Expansion of KPC-producing klebsiella pneumoniae with various mgrB-mutations giving rise to colistin-resistance: the role of ISL3 on plasmids

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University of Groningen

Expansion of KPC-producing klebsiella pneumoniae with various mgrB-mutations giving rise

to colistin-resistance

Giordano, Cesira; Barnini, Simona; Tsioutis, Constantinos; Chlebowicz, Monika A; Scoulica,

Effie V; Gikas, Achilleas; Rossen, John W; Friedrich, Alexander W; Bathoorn, Erik

Published in:

International journal of antimicrobial agents

DOI:

10.1016/j.ijantimicag.2017.10.011

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Giordano, C., Barnini, S., Tsioutis, C., Chlebowicz, M. A., Scoulica, E. V., Gikas, A., Rossen, J. W.,

Friedrich, A. W., & 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. https://doi.org/10.1016/j.ijantimicag.2017.10.011

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Short Communication

Expansion of KPC-producing Klebsiella pneumoniae with various

mgrB mutations giving rise to colistin resistance: the role of ISL3

on plasmids

Cesira 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, Italy}

b_{Department of Internal Medicine, Infectious Diseases Unit, University Hospital of Heraklion, Voutes, 71110, Heraklion, Crete, Greece} c_{School of Medicine, European University Cyprus, 6 Diogenis Street, Engomi, Nicosia 1516, Cyprus}

d_{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 2017 Accepted 21 October 2017 Editor: Dr Seydina Diene

Keywords: KPC Colistin Klebsiella pneumoniae Molecular epidemiology mgrB ISL3 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 other mechanisms. 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 patients were 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 lineages was observed. In two Italian MLST ST512 isolates and eight Greek ST258 isolates, an identical copy of ISL3 was inserted in mgrB nucleotide position 133. ISL3, a transposable restriction–modification system of 8154 nucleotides, was located on pKpQIL-like plasmids and may transpose into the chromosome. In four isolates, chromosomal integration of ISL3 in diverse inner membrane proteins other than mgrB was identified. 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 have

mgrB 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.

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-producing

K. pneumoniae (CPKP) are epidemic and represent a signiﬁcant

pro-portion (ca. 40%) of K. pneumoniae isolates[1–3]. CPKP have become a 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 by CPKP are difficult to treat and are usually associated with high mor-tality rates[5]. Colistin represents a last-line antibiotic choice in the treatment of bacterial infections by carbapenemase-producing Gram-negative isolates[6], representing a valid alternative treatment when carbapenems are not effective. However, increasing colistin use has led to resistance, especially during therapy[7]. In Italy, the rate of colistin-resistant K. pneumoniae rose from 36% in 2011 to 50% in 2015, leading to a worrisome recurrence of clinical outbreaks[8,9]. The majority of known resistance mechanisms in K. pneumoniae involve lipopolysaccharide (LPS) modification mainly occurring at the level of the outer membrane. An increasing number of studies have demonstrated that modifications of the PmrA/B and PhoQ/P two-component systems as well as inactivation of MgrB are able to decrease the affinity of colistin for LPS. PmrA/B and PhoQ/P regulate the pmrHFIJKLM operon, which controls modification of the

* Corresponding author. Department of Medical Microbiology, University of Groningen, 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.2017.10.011

Contents lists available atScienceDirect

International Journal of Antimicrobial Agents

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g

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outer membrane. The 144-bp mgrB gene encodes a small trans-membrane protein that exerts negative feedback on the abovementioned pathway by interaction with the sensor kinase PhoQ at the periplasmic level[10]. Different mutations occurring in the

mgrB gene have already been described as a major cause of

colis-tin resistance in K. pneumoniae. In resistant strains, complete and partial mgrB gene deletions[8,11], non-synonymous single nucleo-tide polymorphisms (SNPs)[12]and inactivation by various insertion sequence (IS) elements have been found[8,12,13]. The latter event has been repeatedly detected in colistin-heteroresistant nosoco-mial isolates and involves mgrB gene disruption by a variety of insertion sequences. However, we have little insight into the prev-alence of these IS elements in the plasmids of clinical isolates and their transposition patterns.

The present study aimed to investigate vertical and horizontal expansion of mutations occurring in mgrB, in particular those oc-curring through insertion of ISL3. Furthermore, the study aimed to identify the positions in the genome that constitute key targets of ISL3 sequences in colistin-resistant CPKP. For this purpose, whole genome sequences of various common nosocomial CPKP lineages isolated 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 colistin were randomly selected from unique patients at Azienda Ospedaliero–Universitaria Pisana (Pisa, Italy) during 2015–2016. Various biological samples were cultured on common isolation media in routine diagnostics and were incubated at 37 °C. Suspected colo-nies were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonics GmbH, Bremen, Germany). Antimicrobial susceptibility testing for amikacin, 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 microdilution assay (Sensititre™; Thermo Fisher, Waltham, MA). Minimum in-hibitory concentrations (MICs) were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.

Colistin-resistant isolates were stored in glycerol at−80 °C for DNA extraction. Total genomic DNA was extracted from fresh cul-tures using an UltraClean®_{Microbial DNA Isolation Kit (MoBio}

Laboratories, Carlsbad, CA) according to the manufacturer’s instruc-tions. The concentration and purity of the extracted DNA were determined with a Qubit®_{2.0 ﬂuorometer using the dsDNA BR Assay}

Kit (Life Technologies, Carlsbad, CA). A DNA library was prepared using Nextera XT v.01 (Illumina Inc., San Diego, CA) according to the 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 4891 alleles were performed using SeqSphere v.3.4.0 (Ridom GmbH, Münster, Germany). Assembled genomes were uploaded to the web tools ResFinder 2.1[15]to identify acquired resistance genes as well as to PlasmidFinder 1.3[16]to detect plasmids. The sequences are available 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 gene mutations associated with colistin resistance. Nucleotide positions refer to the coding sequence of the genes, starting with

the first base of the start codon. PROVEAN software tool was used to predict whether sequence variants at the nucleotide level re-sulted in amino acid substitutions with an impact on the biological function of proteins[17]. IS elements were identified using ISfinder

[18]. The ISL3 reference sequence (GI: NC_009650) was BLASTed against the whole genome sequence of all isolates. The contigs of plasmid sequences of interest, detected by PlasmidFinder, were mapped against complete reference sequences of plasmids pKpQIL-10 (KJ146687.1) and pKPN-IT (JN233704.1). BLAST Ring Image Generator (BRIG) was used to display plasmid sequence compari-sons[19].

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

3. Results

3.1. Isolate characteristics

The 29 isolates from Italy belonged to three different sequence types (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 with meropenem MICs of>8 mg/L. Isolates from ﬁve patients (17%) were resistant to all tested antibiotics, and in 10 other patients (34%) only one antibiotic tested susceptible (tigecycline, fosfomycin or SXT) (Supplementary Table S1). The blaKPC-3carbapenemase gene was

iden-tiﬁed in 28 isolates. In two isolates, both the blaKPC-3and blaVIM-1

carbapenemase genes were detected (Supplementary Table S2). In addition, 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 and aminoglycosides, were detected in all isolates except two. The dfrA12,

dfrA14 and dfrA17 genes associated with resistance to trimethoprim

were detected in 22 isolates.

The plasmid-encoded colistin resistance gene mcr-1.2 associ-ated with an IncX4 plasmid was detected in one isolate. Mutations of selected genes (mgrB, phoP, phoQ, pmrA and pmrB) associated with resistance to colistin are presented inTable 1. The mgrB gene was mutated in 22 colistin-resistant isolates. In 16 isolates, all belong-ing to ST512, there was a deletion of 11 nucleotides (Δ109–119) in

mgrB predicted to lead to a truncated MgrB protein of 37 amino acids

with a G37Y mutation. In two ST37 isolates there was a point mu-tation (A7T) creating a stop codon. Disruption of mgrB by insertion sequences was detected in four isolates: in two ST512 isolates a copy of the complete insertion sequence ISKpn26 (IS5 family) was de-tected at nucleotide position 75; and in another two ST512 the intact insertion sequence ISKpn25 (ISL3 family) was inserted at nucleo-tide position 133. Point mutations in the phoP gene were detected in two isolates. In one of these isolates, the nucleotide mutation T602C leading to amino acid substitution I201T was observed, pre-dicted to have a deleterious impact on the protein function. No mutations 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, no mutations in the abovementioned genes were found in four colistin-resistant isolates with MICs ranging from 4–8 mg/L.

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

The 22 Italian isolates with mutations in the mgrB, together with 19 isolates from the previous Greek study, were selected for wgMLST comparison. The selected isolates from the Greek study with mu-tations in mgrB or phoQ are shown inSupplementary Table S3. The characteristics of these isolates have been reported in a previous publication[12].Fig. 1shows a minimum spanning tree based on allelic mismatch between these isolates. The different MLST types are separated by an intermittent line. ST512 and ST258 are closely related to each other (minimum spanning of 127 alleles differ-ence), whereas ST37 and ST307 are distinct from all presented MLST types (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 clusters of isolates with identical insertions of ISL3 in nucleotide position 133 of mgrB: a cluster of eight isolates among the ST258 isolates from Greece, and a cluster of two isolates among the ST512 iso-lates from Italy. Also, two Greek ST258 isoiso-lates and two Italian ST512 isolates 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 harbouring the 11-nucleotide deletion in mgrB, a cluster of three ST258 iso-lates from Greece with the G110C substitution, and two ST37 isoiso-lates from Italy with the A7T substitution. Among Italian isolates, a clonal cluster of isolates harbouring the 11-nucleotide deletion in mgrB was observed, all belonging to ST512, collected in different hospi-tal wards. Among the Greek isolates, there was a cluster of seven ST258 isolates with disruption of the mgrB by ISL3.

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

ISL3, a transposable insertion sequence of 8154 nucleotides, is referred to as a type I restriction–modiﬁcation system harbouring a transposase (100% identity with ISL3 family transposase

WP_004152342.1), a DNA sequence speciﬁcity subunit (100%

identity with WP_004152344.1), a restriction endonuclease subunit (100% identity with WP_004152343.1) and a DNA methyl transferase subunit (100% identity with WP_004152345.1). As shown inFig. 2, the complete ISL3 sequence was localised on a pKpQIL-like plasmid, which was detected in all ﬁve MLST types. The ISL3 sequence was not detected in the two isolates (ST512 and ST37) in which the pKpQIL-like plasmid was absent. Intact ISL3 was not present on all other detected plasmids, including IncFIB(K) (26 isolates), ColRNAI (22 isolates), IncFII(K) (28 iso-lates), IncX3 (17 isoiso-lates), IncN (2 isoiso-lates), IncQ1 (1 isolate), IncX4 (1 isolate), IncFIA (1 isolate) and IncFIB (AP001918) (2 isolates). Se-quence comparison between the ISL3 on the pKpQIL-like plasmids and ISL3 inserted in mgrB shows a 100% match between the 8154 nucleotides.

Chromosomal integrations of ISL3 in positions other than mgrB were identiﬁed in ﬁve isolates (Table 2). In two Italian ST512/ KPC-3 isolates, ISL3 was inserted in inner membrane protein genes

marC and sbmA, respectively. One Greek ST258 isolate showed two

ISL3 insertions: one in the yidL gene, an AraC family putative tran-scriptional regulator; and one in the inner membrane protein gene

yfdC. 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 the intergenic region between the two-component system genes ycgF and ycgZ.

Table 1

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

Isolate Isolation date Ward MLST COL MIC

(mg/L) [category]

Mutations in

mgrB

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

ISL3-carrying plasmid

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.

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4. Discussion

Clonal expansion of KPC-KP isolates with various mutations in

mgrB in all lineages was shown. De novo acquisition of colistin

re-sistance can occur through transposition of insertion sequences with

mgrB as a target. Genetic comparison of KPC-KP lineages showed

that disruption of the chromosomal mgrB gene may be caused by transposition 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-KP isolates 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 red arrows point out clusters of isolates with identical insertions of insertion sequence ISL3 in nucleotide position 133 of mgrB.

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

Colistin resistance in KPC-KP was sporadically reported in the early days of the KPC-KP outbreak from 2007–2010[9,12]. In recent years, resistance to colistin with MICs varying from 4 mg/L to 256 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 expansion of colistin-resistant KPC-2-KP by mgrB has been reported in a recent study[12], and part of these data are also shown in the present study. The involvement of plasmids in colistin resistance was shown in two recent reports[21,22]. We now add to this the horizontal and ver-tical dissemination of colistin resistance by plasmids carrying ISL3 causing 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 are shown in red in the outer ring plasmid.

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observed that mgrB truncations with identical IS5 insertion ele-ments occurred among clonally unrelated isolates. The present study confirms this observation and identified the plasmid carrying these sequences. 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 assembly protein carried by pKpQIL was detected. Chromosomal targets of IS5 were also detected: in two other Italian isolates, besides the two isolates 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 but also other inner membrane protein-coding sequences such as marC,

yfdC, igaA and sbmA. This frequent insertion of ISL3 in mgrB and other

inner membrane protein sequences suggest that these targets are not random but are hotspots for integration of ISL3. In this study,

mgrB was disrupted by ISL3 at the identical nucleotide position 133

in two different lineages (Italian ST512 and Greek ST258), which shows that this insertion sequence recombines at speciﬁc target se-quence sites. This has also been described for IS5[13]. Thus, insertion sequences may functionally disrupt inner membrane proteins and thereby confer antibiotic resistance.

Transposable insertion sequences may have a general role in antibiotic resistance modiﬁcation in Enterobacteriaceae. For in-stance, mgrB was found truncated by IS5 family in another Klebsiella species, 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 in other Enterobacteriaceae. ISL3 family transposases are present in other Gram-negative isolates, including Escherichia coli (KC999035),

Citrobacter amalonaticus (CP011133.1) Raoultella ornithinolytica

(CP013340.1) and Pantoea ananatis (CP014207.1) with identities of 100%, 96%, 96% and 93%, respectively. These ISL3 sequences appear to have evolved within the same genera. Future studies may give answer to the question whether horizontal transmission of 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 clonal clusters 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 in KPC-KP lineages. Dissemination of ISL3 on pKpQIL plasmids that transpose into the same position in mgrB may explain colistin re-sistance in clonally unrelated isolates with identical mutations in

mgrB. This is cause for serious concern for public health as colistin

is among the few remaining treatment options for infections by multidrug-resistant Gram-negative pathogens.

Funding: This study was partly supported by the Innovation Fund

of the Department of Medical Microbiology of the University Medical Center 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, atdoi:10.1016/j.ijantimicag.2017.10.011.

References

[1]Conte V, Monaco M, Giani T, D’Ancona F, Moro ML, Arena F, et al. Molecular epidemiology of KPC-producing Klebsiella pneumoniae from invasive infections in 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 pneumoniae carbapenemases. Lancet Infect Dis 2013;13:785–96.

[3]Arnold RS, Thom KA, Sharma S, Phillips M, Johnson JK, Morgan DJ. Emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. South Med J 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: what are the treatment options? Drugs 2009;69:1879–901.

[6]Van Boeckel TP, Gandra S, Ashok A, Caudron Q, Grenfell BT, Levin SA, et al. Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales 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 Enterobacteriaceae in South Africa and evidence of in vivo selection of colistin resistance as a consequence of selective decontamination of the gastrointestinal tract. J Clin Microbiol 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-producing

Klebsiella 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 wards of 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 system by a membrane peptide. PLoS Genet 2009;5:e1000788.

[11]Halaby T, Kucukkose E, Janssen AB, Rogers MR, Doorduijn DJ, van der Zanden AG, et al. Genomic characterization of colistin heteroresistance in Klebsiella

pneumoniae during a nosocomial outbreak. Antimicrob Agents Chemother

2016;60:6837–43.

[12]Bathoorn E, Tsioutis C, da Silva Voorham JM, Scoulica EV, Ioannidou E, Zhou K, et al. Emergence of pan-resistance in KPC-2 carbapenemase-producing

Klebsiella pneumoniae in Crete, Greece: a close call. J Antimicrob Chemother

2016;71:1207–12.

[13]Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M, Türkoglu S, et al. The mgrB gene 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 outbreak strain 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 Antimicrob Chemother 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 plasmid multilocus sequence typing. Antimicrob Agents Chemother 2014;58:3895– 903.

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

[18]Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISﬁnder: the reference centre 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. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-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-mediated colistin resistance in a patient infected with Klebsiella pneumoniae. Lancet Infect Dis 2016;16:998–9.

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

[23]Jayol A, Poirel L, Villegas MV, Nordmann P. Modulation of mgrB gene expression as a source of colistin resistance in Klebsiella oxytoca. Int J Antimicrob Agents 2015;46:108–10.

Table 2

ISL3 insertion targets in the chromosome.

Isolate Country Insertion Gene function

1084 Italy marC Inner membrane protein

1235 Italy sbmA Inner membrane protein

554 Greece yidL AraC family putative transcriptional regulator

Greece yfdC Inner membrane protein

546 Greece igaA Inner membrane protein

517 Greece ycgF–ycgZ Two-component system genes for regulation of bioﬁlm and acid resistance 2 isolates Italy mgrB Inner membrane protein