Results and experience of BARN ESBL project

Marina Ivanova and project team

Tallinn

13.05.2013

Aim of the project

Improvement of detection and surveillance of resistance caused by extended spectrum beta-lactamase (ESBL) in Enterobacteriaceae in Baltic region (Estonia, Latvia, Lithuania and St Petersburg area of Russia).

Why ESBL?

1. Beta-lactams are most widely used AB group

2. Incidence is increasing rapidly (compared to MRSA, VRE etc)

Insufficient data on ESBL epidemiology of Baltic region

K. pneumoniae resistant to 3 GC – incidence is increasing

3. Many different types/genes and regional differencies

4. Rapid spread of new types

5. Colonisation of healthy people

24 of 100 travellers were colonised by ESBL pos E. coli and 5 of 21 remained colonised for longer than 6 months after the travel

6. Higher mortality and costs of treatment

Aims of the project in detail

• 1. Introduction and evaluation of phenotypic screening and confirmation algorithms of ESBLs (incl ESBL-A, ESBL-CARBA and ESBL-M)

• 2. Detection of main phenotypic and molecular markers in E. coli and K. pneumoniae strains resistant to 3rd generation cephalosporins in Baltic region

• 3. Creation of culture collection of resistant E. coli and K. pneumoniae strains for future studies

• 4. Improvement of antimicrobial resistance detection and resistance surveillance in Baltic region

• 5. Educational and scientific cooperation between EU (Baltic and Nordic states) and Russia

Project history: activities • Feb 2011 - 1st BARN WS: presentation of project

proposal

• March to Sept 2011: Estonian Pilot study (4 labs)

• Nov 2011: 2nd BARN WS: participants meeting, final protocol

• Jan - May 2012: study period

• Introduction of ESBL screening/confirmation methods

• Collection of strains and data in local labs

• Meetings in St. Petersburg and Vilnius

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Project history: activities • June-Sept 2012:

• Collection of strains and data to study center

• ID- MALDI-TOF

• Extraction of DNA

• Master students training in Stockholm

• 2013

• ECCMID posters preparation and presentation

• Final meeting in Tallinn

• Final report to BARN

• Initiatives for involvement of new countries

• Preparation of publications

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21 labs/hospitals

Collaboration

Strains/data collecting coordinators

Jolanta Miciuleviciene, Lithuania, Vilnius (City) University Hospital Ruta Ambrazaitiene, Vilnius University Hospital Gintaras Makstutis, Siaulai Hospital Arta Balode, Latvia, Pauls Stradins University Dace Rudzite, Riga East Clinical University hospital Tatjana Djundika, Liepaja Hospital Lidia Kaftyreva, Russia, St. Petersburg Pasteur Institute Svetlana Egorova, St. Petersburg Pasteur Institute Nataliya Vedernikova, St. Petersburg Hospital № 4 Lidia Lipskaya, St. Petersburg Hospital № 40 Olga Morozova, St. Petersburg Children's hospital № 1 Tatyana Kurchikova St. Petersburg Children's hospital № 17 Maria Paysetchkaya, St. Petersburg Children's hospital № 5 Marina Smirnova, St. Petersburg Hospital № 16 Irina Konovalenko, St. Petersburg Hospital № 31 Marina Ivanova, Estonia, ITKH Svetlana Rudenko, PERH Kaisa Kirs, LTKH Krista Lõivukene, TÜK Natalja Kamõnina, IVKH

Scientific support and activities Estonia-Sweden-Norway

Barbro Olsson Liljequist, Petra Edquist

– SmittskyddsInstitutet

Paul Naaber – Stavanger University Hospital

Paul Naaber, Epp Sepp, Siiri Kõljalg, Marina Ivanova – Tartu University

Anastasia Pavelkovich, Jana Lillo, Kristiine Pai – Master students

Outputs, results

• Optimization of lab diagnostics and detection of resistance mechanisms

• Sustainable and integrated surveillance network base for ESBLs in Baltic-Nordic region

• Scientific publications about ESBL epidemiology in Baltic region

• National guidelines (for labs and IC - isolation).

Collection of strains

970 ESBL screening positive strains (13 140 screened)

433 strains of E. coli

537 strains of K. pneumoniae January – May 2012

Clinical isolates, 1 per patient

Collection ID check

• All strains were checked with MALDI Biotyper Microflex LT (Bruker Daltonics)

• Only confirmed (EC and KP) strains were included into collection

AST and phenotypical resistance screening

• ESBL A, ESBL M, ESBL CARBA – first EC and KP isolates, meeting the criteria for ESBL phenotypical screening

– Screening (3 gen cefalosporins – ceftazidim, cefotaxim NS)

– Screening (carbapenems – ertapenem, meropenem NS)

Options for ESBL screening

Options Antibiotic(s) Methods Interpretation

(screening positive)

1. recommended Ceftazidime AND

cefotaxime

(ceftriaxone can be

used instead of

cefotaxime)

Disk diffusion or

MIC – Etest or

MIC automated

system

Any non-susceptible (i.e.

Ceftazidime or

cefotaxime or

ceftriaxone)

2. alternative VITEK or other

expert system

e.g. VITEK Non-susceptible

(ceftazidime or

cefotaxime or

ceftriaxone) or VITEK

alert for possible

ESBL/AmpC

3.Carbapenemase

screening

Ertapenem or

Meropenem or

Imipenem

Disk diffusion or

MIC

Any non-susceptible

•

Cefpodoxime < S OR

Cefotaxime and/or Ceftazidime < S (S2)

Ertapenem and/or Meropenem and/or

Imipenem MIK <S (S3)

L1 most common:

E. coli

K. pneumoniae

P. mirabilis

Salmonella spp.

Shigella spp.

L2

K. oxytoca

P. vulgaris

C. koseri

L3

Enterobacter spp.

C. freundii

M. morganii

H. alvei

Serratia spp.

Providencia spp.

ESBL test (T2) AmpC

test

(T1)

NEG or

Non-

detectabl

e

MBL, KPC, AmpC (T4)

ESBL

(R4)

POS

E. coli

Shigella

KR/PL

AmpC

(R1)

K. oxytoca

K. pneumoniae

P. mirabilis

Salmonella spp.

PL AmpC (R2)

KR

AmpC

(R3)

Ceftaz

idime

ESBL

POS

MBL

(R6)

KPC

(R7)

MBL

POS

KPC POS

AmpC NEG

KPC NEG

AmpC POS

AmpC +

porin

(R1/2/3)

KR AmpC

(R3)

Irrespective of

screening results

D-test/

Cefox

(S1)

L3

Enterobacter spp.

C. freundii

M. morganii

H. alvei

Serratia spp.

Providencia spp.

POS

ESBL test (T3)

NEG or

Non-

detectable

POS

ESBL and KR

AmpC (R5)

Comments: S1-S3: Screening tests 1-3; L1-L3: Microbial lists 1-3; T1-T4: Confirmation tests 1-4; R1-7: Lab reports to clinicians and infection control 1-7 KR AmpC: Chromosomal AmpC; PL AmpC: Plasmid AmpC

Algorithm for detection of ESBLA (= ESBL inhibited by clavulanic acid)

CTX-R or CAZ-R

E.coli, K.pneumoniae, P.mirabilis, Salmonella

spp., Shigella spp.

ESBL A-test (clavulanic acid)

Positive = ESBL A

Negative => Perform ESBL M -

test

K.oxytoca, P.vulgaris, C.koseri

Chromosomal beta-lactamase

ESBL A-test only if CAZ-R

Other Enterobacteriaceae

Often chromosomal beta-lactamase

ESBL A-test if resistant to >= 2 non-betalactams

Genotyped by PCR for CTX-M, SHV, TEM or other

Algoritm for detection of aquired (plasmid-mediated) AmpC (=ESBLM)

ESBL A-negative and CTX/CAZ-R; E.coli, K.pneumoniae, (P.mirabilis,

Salmonella spp., Shigella spp.)

ESBL M-test positive (CAZ-CLOX synergy)

E.coli and Shigella spp. : PCR to differentiate between plasmid- or

chromosomally-mediated

K.pneumoniae, P.mirabilis, Salmonella spp. do not possess chrom. AmpC, therefore they have aquired AmpC

ESBL M-test negative (no synergy)

Different mechanism (loss of porins, PBP alterations etc)

Genotyped by PCR for pAmpC; CIT and DHA are most common

Algorithm for detection of carbapenemases (=ESBLCARBA)

Meropenem I/R using DD, or MIC > 0.5 mg/L

Synergy with boronic acid but

not cloxacillin

= KPC (class A carbapenemase)

Synergy with boronic acid and

cloxacillin

= AmpC and loss of porins

Synergy only with dipicolinic acid

(DPA)

= Metallo-betalactamase (e.g.

VIM, IMP, NDM)

No synergy

= ESBL and loss of porin, or OXA-48

ROSCO test evaluation results

Diagnostic tests from Rosco

Diagnostic tests for ESBLM from Rosco

CTX30

CTX30+CLOX

CAZ30

CAZ30+CLOX

Diagnostic tests for ESBLCARBA from Rosco

Molecular analysis of the collection

– Practical training of students in SmittskyddsInstitutet

– ESBL genes detection • ESBLA (CTX-M, SHV, TEM)

• ESBLM (AmpC)

• ESBLCARBA (NDM, KPC…)

ECCMID 2013 posters presented

• Epidemiology of Extended Spectrum Beta Lactamases producing E. coli and K. pneumoniae in the Baltic Sea Region

• Differences in virulence factors of E. coli isolated from the Baltic Sea Region

• The molecular epidemiology of Extended Spectrum Beta Lactamases producing E. coli and K. pneumoniae in the Baltic Sea Region

• Detection of carbapenem non-susceptible E. coli and K. pneumoniae in the Baltic Sea Region

Further analysis of the data

• Sensitivity of different screening agents and criteria (needs additional tests and work)

• Strains without confirmed mechanisms of resistance (phenotypically and/or genes)

• MIC distributions in different ESBL classes?

• Comparison of different fenotypical methods for detecton of resistance mechanisms?