University of Groningen

Expansion of KPC-producing klebsiella pneumoniae with various mgrB-mutations giving riseto colistin-resistanceGiordano, Cesira; Barnini, Simona; Tsioutis, Constantinos; Flissikowska, Monika; Scoulica,Effie V; Gikas, Achilleas; Rossen, Johannes; Friedrich, Alexander; Bathoorn, DerkPublished in:International journal of antimicrobial agents

DOI:10.1016/j.ijantimicag.2017.10.011

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2018

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Giordano, C., Barnini, S., Tsioutis, C., Chlebowicz, M. A., Scoulica, E. V., Gikas, A., ... Bathoorn, E. (2018).Expansion of KPC-producing klebsiella pneumoniae with various mgrB-mutations giving rise to colistin-resistance: the role of ISL3 on plasmids. International journal of antimicrobial agents, 51(2), 260-265. DOI:10.1016/j.ijantimicag.2017.10.011

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 13-07-2018

Short Communication

Expansion of KPC-producing Klebsiella pneumoniae with variousmgrB mutations giving rise to colistin resistance: the role of ISL3on plasmidsCesira Giordano

a, Simona Barnini a, Constantinos Tsioutis b,c, Monika A. Chlebowicz d,

Effie V. Scoulica c, Achilleas Gikas c, John W. Rossen d, Alexander W. Friedrich d,Erik Bathoorn d,*a Bacteriology Unit, Azienda Ospedaliero–Universitaria Pisana, via Paradisa 2, 56124, Pisa, Italyb Department of Internal Medicine, Infectious Diseases Unit, University Hospital of Heraklion, Voutes, 71110, Heraklion, Crete, Greecec School of Medicine, European University Cyprus, 6 Diogenis Street, Engomi, Nicosia 1516, Cyprusd University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, The Netherlands

A R T I C L E I N F O

Article history:Received 8 May 2017Accepted 21 October 2017Editor: Dr Seydina Diene

Keywords:KPCColistinKlebsiella pneumoniaeMolecular epidemiologymgrBISL3

A B S T R A C T

mcr-1 has been reported as the first plasmid-encoded gene conferring colistin resistance. In KPC-producing Klebsiella pneumoniae (KPC-KP), however, colistin resistance is rapidly emerging through othermechanisms. Resistance is frequently due to disruption of the mgrB gene by insertion sequences, e.g. ISL3.The aim of this study was to investigate the expansion of mgrB-mutated KPC-KP isolates. In addition,the localisation and targets of ISL3 sequences within the core and accessory genome of common KPC-KP lineages were identified. A total of 29 clinical K. pneumoniae isolates collected from Italian patientswere randomly selected. Whole genome sequences were analysed for resistance genes, plasmids and in-sertion sequences. In addition, 27 colistin-resistant KPC-KP isolates from a previous study from Crete (Greece)were assessed. Clonal expansion of KPC-KP isolates with various mutations in mgrB among all lineageswas observed. In two Italian MLST ST512 isolates and eight Greek ST258 isolates, an identical copy ofISL3 was inserted in mgrB nucleotide position 133. ISL3, a transposable restriction–modification systemof 8154 nucleotides, was located on pKpQIL-like plasmids and may transpose into the chromosome. Infour isolates, chromosomal integration of ISL3 in diverse inner membrane proteins other than mgrB wasidentified. Colistin resistance is most often explained by clonal expansion of isolates with mutated mgrB.pKpQIL-like plasmids, which are omnipresent in KPC-KP, carry insertion sequences such as ISL3 that havemgrB as a target hotspot for transposition. Transposition of insertion sequences from plasmids and sub-sequent clonal expansion may contribute to the emerging colistin resistance in KPC-KP.

© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Klebsiella pneumoniae has emerged as an important multidrug-resistant nosocomial pathogen worldwide. In some countries, e.g.Italy, Greece and some parts of the USA, carbapenemase-producingK. pneumoniae (CPKP) are epidemic and represent a significant pro-portion (ca. 40%) of K. pneumoniae isolates [1–3]. CPKP have becomea major clinical concern owing to their rapidly increasing resis-tance to nearly all currently available antibiotics [4]. Owing to

extended antibiotic resistance of these strains, infections caused byCPKP are difficult to treat and are usually associated with high mor-tality rates [5]. Colistin represents a last-line antibiotic choice in thetreatment of bacterial infections by carbapenemase-producing Gram-negative isolates [6], representing a valid alternative treatment whencarbapenems are not effective. However, increasing colistin use hasled to resistance, especially during therapy [7]. In Italy, the rate ofcolistin-resistant K. pneumoniae rose from 36% in 2011 to 50% in2015, leading to a worrisome recurrence of clinical outbreaks [8,9].

The majority of known resistance mechanisms in K. pneumoniaeinvolve lipopolysaccharide (LPS) modification mainly occurring atthe level of the outer membrane. An increasing number of studieshave demonstrated that modifications of the PmrA/B and PhoQ/Ptwo-component systems as well as inactivation of MgrB are ableto decrease the affinity of colistin for LPS. PmrA/B and PhoQ/Pregulate the pmrHFIJKLM operon, which controls modification of the

* Corresponding author. Department of Medical Microbiology, University ofGroningen, University Medical Center Groningen, Hanzeplein 1, hpc EB80, 9713 GZ,Groningen, The Netherlands.

E-mail address: d.bathoorn@umcg.nl (E. Bathoorn).

https://doi.org/10.1016/j.ijantimicag.2017.10.0110924-8579/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

International Journal of Antimicrobial Agents 51 (2018) 260–265

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

journal homepage: www.elsevier.com/ locate / i jant imicag

outer membrane. The 144-bp mgrB gene encodes a small trans-membrane protein that exerts negative feedback on theabovementioned pathway by interaction with the sensor kinase PhoQat the periplasmic level [10]. Different mutations occurring in themgrB gene have already been described as a major cause of colis-tin resistance in K. pneumoniae. In resistant strains, complete andpartial mgrB gene deletions [8,11], non-synonymous single nucleo-tide polymorphisms (SNPs) [12] and inactivation by various insertionsequence (IS) elements have been found [8,12,13]. The latter eventhas been repeatedly detected in colistin-heteroresistant nosoco-mial isolates and involves mgrB gene disruption by a variety ofinsertion sequences. However, we have little insight into the prev-alence of these IS elements in the plasmids of clinical isolates andtheir transposition patterns.

The present study aimed to investigate vertical and horizontalexpansion of mutations occurring in mgrB, in particular those oc-curring through insertion of ISL3. Furthermore, the study aimed toidentify the positions in the genome that constitute key targets ofISL3 sequences in colistin-resistant CPKP. For this purpose, wholegenome sequences of various common nosocomial CPKP lineagesisolated in two different regions, namely Pisa (Italy) and Crete(Greece), were analysed and compared.

2. Materials and methods

A total of 29 clinical K. pneumoniae isolates resistant to colistinwere randomly selected from unique patients at AziendaOspedaliero–Universitaria Pisana (Pisa, Italy) during 2015–2016.Various biological samples were cultured on common isolation mediain routine diagnostics and were incubated at 37 °C. Suspected colo-nies were identified by matrix-assisted laser desorption/ionisationtime-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker DaltonicsGmbH, Bremen, Germany). Antimicrobial susceptibility testing foramikacin, amoxicillin/clavulanic acid, ampicillin/sulbactam, cefepime,cefotaxime, ceftazidime, ciprofloxacin, colistin, ertapenem,fosfomycin, gentamicin, imipenem, levofloxacin, meropenem, ni-trofurantoin, piperacillin/tazobactam, tigecycline and trimethoprim/sulfamethoxazole (SXT) were performed by broth microdilutionassay (Sensititre™; Thermo Fisher, Waltham, MA). Minimum in-hibitory concentrations (MICs) were interpreted according toEuropean Committee on Antimicrobial Susceptibility Testing(EUCAST) guidelines.

Colistin-resistant isolates were stored in glycerol at −80 °C forDNA extraction. Total genomic DNA was extracted from fresh cul-tures using an UltraClean® Microbial DNA Isolation Kit (MoBioLaboratories, Carlsbad, CA) according to the manufacturer’s instruc-tions. The concentration and purity of the extracted DNA weredetermined with a Qubit® 2.0 fluorometer using the dsDNA BR AssayKit (Life Technologies, Carlsbad, CA). A DNA library was preparedusing Nextera XT v.01 (Illumina Inc., San Diego, CA) according tothe manufacturer’s instructions and was then run on a MiSeq system(Illumina Inc.) to generate 250-bp paired-end reads. De novo as-sembly was performed using CLC Genomics Workbench v.9.5.2(QIAGEN, Hilden, Germany) after quality trimming (Qs ≥ 20) [14].Multilocus sequence typing (MLST) as well as whole-genome MLST(wgMLST) comparison based on an ad hoc scheme including 4891alleles were performed using SeqSphere v.3.4.0 (Ridom GmbH,Münster, Germany). Assembled genomes were uploaded to the webtools ResFinder 2.1 [15] to identify acquired resistance genes as wellas to PlasmidFinder 1.3 [16] to detect plasmids. The sequences areavailable from the European Nucleotide Archive (study PRJEB19808)The DNA sequences of mgrB (GI: 695277517), phoQ (GI: 378976159),phoP (GI: 378976159), pmrA (HG794234.1:3372–4043) and pmrB(HG794234.1:2271–3368) were used as references for detecting genemutations associated with colistin resistance. Nucleotidepositions refer to the coding sequence of the genes, starting with

the first base of the start codon. PROVEAN software tool was usedto predict whether sequence variants at the nucleotide level re-sulted in amino acid substitutions with an impact on the biologicalfunction of proteins [17]. IS elements were identified using ISfinder[18]. The ISL3 reference sequence (GI: NC_009650) was BLASTedagainst the whole genome sequence of all isolates. The contigs ofplasmid sequences of interest, detected by PlasmidFinder, weremapped against complete reference sequences of plasmids pKpQIL-10 (KJ146687.1) and pKPN-IT (JN233704.1). BLAST Ring ImageGenerator (BRIG) was used to display plasmid sequence compari-sons [19].

In addition, 19 colistin-resistant isolates were selected from apreviously characterised collection of 34 randomly assessed clin-ical KPC-producing K. pneumoniae (KPC-KP) isolates collectedbetween 2010–2014 in Crete (Greece) and deposited at the Euro-pean Nucleotide Archive (study PRJEB10561) for molecularcomparison [12].

3. Results

3.1. Isolate characteristics

The 29 isolates from Italy belonged to three different sequencetypes (2 to ST307, 2 to ST37 and 25 to ST512). All isolates were re-sistant to colistin with MICs ranging from 4 mg/L to >8 mg/L (Table 1).Of the 29 isolates, 27 were resistant to carbapenems withmeropenem MICs of >8 mg/L. Isolates from five patients (17%) wereresistant to all tested antibiotics, and in 10 other patients (34%) onlyone antibiotic tested susceptible (tigecycline, fosfomycin or SXT)(Supplementary Table S1). The blaKPC-3 carbapenemase gene was iden-tified in 28 isolates. In two isolates, both the blaKPC-3 and blaVIM-1

carbapenemase genes were detected (Supplementary Table S2). Inaddition, different combination of β-lactamase genes encoding CTX-M-15, OXA-1, OXA-9, SHV-11, SHV-28 and TEM-1A were detected.Several aminoglycoside resistance genes, including the aac(6’)-Ib-cr gene responsible for low-level resistance to fluoroquinolones andaminoglycosides, were detected in all isolates except two. The dfrA12,dfrA14 and dfrA17 genes associated with resistance to trimethoprimwere detected in 22 isolates.

The plasmid-encoded colistin resistance gene mcr-1.2 associ-ated with an IncX4 plasmid was detected in one isolate. Mutationsof selected genes (mgrB, phoP, phoQ, pmrA and pmrB) associated withresistance to colistin are presented in Table 1. The mgrB gene wasmutated in 22 colistin-resistant isolates. In 16 isolates, all belong-ing to ST512, there was a deletion of 11 nucleotides (Δ109–119) inmgrB predicted to lead to a truncated MgrB protein of 37 amino acidswith a G37Y mutation. In two ST37 isolates there was a point mu-tation (A7T) creating a stop codon. Disruption of mgrB by insertionsequences was detected in four isolates: in two ST512 isolates a copyof the complete insertion sequence ISKpn26 (IS5 family) was de-tected at nucleotide position 75; and in another two ST512 the intactinsertion sequence ISKpn25 (ISL3 family) was inserted at nucleo-tide position 133. Point mutations in the phoP gene were detectedin two isolates. In one of these isolates, the nucleotide mutationT602C leading to amino acid substitution I201T was observed, pre-dicted to have a deleterious impact on the protein function. Nomutations were detected within the phoQ gene. In two ST307 iso-lates, a point mutation G121A was detected in the pmrA gene,resulting in amino acid change A41T that is potentially deleteri-ous. Furthermore, in one of the latter isolates, an additional G385C(amino acid A129P) mutation in the pmrB gene was detected, pre-dicted to be deleterious. In one ST512 isolate a three-nucleotide in-frame insertion in pmrB at position 403 was detected. Finally, nomutations in the abovementioned genes were found in four colistin-resistant isolates with MICs ranging from 4–8 mg/L.

261C. Giordano et al. / International Journal of Antimicrobial Agents 51 (2018) 260–265

3.2. Investigation of clonal expansion by whole-genome multilocussequence typing (wgMLST)

The 22 Italian isolates with mutations in the mgrB, together with19 isolates from the previous Greek study, were selected for wgMLSTcomparison. The selected isolates from the Greek study with mu-tations in mgrB or phoQ are shown in Supplementary Table S3. Thecharacteristics of these isolates have been reported in a previouspublication [12]. Fig. 1 shows a minimum spanning tree based onallelic mismatch between these isolates. The different MLST typesare separated by an intermittent line. ST512 and ST258 are closelyrelated to each other (minimum spanning of 127 alleles differ-ence), whereas ST37 and ST307 are distinct from all presented MLSTtypes (minimum spanning of >3000 alleles difference).

Five clonally related clusters of isolates with identical muta-tions in mgrB were observed. The red arrows point out two clustersof isolates with identical insertions of ISL3 in nucleotide position133 of mgrB: a cluster of eight isolates among the ST258 isolatesfrom Greece, and a cluster of two isolates among the ST512 iso-lates from Italy. Also, two Greek ST258 isolates and two Italian ST512isolates showed identical disruption of mgrB at nucleotide posi-tion 75 by IS5.

Other clusters of isolates with identical mutations in mgrB in-cluded a clonal cluster of 16 ST512 isolates from Italy harbouringthe 11-nucleotide deletion in mgrB, a cluster of three ST258 iso-lates from Greece with the G110C substitution, and two ST37 isolatesfrom Italy with the A7T substitution. Among Italian isolates, a clonalcluster of isolates harbouring the 11-nucleotide deletion in mgrBwas observed, all belonging to ST512, collected in different hospi-tal wards. Among the Greek isolates, there was a cluster of sevenST258 isolates with disruption of the mgrB by ISL3.

3.3. Position of ISL3 in the genome: plasmid position andchromosomal targets

ISL3, a transposable insertion sequence of 8154 nucleotides, isreferred to as a type I restriction–modification system harbouringa transposase (100% identity with ISL3 family transposaseWP_004152342.1), a DNA sequence specificity subunit (100%identity with WP_004152344.1), a restriction endonucleasesubunit (100% identity with WP_004152343.1) and a DNA methyltransferase subunit (100% identity with WP_004152345.1). Asshown in Fig. 2, the complete ISL3 sequence was localised on apKpQIL-like plasmid, which was detected in all five MLST types.The ISL3 sequence was not detected in the two isolates (ST512and ST37) in which the pKpQIL-like plasmid was absent. IntactISL3 was not present on all other detected plasmids, includingIncFIB(K) (26 isolates), ColRNAI (22 isolates), IncFII(K) (28 iso-lates), IncX3 (17 isolates), IncN (2 isolates), IncQ1 (1 isolate), IncX4(1 isolate), IncFIA (1 isolate) and IncFIB (AP001918) (2 isolates). Se-quence comparison between the ISL3 on the pKpQIL-like plasmidsand ISL3 inserted in mgrB shows a 100% match between the 8154nucleotides.

Chromosomal integrations of ISL3 in positions other than mgrBwere identified in five isolates (Table 2). In two Italian ST512/KPC-3 isolates, ISL3 was inserted in inner membrane protein genesmarC and sbmA, respectively. One Greek ST258 isolate showed twoISL3 insertions: one in the yidL gene, an AraC family putative tran-scriptional regulator; and one in the inner membrane protein geneyfdC. In a second Greek isolate, ISL3 was inserted in the inner mem-brane protein gene igaA, and in a third isolate it was inserted in theintergenic region between the two-component system genes ycgFand ycgZ.

Table 1Molecular characteristics of 29 Italian KPC-producing Klebsiella pneumoniae isolates associated with colistin resistance.

Isolate Isolation date Ward MLST COL MIC(mg/L)[category]

Mutations inmgrB

Mutations in phoP, phoQ, pmrA and pmrB andpresence of mcr-1

ISL3-carryingplasmid

1084 July 2015 Pneumology ST512 >8 [R] – – IncFIB(pQil)1091 July 2015 General medicine ST512 >8 [R] – – IncFIB(pQil)1122 Aug. 2015 Emergency medicine ST512 >8 [R] Δ109–119 phoP T602C; pmrB 697 (nnt), G245T* –1123 Feb. 2015 Neurology ST512 4 [R] IS5 75 phoP C55A* IncFIB(pQil)1129 Sept. 2015 General medicine ST307 4 [R] – pmrA G121A; pmrB G385C, A637C*, G766C* IncFIB(pQil)1145 Oct. 2015 Intensive care ST512 >8 [R] Δ109–119 – IncFIB(pQil)1147a Feb. 2015 Intensive care ST512 >8 [R] – – IncFIB(pQil)1196 April 2015 Intensive care ST307 >8 [R] – pmrA G121A; pmrB G245T*, C637A*, G766C* IncFIB(pQil)1201b April 2015 Intensive care ST512 4 [R] – – IncFIB(pQil)1206 Dec. 2015 Burn centre ST512 >8 [R] ISL3 133 – IncFIB(pQil)1043 May 2015 Intensive care ST512 >8 [R] Δ109–119 – IncFIB(pQil)1059 June 2015 Intensive care ST512 >8 [R] Δ109–119 – IncFIB(pQil)1076 June 2015 Burn centre ST512 >8 [R] Δ109–119 – IncFIB(pQil)1079 June 2015 Intensive care ST512 >8 [R] Δ109–119 – IncFIB(pQil)1088 July 2015 Nephrology and dialysis ST512 >8 [R] Δ109–119 – IncFIB(pQil)1126 Sept. 2015 Burn centre ST512 >8 [R] Δ109–119 – IncFIB(pQil)1133 Feb. 2015 General medicine ST512 >8 [R] Δ109–119 – IncFIB(pQil)1136 Sept. 2015 Haematology ST512 >8 [R] Δ109–119 – IncFIB(pQil)1152 Nov. 2015 Intensive care ST512 >8 [R] Δ109–119 – IncFIB(pQil)1168 July 2015 Burn centre ST37 >8 [R] A7T stop codon – IncFIB(pQil)1187 March 2015 General medicine ST512 >8 [R] Δ109–119 – IncFIB(pQil)1195 April 2015 Neurology ST512 >8 [R] Δ109–119 – IncFIB(pQil)1203 April 2015 Intensive care ST512 >8 [R] Δ109–119 – IncFIB(pQil)1140 Sept. 2015 Paediatric haematology ST512 >8 [R] – mcr-1.2 IncFIB(pQil)1147b Oct. 2015 Haematology ST512 >8 [R] Δ109–119 – IncFIB(pQil)1208 Jan. 2016 Burn centre ST512 >8 [R] ISL3 133 – IncFIB(pQil)1235 Feb. 2016 Infectious diseases ST512 >8 [R] IS5 75 – IncFIB(pQil)1236 Feb. 2016 Cardiology ST37 >8 [R] A7T stop codon pmrA C121T IncFIB(pQil)1307 June 2016 General medicine ST512 >8 [R] Δ109–119 – IncFIB(pQil)

MLST, multilocus sequence typing; COL, colistin; MIC, minimum inhibitory concentration; R, resistant.Non-synonymous nucleotide mutations and their positions in the reference coding sequence are presented.

* Neutral mutation predicted not to cause functional changes to the protein.

262 C. Giordano et al. / International Journal of Antimicrobial Agents 51 (2018) 260–265

4. Discussion

Clonal expansion of KPC-KP isolates with various mutations inmgrB in all lineages was shown. De novo acquisition of colistin re-sistance can occur through transposition of insertion sequences withmgrB as a target. Genetic comparison of KPC-KP lineages showed

that disruption of the chromosomal mgrB gene may be caused bytransposition of the ISL3 insertion sequence carried by pKpQIL-like plasmids. This ISL3-carrying plasmid is omnipresent in KPC-KP, both in KPC-2-KP lineages isolated in Greece and in KPC-3-KPisolates originating from Italy. Horizontal dissemination of plas-mids harbouring insertion sequences, e.g. ISL3, could facilitate the

Fig. 1. Minimum spanning tree based on allelic mismatch between 22 Italian and 27 Greek KPC-producing Klebsiella pneumoniae isolates with mutations in mgrB. The span-ning tree is based on 4891 alleles, pairwise ignoring missing values. The colour of the spots represent mutations in mgrB identified in the sequence of the isolate. The redarrows point out clusters of isolates with identical insertions of insertion sequence ISL3 in nucleotide position 133 of mgrB.

263C. Giordano et al. / International Journal of Antimicrobial Agents 51 (2018) 260–265

acquisition of colistin resistance de novo. The observed subse-quent clonal expansion contributes to the emergence of colistinresistance in KPC-KP in Southern Europe.

Colistin resistance in KPC-KP was sporadically reported in theearly days of the KPC-KP outbreak from 2007–2010 [9,12]. In recentyears, resistance to colistin with MICs varying from 4 mg/L to256 mg/L has been reported in studies from Greece and Italy [12,20].Insertional inactivation of mgrB by insertion sequences was most

often associated with colistin resistance. Vertical clonal expansionof colistin-resistant KPC-2-KP by mgrB has been reported in a recentstudy [12], and part of these data are also shown in the present study.The involvement of plasmids in colistin resistance was shown in tworecent reports [21,22]. We now add to this the horizontal and ver-tical dissemination of colistin resistance by plasmids carrying ISL3causing disruption of mgrB. A plasmid origin of insertion se-quences targeting mgrB has been suggested by Poirel et al [13]. They

Fig. 2. Genome comparison of ISL3-carrying pKpQIL-like plasmids in Italian and Greek KPC-producing Klebsiella pneumoniae isolates. Plasmids are grouped by colour ac-cording to different multilocus locus sequence typing (MLST) types and countries where the isolates have been detected. The subunits of the complete ISL3 sequence areshown in red in the outer ring plasmid.

264 C. Giordano et al. / International Journal of Antimicrobial Agents 51 (2018) 260–265

observed that mgrB truncations with identical IS5 insertion ele-ments occurred among clonally unrelated isolates. The present studyconfirms this observation and identified the plasmid carrying thesesequences. We focused on ISL3, however IS5 was also frequently de-tected on the same pKpQIL-like plasmids. In five isolates from Italy,disruption by IS5 of the traE gene coding for the pilus assemblyprotein carried by pKpQIL was detected. Chromosomal targets ofIS5 were also detected: in two other Italian isolates, besides the twoisolates with disrupted mgrB, IS5 was found to disrupt a chromo-somal hypothetical protein.

In this study, it was shown that ISL3 not only targets mgrB butalso other inner membrane protein-coding sequences such as marC,yfdC, igaA and sbmA. This frequent insertion of ISL3 in mgrB and otherinner membrane protein sequences suggest that these targets arenot random but are hotspots for integration of ISL3. In this study,mgrB was disrupted by ISL3 at the identical nucleotide position 133in two different lineages (Italian ST512 and Greek ST258), whichshows that this insertion sequence recombines at specific target se-quence sites. This has also been described for IS5 [13]. Thus, insertionsequences may functionally disrupt inner membrane proteins andthereby confer antibiotic resistance.

Transposable insertion sequences may have a general role inantibiotic resistance modification in Enterobacteriaceae. For in-stance, mgrB was found truncated by IS5 family in another Klebsiellaspecies, namely Klebsiella oxytoca, resulting in colistin resistance,which is similar to reports in K. pneumoniae [13,23]. In this study,a BLASTn search was performed to identify the presence of ISL3 inother Enterobacteriaceae. ISL3 family transposases are present inother Gram-negative isolates, including Escherichia coli (KC999035),Citrobacter amalonaticus (CP011133.1) Raoultella ornithinolytica(CP013340.1) and Pantoea ananatis (CP014207.1) with identitiesof 100%, 96%, 96% and 93%, respectively. These ISL3 sequencesappear to have evolved within the same genera. Future studiesmay give answer to the question whether horizontal transmissionof insertion sequences may occur among different genera.

In conclusion, an international collaborative study on the mo-lecular epidemiology of KPC-KP resulted in identification of clonalclusters with various mutations in mgrB leading to resistance to co-listin. Expansion of a pKpQIL-like plasmid was demonstrated.Insertion sequences originate from this plasmid and target mgrB inKPC-KP lineages. Dissemination of ISL3 on pKpQIL plasmids thattranspose into the same position in mgrB may explain colistin re-sistance in clonally unrelated isolates with identical mutations inmgrB. This is cause for serious concern for public health as colistinis among the few remaining treatment options for infections bymultidrug-resistant Gram-negative pathogens.

Funding: This study was partly supported by the Innovation Fundof the Department of Medical Microbiology of the University MedicalCenter Groningen (Groningen, The Netherlands).

Competing interests: None declared.Ethical approval: Not required.

Appendix. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.ijantimicag.2017.10.011.

References

[1] Conte V, Monaco M, Giani T, D’Ancona F, Moro ML, Arena F, et al. Molecularepidemiology of KPC-producing Klebsiella pneumoniae from invasive infectionsin Italy: increasing diversity with predominance of the ST512 clade II sublineage.J Antimicrob Chemother 2016;71:3386–91.

[2] Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M,et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniaecarbapenemases. Lancet Infect Dis 2013;13:785–96.

[3] Arnold RS, Thom KA, Sharma S, Phillips M, Johnson JK, Morgan DJ. Emergenceof Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. South MedJ 2011;104:40–5.

[4] Giacobbe DR, Del Bono V, Trecarichi EM, De Rosa FG, Giannella M, Bassetti M,et al. Risk factors for bloodstream infections due to colistin-resistant KPC-producing Klebsiella pneumoniae: results from a multicenter case–control–control study. Clin Microbiol Infect 2015;21:1106–8.

[5] Giamarellou H, Poulakou G. Multidrug-resistant Gram-negative infections: whatare the treatment options? Drugs 2009;69:1879–901.

[6] Van Boeckel TP, Gandra S, Ashok A, Caudron Q, Grenfell BT, Levin SA, et al. Globalantibiotic consumption 2000 to 2010: an analysis of national pharmaceuticalsales data. Lancet Infect Dis 2014;14:742–50.

[7] Brink AJ, Coetzee J, Corcoran C, Clay CG, Hari-Makkan D, Jacobson RK, et al.Emergence of OXA-48 and OXA-181 carbapenemases among Enterobacteriaceaein South Africa and evidence of in vivo selection of colistin resistance as aconsequence of selective decontamination of the gastrointestinal tract. J ClinMicrobiol 2013;51:369–72.

[8] Giani T, Arena F, Vaggelli G, Conte V, Chiarelli A, Henrici De Angelis L, et al.Large nosocomial outbreak of colistin-resistant, carbapenemase-producingKlebsiella pneumoniae traced to clonal expansion of an mgrB deletion mutant.J Clin Microbiol 2015;53:3341–4.

[9] Mammina C, Bonura C, Di Bernardo F, Aleo A, Fasciana T, Sodano C, et al.Ongoing spread of colistin-resistant Klebsiella pneumoniae in different wardsof an acute general hospital, Italy, June to December 2011. Euro Surveill 2012;17.20248 [pii].

[10] Lippa AM, Goulian M. Feedback inhibition in the PhoQ/PhoP signaling systemby a membrane peptide. PLoS Genet 2009;5:e1000788.

[11] Halaby T, Kucukkose E, Janssen AB, Rogers MR, Doorduijn DJ, van der ZandenAG, et al. Genomic characterization of colistin heteroresistance in Klebsiellapneumoniae during a nosocomial outbreak. Antimicrob Agents Chemother2016;60:6837–43.

[12] Bathoorn E, Tsioutis C, da Silva Voorham JM, Scoulica EV, Ioannidou E, ZhouK, et al. Emergence of pan-resistance in KPC-2 carbapenemase-producingKlebsiella pneumoniae in Crete, Greece: a close call. J Antimicrob Chemother2016;71:1207–12.

[13] Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M, Türkoglu S, et al. The mgrBgene as a key target for acquired resistance to colistin in Klebsiella pneumoniae.J Antimicrob Chemother 2015;70:75–80.

[14] Zhou K, Lokate M, Deurenberg RH, Arends J, Lo-Ten Foe J, Grundmann H, et al.Characterization of a CTX-M-15 producing Klebsiella pneumoniae outbreakstrain assigned to a novel sequence type (1427). Front Microbiol 2015;6:1250.

[15] Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al.Identification of acquired antimicrobial resistance genes. J AntimicrobChemother 2012;67:2640–4.

[16] Carattoli A, Zankari E, Garcia-Fernandez A, Voldby Larsen M, Lund O, Villa L,et al. In silico detection and typing of plasmids using PlasmidFinder and plasmidmultilocus sequence typing. Antimicrob Agents Chemother 2014;58:3895–903.

[17] Choi Y, Chan AP. PROVEAN web server: a tool to predict the functionaleffect of amino acid substitutions and indels. Bioinformatics 2015;31:2745–7.

[18] Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the referencecentre for bacterial insertion sequences. Nucleic Acids Res 2006;34:D32–6.Database issue.

[19] Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator(BRIG): simple prokaryote genome comparisons. BMC Genomics 2011;12:402.

[20] Cannatelli A, D’Andrea MM, Giani T, Di Pilato V, Arena F, Ambretti S, et al. Invivo emergence of colistin resistance in Klebsiella pneumoniae producingKPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother 2013;57:5521–6.

[21] Nicolet S, Goldenberger D, Schwede T, Page M, Creus M. Plasmid-mediatedcolistin resistance in a patient infected with Klebsiella pneumoniae. Lancet InfectDis 2016;16:998–9.

[22] Antonelli A, D’Andrea MM, Giani T, Rossolini GM. Plasmid-mediated orchromosomally mediated colistin resistance in Klebsiella pneumoniae? LancetInfect Dis 2017;17:26–7.

[23] Jayol A, Poirel L, Villegas MV, Nordmann P. Modulation of mgrB gene expressionas a source of colistin resistance in Klebsiella oxytoca. Int J Antimicrob Agents2015;46:108–10.

Table 2ISL3 insertion targets in the chromosome.

Isolate Country Insertion Gene function

1084 Italy marC Inner membrane protein1235 Italy sbmA Inner membrane protein554 Greece yidL AraC family putative transcriptional

regulatorGreece yfdC Inner membrane protein

546 Greece igaA Inner membrane protein517 Greece ycgF–ycgZ Two-component system genes for

regulation of biofilm and acid resistance2 isolates Italy mgrB Inner membrane protein10 isolates Greece mgrB Inner membrane protein

265C. Giordano et al. / International Journal of Antimicrobial Agents 51 (2018) 260–265