J Antimicrob Chemother 2014; 69: 969 – 972 doi:10.1093/jac/dkt464 Advance Access publication 19 November 2013

Single origin of three plasmids bearing blaCTX-M-15 from different Klebsiella pneumoniae clones Juyoun Shin and Kwan Soo Ko* Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea

Received 5 August 2013; returned 9 September 2013; revised 21 October 2013; accepted 28 October 2013 Objectives: To determine and compare the complete nucleotide sequences of plasmids carrying blaCTX-M-15 from three different Klebsiella pneumoniae clones. Methods: IncFII-type plasmids pKP02022, pKP09085 and pKP007 were extracted from three K. pneumoniae strains. These strains belong to sequence types (STs) ST15, ST48 and ST23, respectively, and were isolated in Korea. Plasmids were sequenced using the 454 Genome Sequencer FLX system. Results: The three plasmids, pKP02022 (203577 bp), pKP09085 (213019 bp) and pKP007 (246176 bp), all exhibited a very similar structure, with a pKPN3-like backbone and a resistance region including blaOXA-1, aac(6 ′ )-Ib-cr and cat genes as well as blaCTX-M-15. They were also very similar to pUUH239.2, previously isolated in Sweden. Iron (III) uptake-related genes were found in pKP007 from the ST23 strain, which has been reported to be associated with liver abscesses. The resistance region contained several insertion sequences, such as IS26, which may play an important role in structural rearrangements of plasmids. Conclusions: The very similar structure of the three plasmids, extracted from different clones, suggests that the spread of CTX-M-producing K. pneumoniae isolates might result from the horizontal transfer of plasmids and subsequent integration and recombination. Keywords: extended-spectrum b-lactamases, ESBLs, IncFII, Enterobacteriaceae

Introduction Global spread of CTX-M-type extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae is a major concern. In particular, CTX-M-15 has increased rapidly and is now the most common ESBL in the world as well as in Asia.1,2 Although the spread of CTX-M-15 has been mainly associated with a high-risk clone of Escherichia coli, ST131,3 CTX-M-15-producing Klebsiella pneumoniae isolates are also disseminating worldwide.2,4 In particular, the IncF-type plasmid is considered to contribute to the dissemination of blaCTX-M-15 worldwide.5 In our previous study, blaCTX-M-15 was identified in diverse K. pneumoniae clones in Korea.1 Our replicon sequence typing study indicated that diverse IncF-type plasmids have incorporated into diverse strains of K. pneumoniae, contributing to the spread of the CTX-M-15 type.6 However, the intrinsic variability of replicon sequences has been reported, indicating the need for further characterization.7 Whole-plasmid sequencing and comparative analyses have provided significant information on the emergence and spread of antibiotic-resistant plasmids within and among Enterobacteriaceae.8,9 In the present study, we determined the complete sequences of plasmids carrying blaCTX-M-15 from three

different K. pneumoniae clones, in order to investigate the contribution of the IncFII-type plasmid to the spread of blaCTX-M-15.

Materials and methods Strains and plasmid extraction Plasmids containing blaCTX-M-15, which were designated pKP007, pKP02022 and pKP09085, were extracted from K. pneumoniae isolates K06-007, K01-02022 and K01-09085, respectively (Table 1). These isolates were from different patients with bacteraemia in Korea. These isolates have been characterized to produce CTX-M-15-type ESBLs and belonged to different sequence types (STs): ST23, ST15 and ST48.1 In our previous study, they belonged to the same incompatibility replicon group, IncFII, but showed different plasmid restriction fragment length polymorphism (pRFLP) patterns.6 Plasmids bearing blaCTX-M-15 were extracted by the conjugation method using the three K. pneumoniae isolates as donors and streptomycin-resistant E. coli DH5a as a recipient. Transconjugants were selected by plating on MacConkey agar containing ceftriaxone (30 mg/L) and streptomycin (300 mg/L). Plasmid DNA samples from the isolates and their transformants were extracted using a Qiagen Plasmid Midi Kit

# The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]

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*Corresponding author. Tel: +82-31-299-6223; Fax: +82-31-299-6229; E-mail: [email protected]

Shin and Soo Ko

Table 1. Characterization of three IncFII CTX-M-15-carrying plasmids from K. pneumoniae isolates Plasmid ST of donor isolate

name

size (bp)

no. of ORFs

resistance genes

addiction systems

K01-09085

48

pKP09085

213019

204

hok-mok, vagCD, pemKI

K01-02022

15

pKP02022

203577

191

K06-007

23

pKP007

246176

215

blaCTX-M-15, blaTEM-1, blaOXA-1, mphR, mrx, mph(A), dhfrXII, sul1, aac(6 ′ )-Ib-cr, cat-trunc blaCTX-M-15, blaTEM-1, blaOXA-1, cat, aac(6 ′ )-Ib-cr, tetA, tetR blaCTX-M-15, blaOXA-1, cat, aac(6 ′ )-Ib-cr, tetA, tetR

Isolate

Plasmid sequencing and bioinformatics Whole-plasmid sequencing of the blaCTX-M-15-carrying plasmids was performed with a Roche 454 Genome Sequencer FLX system (v 2.6). A total of 38477, 23630 and 24227 sequence reads were generated, yielding a mean sequence coverage of 9, 22 and 10 times for pKP007, pKP02022 and pKP09085, respectively. Sequencing reads were assembled into consensus de novo assembly contigs using the Roche 454 Genome Sequencer FLX software GSA Assembler (v 2.6), yielding 7, 9 and 14 contigs, respectively. Gaps between contigs were closed by PCR and standard Sanger sequencing. Open reading frames (ORFs) were predicted and annotated using the Glimmer 3.0 System (http://ccb.jhu.edu/software/glimmer/index.shtml) and confirmed with DNAMAN 5.2.10 software (Lynnon BioSoft, Lynnon Corporation; http://www.lynnon.com). Each predicted protein was compared against an all-protein database using BlastP (http://blast.ncbi.nlm.nih. gov/Blast.cgi) with a minimum cut-off of 30% identity and .80% length coverage. Gene sequences were further compared and aligned using cross_ match 1.080812 software and CLUSTALW (http://www.ebi.ac.uk/Tools/msa/ clustalw2) with reference plasmids pKPN3 (GenBank accession number NC_009649.1) and pUUH239.2 (GenBank accession number NC_016966.1).

Nucleotide sequence accession numbers The annotated sequences of pKP09085, pKP007 and pKP02022 have been submitted to the GenBank nucleotide sequence database (GenBank accession numbers KF719970, KF719971 and KF719972, respectively).

Results and discussion In this study, we determined whole sequences of plasmids bearing blaCTX-M-15 from three K. pneumoniae isolates, which showed different STs in multilocus sequence typing (MLST). One isolate, K01-02022, belongs to ST15, which has been reported as the main CTX-M-15-producing K. pneumoniae clone worldwide, including in Korea.4,10 ST23, to which K06-007 belongs, has been associated with invasive liver abscesses in Asian countries11 and ST48, to which K01-09085 belongs, is known as an international CTX-M-producing K. pneumoniae clone.12 Replicon typing showed that the three blaCTX-M-15-carrying plasmids from these isolates belong to the same incompatibility replicon group, IncFII.6 The three plasmids, pKP09085, pKP02022 and pKP007, were determined to be 213019, 203577 and 246176 bp in size, with average GC contents of 52.5%, 51.7% and 52.4%, respectively.

970

hok-mok, vagCD, pemKI

They contained 204, 191 and 215 predicted ORFs, respectively (Table 1). As shown in Figure 1, the overall structure of the three plasmids is highly similar. They encode typical IncFII plasmid replication functions and are common in having three addiction systems: the toxic killing protein hok-mok system, the toxin – antitoxin system pemIK and the vagCD virulence-associated genes. They are assumed to be highly transmissible because they were extracted by transconjugation into E. coli DH5a and they conserve the transfer region, traM to traC. In addition, they share antibiotic resistance genes, including blaCTX-M-15 and blaOXA-1 (b-lactams), aac(6 ′ )-Ib-cr (aminoglycosides and fluoroquinolones) and cat (chloramphenicol). The blaTEM-1 (b-lactams) gene is shared between pKP09085 and pKP02022, while the tetA (tetracyclines) and tetR (tetracyclines) genes are shared between pKP02022 and pKP007. The mphR and mph(A) (macrolides), mrx (macrolides), dhfrXII (trimethoprim) and sul1 (sulphonamide) genes are found only in pKP09085 (Table 1). As shown in Figure 1, the three plasmids in this study and two other retrieved plasmids, pUUH239.2 and pKPN3, can be divided into two regions: a pKPN3-like backbone and a resistance region. The pKPN3-like backbone, which includes an origin of replication and the tra locus, is a preserved region common to all five plasmids; the resistance region includes several antibiotic resistance genes and is rather variable. The pKPN3-like backbone (locus ptx to repB-repA in Figure 1) was identified in plasmid pKPN3 (GenBank accession number CP000648); this plasmid was isolated from a multidrug-resistant K. pneumoniae strain (MGH 78578) from Massachusetts (USA) in 1994. The pKPN3-like backbone was also found in other K. pneumoniae plasmids, such as pBK32179, carrying blaKPC-2, from New York City and pKPN-IT, carrying blaKPC-3, from Italy.13,14 The backbones of pKP02022, pKP007 and pUUH239.2 are different from that of plasmid pKPN3 in that two genes of addiction systems, vagD and hok, are inserted between psiAB and the tra locus (Figure 1). Although pKP09085 also has a backbone very similar to the other plasmids, the ydeA, parB and yefA genes were inserted between the ssb and psiAB genes; the positions of two addiction systems, vagCD and hok-mok, were also inverted in this plasmid. The three plasmids in this study are very similar to pUUH239.2 (GenBank accession number NC_016966.1), carrying blaCTX-M-15, from Sweden,9 including the resistance region (Figure 1). Particularly, pUUH239.2 and pKP09085 show very similar resistance regions; in addition to blaCTX-M-15, they share several antibiotic resistance genes, such as blaTEM-1, blaOXA-1, mphR, mph(A), mrx, qacED1, sul1, dhfrXII, and aac(6 ′ )-Ib-cr. Although there is rearrangement

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(Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The presence of blaCTX-M-15 in extracted plasmids was confirmed using PCR.

hok-mok, vagCD, pemKI

Whole sequences of plasmids bearing blaCTX-M-15 in K. pneumoniae

Figure 1. Major structural features of pKP09085, pKP02022 and pKP007 compared with those of IncFII-type plasmids pUUH239.2 (GenBank accession number NC_016966.1) and pKPN3 (GenBank accession number CP000648). The pKPN3-like backbone and resistance region are indicated. White boxes indicate plasmid scaffold regions that are in common among the plasmids. The tra locus is indicated within the white boxes with capital letters. Resistance genes are indicated by orange boxes, and transposon-related genes, integrase and insertion sequences are indicated by red boxes. Other genes are indicated by coloured boxes as follows: replicase genes as violet, restriction enzyme and DNA methylase genes as grey, and addiction system genes as green. Light grey shading denotes shared regions of homology. Arrows indicate blaCTX-M-15 genes in the plasmids. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

JAC

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Shin and Soo Ko

Acknowledgements The K. pneumoniae strains used in this study were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID, Seoul, Korea).

Funding This research was supported by the Basic Science Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0004848). J. Shin was supported partly by the Basic Science Research Program through the NRF funded by the Ministry of Education (2013R1A12062884).

Transparency declarations None to declare.

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References 1 Shin J, Kim DH, Ko KS. Comparison of CTX-M-14- and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae isolates from patients with bacteremia. J Infect 2011; 63: 39– 47. 2 Lee MY, Ko KS, Kang CI et al. High prevalence of CTX-M-15-producing Klebsiella pneumoniae isolates in Asian countries: diverse clones and clonal dissemination. Int J Antimicrob Agents 2011; 38: 160–3. 3 Woodford N, Turton JF, Livermore DM. Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 2011; 35: 736– 55. 4 Coelho A, Gonza´lez-Lo´pez J, Miro´ E et al. Characterization of the CTX-M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration. Int J Antimicrob Agents 2010; 36: 73–8. 5 Villa L, Garcı´a-Ferna´ndez A, Fortini D et al. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J Antimicrob Chemother 2010; 65: 2518– 29. 6 Shin J, Choi MJ, Ko KS. Replicon sequence typing of IncF plasmids and the genetic environments of blaCTX-M-15 indicate multiple acquisitions of blaCTX-M-15 in Escherichia coli and Klebsiella pneumoniae isolates from South Korea. J Antimicrob Chemother 2012; 67: 1853 –7. 7 Partridge SR, Zong Z, Iredell JR. Recombination in IS26 and Tn2 in the evolution of multiresistance regions carrying blaCTX-M-15 on conjugative IncF plasmids from Escherichia coli. Antimicrob Agents Chemother 2011; 55: 4971 –8. 8 Woodford N, Carattoli A, Karisik E et al. Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone. Antimicrob Agents Chemother 2009; 53: 4472– 82. 9 Sandegren L, Linkevicius M, Lytsy B et al. Transfer of an Escherichia coli ST131 multiresistance cassette has created a Klebsiella pneumoniaespecific plasmid associated with a major nosocomial outbreak. J Antimicrob Chemother 2012; 67: 74 –83. 10 Damjanova I, To´th A, Pa´szti J et al. Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type b-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? J Antimicrob Chemother 2008; 62: 978–85. 11 Chung DR, Lee HR, Lee SS et al. Evidence of clonal dissemination of the serotype K1 Klebsiella pneumoniae strain causing invasive liver abscesses in Korea. J Clin Microbiol 2008; 46: 4061 –3. 12 An S, Chen J, Wang Z et al. Predominant characteristics of CTX-M-producing Klebsiella pneumoniae isolates from patients with lower respiratory tract infection in multiple medical centers in China. FEMS Microbiol Lett 2012; 332: 137– 45. 13 Chen L, Chavda KD, Melano RG et al. Complete sequence of a blaKPC-2-harboring IncFIIK1 plasmid from a Klebsiella pneumoniae sequence type 258 strain. Antimicrob Agents Chemother 2013; 57: 1542– 5. 14 Garcı´a-Ferna´ndez A, Villa L, Carta C et al. Klebsiella pneumoniae ST258 producing KPC-3 identified in Italy carries novel plasmids and OmpK36/OmpK35 porin variants. Antimicrob Agents Chemother 2012; 56: 2143– 5.

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within the pKPN3-like backbone of pKP09085, pUUH239.2 and pKP09085 might have derived from the same plasmid. pKP02022 and pKP007 also showed similar resistance region characteristics with pUUH239.2, as shown in Figure 1. In particular, the pKPN3-like backbones of the three plasmids are the same. pKP007 has an inserted region including the LAC locus encoding b-D-galactosidase and iron (III) uptake-related genes (locus Fec) within its resistance region. The iron (III) uptake system may contribute the virulence of a bacterium, as it is likely involved in the acquisition of iron in the human host.14 It is interesting that ST23, to which pKP007 belongs, is the main clone associated with K. pneumoniae liver abscesses in Asian countries.11 Although ESBL-producing K. pneumoniae ST23 isolates have been rarely reported,11 our result suggests that the emergence of CTX-M-15-producing ST23 isolates might result from the horizontal transfer of a plasmid common in other K. pneumoniae clones. The resistance regions of pUUH239.2 and our three plasmids showed very similar characteristics to the blaCTX-M-15-carrying E. coli plasmids pEK499 and pC15-1a.8,9 Although rearrangements of the resistance regions of plasmids might occur due to some role of IS26 elements,7 addiction systems and several antibiotic resistance genes are shared between these K. pneumoniae and E. coli plasmids. Addiction systems have been known to ensure the maintenance of plasmids in the absence of any antibiotic pressure.8 In summary, this study described the complete sequences of three blaCTX-M-15-containing IncFII plasmids from K. pneumoniae strains belonging to different clones. They showed very similar structures, suggesting a common origin to pUUH239.2 from Sweden. Based on comparison of plasmid structures, pUUH239.2 and three plasmids from different K. pneumoniae clones might be composites of the pKPN3-like backbone prevailing in K. pneumoniae strains and resistance regions from ST131 E. coli plasmids carrying blaCTX-M-15. Plasmids with a pKPN3-like backbone might be successful since they possess several metal resistance genes and addiction systems. This study contributes to the understanding of the dissemination of CTX-M-15-producing K. pneumoniae isolates.

Single origin of three plasmids bearing blaCTX-M-15 from different Klebsiella pneumoniae clones.

To determine and compare the complete nucleotide sequences of plasmids carrying blaCTX-M-15 from three different Klebsiella pneumoniae clones...
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