

1463

47 p

2

ASM

1316

86 p

1

NUHL

2483

5 p1

MG

H111

p5

pAsa

3 NC

004

924

pKPS

77 K

F954

150

pYDC

676

KT22

5462

p58

FN64

9416

pPM

K1−N

DM

ST15

p1

pWSZ

BR

H112

2 p3

pK15

ENV

A HG

9180

41

K47

p1

pNDN

−KN

JN15

7804

pNDM

5−X3

KU7

6132

8

pOXA

−48

JN62

6286

pK A

Y079

200

pKP0

9085

KF7

1997

0

PittN

DM01

pla

smid

2

C168

6 p1

pA17

2 EU

3314

25

44 p

1

C169

3 p1

pNDM

−MAR

JN4

2033

6

pKPN

−CZ

JX42

4424

ST15

p7

pSZE

CL e

KU3

0280

7

UCI9

6 p1

pKpn

2341

2−36

2

pKPN

−852

CP0

0987

8

K47

p2

ColA

M37

402

C169

4 p2

C168

6 p3

ASM

1463

47 p

1

ST15

p2

ASM

1316

86 p

3

pPM

K1−B

C169

4 p1

pKPN

6 CP

0006

51

PLO

2 AM

2387

00

R27

AF25

0878

pKV2

CP0

0927

6

pmcr

1 X4

KU7

6132

7

KP61

7 p1

44 p

2

pKP0

07 K

F719

971

ASM

1316

86 p

2

pCTX

−M−3

AF5

5041

5

MG

H111

p1

C169

9 p2

H112

2 p1

MG

H111

p2

44 p

3

pKP3−A

JN2

0580

0

pKPH

S2 C

P003

224

MG

H111

p3

R471

KM

4064

89

pTKH

11 Y

1771

6

pKPH

S1 C

P003

223

pC06

114

4 CP

0160

38KP

617

p2

pPvu

1 AF

3056

15

pMM

1 EU

4104

82

pKpQ

IL G

U595

196

ASM

1463

47 p

3

1208

2 2

21 p

1

pPM

K1−A

pIP1

202

CP00

0603

pRYC

11 L

K391

770

H111

9 p1

H111

9 p2

pc15−k

HQ

2022

66

pBK3

2179

JX4

3044

8

PittN

DM01

pla

smid

3

ST15

p8

SL15

1 pu

2 CP

0170

59

C169

4 p3

UCI4

p1

ASM

1316

86 p

4 pNK2

45 D

Q44

9578

pR55

JQ

0109

84

pVCM

01 J

X133

088

pPM

K1−C

PittN

DM01

pla

smid

1

pKP0

2022

KF7

1997

2

1208

2 2

21 p

4

pKP1

780J

X424

614

pRA1

FJ7

0580

7

C168

6 p2

pH20

5 EU

3314

26

H49

p2

pEC3

4A H

Q62

2575

pVR5

0H C

P011

142

pSZE

CL a

KU3

0280

3

H49

p1

5

100

100

90

100

93

74

47

52

41

45

89

40

35 24

1008337 19 5 3 1

98

1 1 7 3 6 9 7 2 228 26 2

591

5471

59

8195

389835

6164

9970

100

41 45 7287

74

29100 51

100

100 4954

10069

90 35

38436238

8596

5263

100

100

97100

100

100

100

61

95

6095

49 31

99

8799

100

5599

52

91

5345

100

8589

76

10076

6960

73

6286

9773

66

89100

100

7081

100

100

100

100

100 75

100

100

100

100

100

100

6642

8271

99

100

100

IncH

I1B

-FIB

(pN

DM

-MA

R)/M

OB

H11

IncH

I2B

/MO

BH

11

IncA

/C2/M

OB

H12

1

IncF

IIA

(pC

15-1

a)/M

OB

F12

Col

E-lik

e/M

OB

P5

IncX

3/M

OB

P3

IncX

4/M

OB

P3

IncL

/MO

BP1

3 In

cM/M

OB

P13

IncF

IB (p

KPH

S1)/-

IncF

/MO

BF1

2 In

cFIB

(pK

PN3)

–(I

ncFI

I K)

IncF

IIK

(no

tole

ranc

e ge

nes

to m

etal

s)

IncF

IB(p

KPN

3) (n

o to

lera

nce

gene

s to

met

als)

(col

KP3

)

[col

(pV

C)]

Figu

re 7

. Hie

rarc

hica

l clu

ster

ing

dend

rogr

am o

f Kp

CG

14 a

nd C

G15

pla

smid

s (in

dica

ted

by a

ster

isk)

incl

udin

g re

fere

nce

plas

mid

s.

The

Nei

ghbo

r-Jo

inin

g de

ndro

gram

was

bas

ed o

n pr

otei

n cl

uste

r ana

lysi

s us

ing

60%

seq

uenc

e id

entit

y an

d 80

% c

over

age.

Diff

eren

t col

our b

ackg

roun

ds a

re s

how

n to

em

phas

ize

bran

ches

of r

elat

ed p

lasm

ids

and

the

Inc

and

MO

B ty

pes

are

indi

cate

d.

IncR

-(In

cFIA

[HI1

])/-

*

* *

* *

* *

*

* *

*

* *

* *

* *

* * *

* *

* *

* *

* *

*

* *

* *

* * * * *

* *

* *

* *

*

*

*

* * * *

* *

* *

* *

* *

*

* *

* * *

* B

acte

rioph

ages

IncF

IB(S

L151

_p2)

/MO

BF1

2


275

Table S1. Molecular size of the plasmids estimated by PLACNET, PlasmidSPAdes and S1-

PFGE and reconstruction in the four strains sequenced in this study.

Strain PLACNET (kb) PlasmidSPAdes (kb) S1-PFGE (kb)

H1122 2, 4, 39, 219 2, 4, 236 40, 219

H49 119, 167 167 119, 167

44 32, 63, 202 32, 63, 208 32, 61, 202

C1686 42, 55, 204, 305 42, 55, 507 42, 55, 204, 307

C1693 212 201 211

H1119 3, 45, 211 2, 247 44, 211

C1699 68, 198 230 64, 196

C1694 3, 74, 204 3, 195 75, 205

K47 3, 485 2, 485 486


276

Pla

smid

nam

eS

ize (

kb

)S

pecie

sG

en

Ban

k a

cess

ion

nr.

Pla

smid

Fam

ily (

Inc/M

OB

)P

lasm

id n

am

eS

train

ST

K l

ocu

sS

ize (

kb

)P

lasm

id f

am

ily (

Inc/M

OB

)pN

DM

-Mar

267,2

42

Kle

bsi

ell

a p

neum

onia

eJN

420336

IncH

I1B

+F

IB(p

ND

M-M

AR

)/M

OB

H11

44_p1

44

15

K24

31,8

77

IncX

4/M

OB

P3

pK

pn23412-3

62

361,9

64

Kle

bsi

ell

a p

neum

onia

eC

P011314

IncH

I1B

+F

IB(p

ND

M-M

AR

)/M

OB

H11

NU

HL

24835_p2

NU

HL

24835

14

K2

46,1

61

IncX

3/M

OB

P3

pK

15 E

NV

A253,9

84

Kle

bsi

ell

a p

neum

onia

eH

G918041

IncH

I1B

+F

IB(p

ND

M-M

AR

)/M

OB

H11

C1686_p2

C1686

15

K24

55,3

28

phage-r

ela

ted

p1(S

KG

H01)

281,1

9K

lebsi

ell

a p

neum

onia

eC

P015501

IncH

I1B

+F

IB(p

ND

M-M

AR

)/M

OB

H11

44_p2

44

15

K24

62,8

52

IncL

/MO

BP

13

R478

274,7

62

Serr

ati

a m

arc

esc

ens

BX

664015

IncH

I2/M

OB

H11

AS

M147347_p1

AS

M147347

14

K2

64,5

18

IncL

/MO

BP

13

R27

180,4

61

Salm

onell

a T

yphi

AF

250878

IncH

I1/M

OB

H11

ST

15_p6

ST

15

15

KL

112

62,9

4In

cF

IIA

(pC

15-a

)/M

OB

F12

pR

55

170,8

1K

lebsi

ell

a p

neum

onia

eJQ

010984

IncA

/C2/M

OB

H121

12082_2_21_p4

12082_2_21

15

K24

64,6

71

IncF

IIA

(pC

15-a

)/M

OB

F12

pN

DM

-KN

162,7

46

Kle

bsi

ell

a p

neum

onia

eJN

157804

IncA

/C2/M

OB

H121

pP

MK

1-C

PM

K1

15

KL

112

69,9

47

IncF

II_re

pA

_su

perf

am

ily/-

pR

A1

143,9

63

Aero

monas

hydro

phil

a

FJ7

05807

IncA

/C2/M

OB

H121

MG

H111_p3

MG

H111

15

K24

95,8

58

-pA

SP

-a58

198,3

07

Aero

monas

vero

nii

C

P014775

IncA

/C2/M

OB

H121

MG

H111_p4

MG

H111

15

K24

185,5

08

IncF

IIK

1-I

nH

I1B

-IncR

/-pE

A1509_A

162,2

02

Ente

robacte

r aero

genes

NC

_020180

IncA

/C2/M

OB

H121

C1686_p4

C1686

15

K24

304,7

54

IncH

I1B

-IncF

IB(p

ND

M-M

AR

)/M

OB

H11

pIP

1202

182,9

13

Yers

inia

pest

isC

P000603

IncA

/C2/M

OB

H121

Pit

tND

M01_pla

smid

1P

ittN

DM

01

14

K2

283,3

71

IncH

I1B

-IncF

IB(p

ND

M-M

AR

)/M

OB

H11

SL

151 p

lasm

id u

nnam

ed2

171,2

33

Cit

robacte

r fr

eundii

CP

017059

-K

P617-p

lasm

id1

KP

617

14

K2

273,6

28

IncH

I1B

-IncF

IB(p

ND

M-M

AR

)/M

OB

H11

pE

K516

64,4

71

Esc

heri

chia

coli

EU

935738

IncF

IIA

/MO

BF

12

pP

MK

1-N

DM

PM

K1

15

KL

112

304,5

26

IncH

I1B

-IncF

IB(p

ND

M-M

AR

)/M

OB

H11

pC

15-1

a92,3

53

Esc

heri

chia

coli

AY

458016

IncF

IIA

/MO

BF

12

ST

15_p9

ST

15

15

KL

112

292,5

17

IncH

I1B

-IncF

IB(p

ND

M-M

AR

)/M

OB

H11

pU

TI8

9114,2

3E

scheri

chia

coli

CP

000244

IncF

IIA

/MO

BF

12

AS

M131686_p5

AS

M131686

14

K2

261,2

64

IncH

I1B

-IncF

IB(p

ND

M-M

AR

)/M

OB

H11

pO

XA

-48

61,8

81

Kle

bsi

ell

a p

neum

onia

eJN

626286

IncL

/MO

BP

13

pK

47_2

K47

15

KL

110

484,6

66

IncH

I2/M

OB

H11

R471

86,7

48

Serr

ati

a m

arc

esc

ens

KM

406489

IncM

/MO

BP

13

MG

H111_p5

MG

H111

15

K24

188,8

58

IncA

/C2/M

OB

H121

pC

TX

-M-3

89,4

68

Cit

robacte

r fr

eundii

AF

550415

IncM

2/M

OB

P13

pH

49_1

H49

14

K2

181,0

38

IncA

/C2/M

OB

H121

pK

PN

_C

Z207,8

19

Kle

bsi

ell

a p

neum

onia

eJX

424424

IncF

IIK

8+

FIB

(pK

PN

3)/

MO

BF

12

H49_p2

H49

14

K2

104,4

92

IncF

IB(K

pn3)/

-pK

PN

-IT

208,1

91

Kle

bsi

ell

a p

neum

onia

eJN

233704

IncF

IIK

1+

FIB

(pK

PN

3)/

MO

BF

12

pP

MK

1-A

PM

K1

15

KL

112

187,5

71

IncF

IIK

1-I

ncF

IB(K

pn3)/

MO

BF

12

pB

K32179

165,2

95

Kle

bsi

ell

a p

neum

onia

eJX

430448

IncF

IIK

1+

FIB

(pK

PN

3)/

MO

BF

12

ST

15_p8

ST

15

15

KL

112

224,0

41

IncF

IIK

1-I

ncF

IB(K

pn3)/

MO

BF

12

pK

P09085

213,0

19

Kle

bsi

ell

a p

neum

onia

eK

F719970

IncF

IIK

5+

FIB

(pK

PN

3)/

MO

BF

12

C1694_p3

C1694

15

K19

211,5

12

IncF

IIK

5-I

ncF

IB(K

pn3)/

MO

BF

12

pK

P02022

203,5

77

Kle

bsi

ell

a p

neum

onia

eK

F719972

IncF

IIK

5+

FIB

(pK

PN

3)/

MO

BF

12

H1122_p4

H1122

14

K16

226,2

69

IncF

IIK

1-I

ncF

IB(K

pn3)/

MO

BF

12

pK

P007

246,1

76

Kle

bsi

ell

a p

neum

onia

eK

F719971

IncF

IIK

5+

FIB

(pK

PN

3)/

MO

BF

12

H1119_p3

H1119

15

K24

210,7

31

IncF

IIK

1-I

ncF

IB(K

pn3)/

MO

BF

12

pU

UH

239.2

220,8

24

Kle

bsi

ell

a p

neum

onia

eC

P002474

IncF

IIK

5+

FIB

(pK

PN

3)/

MO

BF

12

12082_2_21_p5

12082_2_21

15

K24

209,9

21

IncF

IIK

1-I

ncF

IB(K

pn3)/

MO

BF

12

pc15-k

95,6

26

Kle

bsi

ell

a p

neum

onia

eH

Q202266

IncF

IIK

2/M

OB

F12

UC

I96_p1

UC

I96

15

K24

196,9

22

IncF

IB/M

OB

F12

pK

pQ

IL113,6

37

Kle

bsi

ell

a p

neum

onia

eG

U595196

IncF

IIK

2/M

OB

F12

UC

I4_p1

UC

I414

K16

185,4

03

IncF

IB/M

OB

F12

pK

PN

-4107,5

76

Kle

bsi

ell

a p

neum

onia

eC

P000649

IncF

IIK

1/M

OB

F12

C1699_p2

C1699

15

KL

112

200,0

79

IncF

IIK

9-I

ncF

IB(K

pn3)/

MO

BF

12

pK

PH

S02

111,1

95

Kle

bsi

ell

a p

neum

onia

eC

P003224

IncF

IIK

2+

R/M

OB

F12

AS

M147347_p3

AS

M147347

14

K2

274,7

57

IncF

IIK

5-I

ncF

IB(K

pn3)/

MO

BF

12

pK

P048

151,1

88

Kle

bsi

ell

a p

neum

onia

eF

J628167

IncF

IIK

5/M

OB

F12

C1686_p3

C1686

15

K24

210,4

52

IncF

IIK

5-I

ncF

IB(K

pn3)/

MO

BF

12

pK

DO

1127,5

08

Kle

bsi

ell

a p

neum

onia

eJX

424423

IncF

IIK

77M

OB

F12

44_p3

44

15

K24

210,1

13

IncF

IIK

5-I

ncF

IB(K

pn3)/

MO

BF

12

pR

YC

11

94,8

93

Kle

bsi

ell

a p

neum

onia

eL

K391770

IncF

IIK

2/M

OB

F12

C1693_p1

C1693

15

K24

212,6

89

IncF

IIK

5-I

ncF

IB(K

pn3)/

MO

BF

12

pK

F3-9

494,2

19

Kle

bsi

ell

a p

neum

onia

eF

J876826

IncF

IIK

2/M

OB

F12

NU

HL

24835_p1

NU

HL

24835

14

K2

101,0

3In

cF

IIK

2/M

OB

F12

pK

PH

S1

122,7

99

Kle

bsi

ell

a p

neum

onia

eC

P003223

FIB

(pK

PH

S01)/

- [p

hage-r

ela

ted]

AS

M131686_p4

AS

M131686

14

K2

95,7

73

IncF

IB (

Kpn3)/

MO

BF

12

pN

K245

98,2

64

Kle

bsi

ell

a p

neum

onia

eD

Q449578

IncR

-IncF

IA(H

I1)/

-P

ittN

DM

01_pla

smid

2P

ittN

DM

01

14

K2

103,6

94

IncF

IB (

Kpn3)/

MO

BF

12

pK

PS

77

45,8

67

Kle

bsi

ell

a p

neum

onia

eK

F954150

IncR

/-C

1686_p1

C1686

15

K24

42,5

43

IncF

II_re

pA

_su

perf

am

ily/-

pK

P1780

49,7

7K

lebsi

ell

a p

neum

onia

eJX

424614

IncR

/-A

SM

147347_p2

AS

M147347

14

K2

110,9

59

IncF

IB(p

KP

HS

1)/

- [p

hage-r

ela

ted]

pY

DC

676

50,1

82

Kle

bsi

ell

a p

neum

onia

eK

T225462

IncR

/-S

T15_p7

ST

15

15

KL

112

111,7

69

IncF

IB(p

KP

HS

1)/

- [p

hage-r

ela

ted]

pK

PN

-852

51,6

22

Kle

bsi

ell

a p

neum

onia

eC

P009878

phage-r

ela

ted p

lasm

idpP

MK

1-B

PM

K1

15

KL

112

111,6

93

IncF

IB(p

KP

HS

1)/

- [p

hage-r

ela

ted]

pK

V2

54,7

15

Kle

bsi

ell

a v

ari

icola

CP

009276

phage-r

ela

ted p

lasm

idpH

1122_3

H1122

14

K16

39,1

89

IncF

IA(H

I1)/

-pC

RO

D2

39,2

65

Cit

robacte

r ro

denti

um

FN

543504

IncX

4/M

OP

312082_2_21_p3

12082_2_21

15

K24

34,7

56

IncR

/-pS

H146_32

32,4

47

Salm

onell

a e

nte

rica

JX258655

IncX

4/M

OP

3pH

1119_p2

H1119

15

K24

45,3

42

IncR

/-pm

cr1

_In

cX

433,2

87

Kle

bsi

ell

a p

neum

onia

eK

U761327

IncX

4/M

OP

3pW

SZ

BR

BR

15

KL

112

76,2

51

IncR

/-

pIn

cX

-SH

V43,3

8K

lebsi

ell

a p

neum

onia

eJN

247852

IncX

3/M

OB

3C

1694_p2

C1694

15

K19

65,3

51

IncR

-IncF

IA(H

I1)/

-pE

C14_35

34,9

45

Esc

heri

chia

coli

JN935899

IncX

3/M

OB

3A

SM

131686_p3

AS

M131686

14

K2

45,4

82

IncR

/-pN

DM

5_In

cX

346,1

61

Kle

bsi

ell

a p

neum

onia

eK

U761328

IncX

3/M

OB

3P

ittN

DM

01_pla

smid

3P

ittN

DM

01

14

K2

70,8

14

IncR

-IncF

IA(H

I1)/

-C

olA

6,7

2E

scheri

chia

coli

M37402

ColE

-lik

e/M

OB

P5

C1699_p1

C1699

15

KL

112

69,3

26

IncR

-IncF

IA(H

I1)/

-C

olE

16,6

46

Esc

heri

chia

coli

CE

1C

G13

ColE

-lik

e/M

OB

P5

AS

M131686_p2

AS

M131686

14

K2

7,8

5C

olK

P3 (

ColE

-lik

e)/

MO

BP

5pL

O2

7,9

41

Lis

tonell

a a

nguil

laru

mA

M238700

ColE

-lik

e/M

OB

P5

Pit

tND

M01_pla

smid

4P

ittN

DM

01

14

K2

6,1

41

ColK

P3 (

ColE

-lik

e)/

MO

BP

5pA

SA

35,6

16

Aero

monas

salm

onic

ida

NC

_004924

ColE

-lik

e/M

OB

P5

KP

617_p2

KP

617

14

K2

6,1

41

ColK

P3 (

ColE

-lik

e)/

MO

BP

5pP

vu1

4,6

75

Pro

teus

vulg

ari

sA

F305615

ColE

-lik

e/M

OB

P5

ST

15_p5

ST

15

15

KL

112

5,7

37

ColE

-lik

e/M

OB

P5

pS

ZE

CL

_e

4,8

63

Ente

robacte

r clo

acae

KU

302807

ColE

-lik

e/M

OB

P5

ST

15_p4

ST

15

15

KL

112

5,3

32

ColE

-lik

e/M

OB

P5

pK

PN

-64,2

59

Kle

bsi

ell

a p

neum

onia

eC

P000651

ColE

-lik

e/M

OB

P5

pH

1122_2

H1122

14

K16

3,6

71

ColE

1-l

ike/M

OB

P5

fosm

id_4D

12

10,1

58

Kle

bsi

ell

a p

neum

onia

eH

Q419283

ColE

-lik

e/M

OB

P5

ST

15_p2

ST

15

15

KL

112

3,7

53

ColE

-lik

e/M

OB

P5

pT

KH

11

8,1

93

Kle

bsi

ell

a o

xyto

ca

Y17716

ColE

-lik

e/M

OB

P5

ST

15_p1

ST

15

15

KL

112

3,7

03

ColE

-lik

e/M

OB

P5

pH

205

8,1

95

Kle

bsi

ell

a p

neum

onia

eE

U331426

ColE

-lik

e/M

OB

P5

MG

H111_p1

MG

H111

15

K24

2,1

55

ColE

-lik

e/M

OB

P5

pA

172

8,1

97

Salm

onell

a e

nte

rica

EU

331425

ColE

-lik

e/M

OB

P5

12082_2_21_p2

12082_2_21

15

K24

2,7

32

ColE

-lik

e/M

OB

P5

pK

P3-A

(colK

P3)

7,6

05

Kle

bsi

ell

a p

neum

onia

eJN

205800

ColK

P3/M

OB

P5

AS

M131686_p1

AS

M131686

14

K2

2,6

55

ColE

-lik

e/M

OB

P5

pC

06114_4 (

colK

P3)

6,1

41

Kle

bsi

ell

a p

neum

onia

eC

P016038

ColK

P3/M

OB

P5

C1694_p1

C1694

15

K19

2,8

11

ColE

-lik

e/M

OB

P5

p9956

5,6

74

Acti

nobacil

lus

ple

uro

pneum

onia

eA

Y362554

ColE

-lik

e/M

OB

P5

H1122_p1

H1122

14

K16

2,3

65

ColE

-lik

e/M

OB

P5

pE

C34A

3,7

7E

scheri

chia

coli

HQ

622575

ColE

-lik

e/M

OB

P5

K47_p1

K47

15

KL

110

2,5

64

Col(

pV

C)

/MO

BP

5pK

P13a

1,9

68

Kle

bsi

ell

a p

neum

onia

eC

P003996

ColE

-lik

e/M

OB

P5

H1119_p1

H1119

15

K24

3,3

57

Col(

pV

C)

/MO

BP

5pK

4,2

45

Salm

onell

a E

nte

riti

dis

AY

079200

ColE

-lik

e/M

OB

P5

MG

H111_p2

MG

H111

15

K24

2,1

55

Col(

pV

C)

/MO

BP

5pM

M1

2,1

4P

seudom

onas

aeru

gin

osa

EU

410482

ColE

-lik

e/M

OB

P5

12082_2_21_p1

12082_2_21

15

K24

2,2

75

Col(

pV

C)

/MO

BP

5pS

ZE

CL

_a (

colp

VC

)2,0

58

Ente

robacte

r clo

acae

KU

302803

Col(

pV

C)/

MO

BP

5S

T15_p3

ST

15

15

KL

112

3,8

67

ColE

-lik

e/M

OB

P5

pV

CM

01 (

colp

VC

)1,9

81

Salm

onell

a e

nte

rica

JX133088

Col(

pV

C)/

MO

BP

5pV

R50H

(colP

V)

1,7

83

Esc

heri

chia

coli

CP

011142

Col(

pV

C)/

MO

BP

5p58

5,8

Esc

heri

chia

coli

FN

649416

ColE

-lik

e/M

OB

P5

Pla

smid

s fr

om

CG

14 a

nd

CG

15 s

tud

y c

oll

ecti

on

Refe

ren

ce p

lasm

ids

Tabl

e S2

. Cha

ract

eris

tics

[siz

e (k

b), I

nc a

nd M

OB

gro

up] o

f ref

eren

ce p

lasm

ids

(A) a

nd p

lasm

ids

from

Kp

CG

14 a

nd C

G15

(B) u

sed

for p

lasm

id p

rote

ome

anal

ysis

.

A)

B)

ST, s

eque

nce

type


277

Table S3. Assembly statistics for the nine ST14 and ST15 genomes sequenced in this study.

Strain No. of

contigs

Total

Length (bp)

CG (%) N50 Largest

Contig (bp)

K-mer

H49 88 5,592,864 57.32 350,404 701,584 101,111,121,127

H1122 106 5,587,835 57.21 1,013,553 399,091 101,111,121,127

44 120 5,748,666 56.95 210,873 584,875 101,111,121,127

C1686 148 6,054,170 56.46 210,874 472,869 101,111,121,127

C1693 140 5,650,579 57.12 184,651 371,151 101,111,121,127

H1119 124 5,530,501 57.26 162,787 429,762 101,111,121,127

C1699 121 5,583,524 57.05 220,754 472,869 101,111,121,127

C1694 104 5,458,733 57.17 203,303 584,887 101,111,121,127

K47 144 5,745,435 56.63 204,524 584,887 101,111,121,127


278

Table S4. Reference strains used in the comparison of the cps operon.

NA, allele number not attributed.

Strain GenBank accession no.

wzi/K wzc/K Kaptive Reference (PMID or DOI)

K. pneumoniae ATCC 43816 KJ541664 wzi2/K2 wzc2:14/K2 KL2 25203254 K. pneumoniae 2069/49 AB742228 wzi16/K16 wzc16:85/K16 KL16 24349011 Klebsiella sp. 1680/49 AB924562 wzi24/K24 wzc25:4/K24 KL24 26493302 K. pneumoniae 7749 AB742230 wzi39/K39 wzc39:21/K39 KL39 24349011 Klebsiella sp. 1754/49 AB924559 wzi19/K19 wzc20:46/K19 KL19 26493302 Klebsiella sp. 4463/52 AB924597 wzi60/K60 wzc60:37/K60 KL60 26493302 K. pneumoniae 484 LT174581 wzi93/K60 wzc923:66/K- KL112 https://doi.org/10.1101/071415 K. pneumoniae ST15 LN714331 wzi93/K60 wzc923:66/K- KL112 https://doi.org/10.1101/071415 Klebsiella sp. 7824 AB924568 wzi30/K30 wzc903:18/K30 KL30 26493302 K. pneumoniae 214 LT174579 wzi89/- NA KL110 https://doi.org/10.1101/071415 K. pneumoniae 85/08 LT603721 wzi398/- NA KL146 https://doi.org/10.1101/071415


279

Figure S1. BRIG comparative analysis of all coding sequences (CDS) identified in the variable region of the cps locus from Kp CG14 and CG15 representative isolates.


280

Figure S2. BRIG comparative analysis of IncFIIA/MOBF12 using plasmid pC15-1a as inner reference.


281

Figure S3. BRIG comparative analysis of IncFIB/MOBF12 using plasmid pCAV174-101 as inner reference.


282

Figure S4. BRIG comparative analysis of IncF/MOBF12 using plasmid pUUH239 as inner reference.

FIB(pKPN3) no metal tolerance genes

FIB(pKPN3) with or without IncFIIK

IncFIIK , no metal tolerance genes


283

Figure S5. BRIG comparative analysis of IncFIB phage-related plasmids using plasmid pKPHS1 as inner reference.


284

Figure S6. BRIG comparative analysis of IncR plasmids using plasmid PittNDM01_p3 as inner reference.


285

Figure S7. BRIG comparative analysis of IncHIB.-IncFIB/MOBH11 plasmids using pNDM-MAR as inner reference.


286

Figure S8. BRIG comparative analysis of IncX3/MOBP3 plasmids using pNDM5_X3 as inner reference.


287

Figure S9. BRIG comparative analysis of IncX4/MOBP3 plasmids using pCROD2 as inner reference.


288

Figure S10. BRIG comparative analysis of IncL/MOBP13 plasmids using pOXA-48 as inner reference.


289

Figure S11. BRIG comparative analysis of IncA/C2/MOBH121 plasmids using pEA1509_A as inner reference.


290

Figure S12. BRIG comparative analysis of phage-like plasmids using pKV2 as inner reference.

“Nobody said it was easy

No one ever said it would be this hard.”

Coldplay, “The Scientist”

08 Fall

Conclusions

Chapter 4

Chapter 4 - Conclusions

293

The General conclusions that can be extracted from the achievement of the

specific aims proposed are summarized as follows:

1. The landscape of clinical E. coli and K. pneumoniae resistant to extended-

spectrum β-lactams suffered significant changes in the period analysed (2006-

2016). Although CTX-M-15 is still the most prevalent enzyme among extended-

spectrum β-lactamase (ESBL)-producing E. coli, the increase in the diversity of CTX-M enzymes observed more recently suggests a non-clinical reservoirs’

origin. Conversely, the increase in the proportion of ESBL-producing K.

pneumoniae (a ca. five-fold increase in 3 years) is remarkable with a

noteworthy expansion of CTX-M-15 and in a lesser extent SHV-12,

suggesting a higher involvement of nosocomial transmission. Acquired carbapenemases (KPC-3, VIM-34) were detected since 2011 in our

geographic area, exclusively among K. pneumoniae isolates, either sporadically

(VIM-34, described for the first time in this study), or rapidly disseminating

(KPC-3) in isolates causing infections among hospitalized patients in different

hospitals and also residents of long-term care facilities and nursing homes, a

situation of high concern that requires reinforced surveillance and a concerted

action between the different stakeholders.

2. In the collection tested (ESBL- or carbapenemase-producing isolates), resistance

rates to last resource antibiotics are still low (9.3% for fosfomycin or 0% for

colistin), preserving these antibiotics as possible therapeutic choices to treat

infections caused by extended-spectrum β-lactam-resistant E. coli or K.

pneumoniae. However, the detection of the first clinical case involving one E.

coli isolate harbouring a plasmid-mediated fosfomycin resistance gene (fosA3) in Portugal is of particular concern and brings awareness to the need of

further surveillance and monitoring of fosfomycin resistance trends in Portugal

and other European countries. Moreover, the identification of this gene in a

patient that had travelled to Asia highlights the relevance of human mobility in

the dissemination of antibiotic resistance genes.

3. Interestingly, the amplification of CTX-M-15 in both species occurred mainly

throughout clonal expansion of certain worldwide epidemic clones and different

plasmid types among E. coli (IncFII plasmids and ST131) and K.

pneumoniae (IncR plasmids and ST15), revealing independent capture events of blaCTX-M-15 and subsequent expansion in each species.


294

4. Different lineages of B2-ST131 were identified among the faecal flora of

nursing home residents (O25b:H4-CTX-M-15-fimH30-virotype C and O25b:H4-

CTX-M-14-fimH22-virotype D) and healthy volunteers (O25b:H4-CTX-M-27-

non-Rx-fimH30-virotype C), reflecting the importance of subtyping ST131 isolates. Moreover, these data anticipate variability on ST131 isolates causing

human infections in our country and furthermore suggest differential adaptation

of these lineages to different human host populations.

5. No carbapenemase producers were by far identified colonizing long-term care

facilities or nursing home residents or healthy volunteers, but the rates of faecal carriage of K. pneumoniae or E. coli resistant to extended-spectrum

cephalosporins (ESC) (40%, 20%, 2% respectively) and/or the carriage of

clones with enhanced ability to persist and/0r to cause infection is

disconcerting due to the risk of infection for carriers and also of subsequent

dissemination of these multidrug resistant (MDR) bacteria to healthcare

professionals, residents’ or healthy adults families, and to the community.

6. This thesis provides a comprehensive overview of the molecular basis for ESC

and carbapenem resistance among K. pneumoniae from different clinical and

non-clinical human niches in Portugal. ESBL or carbapenemases are encoded by

plasmids from a few families, IncR and IncFIIK or IncFIA and IncN,

respectively, most of them apparently well adapted to this species. The increase

in frequency of these β-lactamases was driven by clonal expansion of certain

worldwide disseminated high-risk clones (ST11, ST15, ST147) able to

carry variable ESBL/carbapenemase encoding plasmids, which reflects the open

genome of these K. pneumoniae lineages. Some of the K. pneumoniae MDR

clones studied presented an enrichment in specific virulence factors (types 1 and 3 fimbriae, yersiniabactin and Kfu system), which is worrisome due

to the possible clinical impact of combined multidrug resistance with virulence.

7. Specific capsular types were identified among main MDR K. pneumoniae clones

circulating in Portugal belonging to ST15 (K19, K24, KL110, KL112) and ST147 (K64), which were found either causing infections in clinical institutions

or colonizing long-term care facilities residents from the same geographic area. Moreover, the diversity on genotypic features and capsule types observed among

some of them (especially ST11 or ST15) supports a suboptimal resolution of multilocus sequence typing (MLST)-based typing scheme for K.


295

pneumoniae and the need for alternative methods for delineation of

phylogenetic boundaries.

8. It is demonstrated for the first time the ability of Fourier transform infrared

(FTIR) spectroscopy to differentiate K. pneumoniae capsular types,

highlighting the potential of this methodology as a fast and accurate alternative

K-typing tool. The establishment of stable associations between capsular types and specific MDR K. pneumoniae clones/lineages in different

geographic regions over time opens the possibility of using FTIR for K.

pneumoniae typing, as demonstrated by the application of this approach in a

real-time context. Given the very good performance of this methodology for

typing of different bacterial species (accurate, simple, low cost, quick), it is

justified the investment in instruments, robust databases and procedure

automation to approximate FTIR to routine microbiology laboratories.

9. Conversely, matrix assisted laser desorption ionization-time of flight mass

spectrometry (MALDI-TOF MS) demonstrated a limited potential to

discriminate MDR clinically relevant K. pneumoniae clones, apparently

explained by the high degree of conservation of K. pneumoniae subsp.

pneumoniae ribosomal proteins. The variable typeability potential of FTIR

and MALDI-TOF MS is linked to the different molecular key features depicted

by the different methodologies.

10. Comparative genomic analysis of CG14 and CG15 K. pneumoniae allowed the

precise definition of diverse lineages carrying specific capsular types in

different geographic regions for several years. Comprehensive analysis of the

plasmidome of CG14 and CG15 revealed turbulent flux of plasmids of disparate families containing a wide number of adaptive traits,

including antibiotic resistance genes, with IncF/MOBF12 and IncR plasmids

being pervasive and greatly contributing to the CG14/15 pangenome.

11. Finally, the high congruence observed between genotypic, genomics and

metabolomics approaches for K. pneumoniae typing points to a great

relevance of surface bacterial components (and particularly the capsule) on the

evolution, host adaptation and/or virulence features in this species that deserves

to be further explored.

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