and IncN3-type plasmids in a Klebsiella pneumoniae isolate from Korea

Transcription

1 AAC Accepted Manuscript Posted Online 7 December 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Complete sequences of bla KPC-2 -harboring plasmid with a mosaic of IncN1- and IncN3-type plasmids in a Klebsiella pneumoniae isolate from Korea Short title: A plasmid harboring bla KPC-2 from Korea So Yeon Kim, 1 Ji-Young Rhee, 2 and Kwan Soo Ko 1 * 1 Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon , Korea 2 Division of Infectious Diseases, Department of Medicine, Dankook University, Cheonan , Korea * Corresponding author: Kwan Soo Ko, PhD Department of Molecular Cell Biology Sungkyunkwan University School of Medicine Suwon , Korea Phone: , Fax: , ksko@skku.edu -1-

2 A carbapenem-resistant Klebsiella pneumoniae isolate was isolated from the pus of a patient in a Korean hospital in September The isolate belonged to ST1612, which has been found in Japan (1). Because the Japanese ST1612 isolates did not produce KPC-2, the Korean isolate did probably acquire the plasmid bearing bla KPC-2 from other species or clone. In the present study, we obtained complete sequences of a plasmid carrying bla KPC-2 from the Korean K. pneumoniae isolate to understand the emergence of a new KPC-2-producing clone, and the spread of the bla KPC-2 gene because of the mobility of Tn4401 was shown. A plasmid carrying bla KPC-2 was extracted by the conjugation method using the sodium azide-resistant Escherichia coli strain J53 as a recipient (Table 1) (2). Complete sequencing of the bla KPC-2 -carrying plasmid was performed using a Roche 454 GS-FLX system (v 2.9) (GenBank accession number, KR091915). A total of 17,297 sequence reads were generated, yielding a mean sequence coverage of 215 for the plasmid pkpc-dk05. Gaps between obtained two contigs were closed by PCR and standard Sanger sequencing, using a primer set (forward, 5 -GAA AGA ACG CGC TCA GGC CTT GCA-3 ; reverse, 5 - CGC TTC CCG TTG CTG TTC ACG CTG-3 ). Open reading frames were predicted and annotated using the Glimmer 3.0 System ( and confirmed in the DNAMAN software (Lynnon BioSoft, Lynnon Corporation; The plasmid pkpc-dk05 was found to be 56,728 bp in size, with the average GC content of 50.5%. It contains 45 predicted open reading frames. As shown in Fig. 1A, replicon of the plasmid has structure highly similar to that of the IncN1-type pr46 plasmid of Salmonella enterica R46 (GenBank, AY046276), but its tra locus homologous to the IncN3- type plasmid pn-cit of Citrobacter freundii CFSTE (JQ ). Thus, it is likely that pkpn-dk05 is a mosaic of plasmids with two different types. The pkpc-dk05 plasmid also contains two regions that we predicted to be involved in the conjugative transfer of plasmid -2-

3 DNA. The transfer region containing tra systems may contribute to the high transmissibility of the plasmid. The genes that are located upstream of the trakji regions were predicted to play a role in plasmid stability (the gray region in Fig. 1A). It is known that the stbabc operon stabilizes single-stranded DNA during conjugation (3, 4). The carbapenem resistance of the isolate that we uncovered in this study may not be due to the horizontal transfer of the plasmid and clonal spread because similar plasmids carrying bla KPC-2 have not been reported and because the K. pneumoniae isolate carrying pkpc-dk05 has a novel genotype unknown to produce KPC (5-8). Thus, we can speculate that our carbapenemase-producing K. pneumoniae isolate emerged via incorporation of Tn4401 into the prevailing plasmid. The genetic structure of Tn4401 was compared with several bla KPC -carrying IncN plasmids (Fig. 1B) (9, 10). Our pkpc-dk05 plasmid shows structure very similar to that of pkox105 of K. oxytoca from Italy and pkpc_fcf13/05 of K. pneumoniae ST442 from Brazil. This result suggests that KPC production by our strain is likely to be the result of incorporation of a Tn4401 into a plasmid showing features similar to those of a C. freundii plasmid. In summary, we described the complete sequence of a recombinant bla KPC-2 -carrying plasmid from a Korean K. pneumoniae strain. This plasmid might have emerged via incorporation of a Tn4401 into a plasmid with an origin of the mosaic plasmid with IncN1- typed replicon and IncN3-typed tra system. Acknowledgements -3-

4 This work was supported in part by the Basic Science Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A ) Downloaded from on September 6, 2018 by guest -4-

5 References 1. Ito R, Shindo Y, Kobayashi D, Ando M, Jin W, Wachino I, Yamada K, Kimura K, Yagi T, Hasegawa Y, Arakawa Y Molecualr epidemiological characteristics of Klebsiella pneumoniae associated with bacteremia among patients with pneumonia. J Clin Microbiol 53 : Shin J, Ko KS Single origin of three plasmids bearing bla CTX-M-15 from different Klebsiella pneumoniae clones. J Antimicrob Chemother 69: Chen L, Mathema B, Chavda KD, DeLeo FR, Bonomo RA, Kreiswirth BN Carbapenemas-producing Klebsiella pneumoniae: molecular and genetic decoding. Trend Microbiol 22: Chen L, Chavda KD, Fraimow HS, Mediavilla JR, Melano RG, Jacobs MR, Bonomo RA, Kreiswirth BN Complete nucleotide sequences of bla KPC-4 and bla KPC-5 -harboring IncN and IncX plasmids from Klebsiella pneumoniae strains isolated in New Jersey. Antimicrob Agents Chemother 57: Rhee JY, Park YK, Shin JY, Choi JY, Lee MY, Peck KY, Song JH, Ko KS KPCproducing extreme drug-resistant Klebsiella pneumoniae isolate from a patient with diabetes mellitus and chronic renal failure on hemodialysis in South Korea. Antimicrob. Agents Chemother. 54: Yoo JS, Kim HM, Yoo JI, Yang JW, Kim HS, Chung GT, Lee YS Detection of clonal KPC-2-producing Klebsiella pneumoniae ST258 in Korea during nationwide surveillance in J. Med. Microbiol. 62: Hong SK, Yong D, Kim K, Hong SS, Hong SG, Khosbayar T, Song W, Roh KH, Jeong SH, Lee K, Chong Y First outbreak of KPC-2-producing Klebsiella pneumoniae sequence type 258 in a hospital in South Korea. J. Clin. Microbiol. 51: Lee Y, Kim BS, Chun J, Yong JH, Lee SY, Yoo JS, Yong D, Hong SG, D Souza R, Thomson KS, Lee K, Chong Y Clonality and resistome analysis of KPCproducing Klebsiella pneumoniae strain isolated in Korea using whole genome sequencing. BioMed Res. Int. 2014:

6 Bryant KA, Van Schooneveld TC, Thapa I, Bastola D, Williams LO, Safranek TJ, Hinrichs SH, Rupp ME, Fey PD KPC-4 Is Encoded within a truncated Tn4401 in an IncL/M plasmid, pne1280, isolated from Enterobacter cloacae and Serratia marcescens. Antimicrob Agents Chemother 57: Naas T, Cuzon G, Truong HV, Nordmann P Role of ISKpn7 and Deletions in bla KPC Gene Expression. AntimicrobAgents Chemother 56: Downloaded from on September 6, 2018 by guest -6-

7 Table 1. Minimal inhibitory concentrations (MICs) of a KPC-2-producing Klebsiella pneumoniae isolate (KPC-DK05) and its transconjugant bearing a bla KPC-2-carrying plasmid. MIC (mg/l) Antimicrobial agents Isolate Recipient Transconjugant* KPN-DK05 J53 TCS KPN-DK05 Imipenem Meropenem Cefotaxime > >128 Ceftazidime >64 2 >64 Cefepime > >64 Ceftriaxone > >128 Ampicillin >64 8 >64 Gentamicin Amikacin Aztreonam > >64 Ciprofloxacin Tetracycline Tigecycline Rifampicin Colistin Polymyxin B Trimethoprim/sulfamethoxazole 0.5/ / /2.375 Piperacillin/tazobactam 256/4 4/4 256/4 * MICs that are greater in the transconjugant than in the recipient (E. coli J53) are shown in boldface. -7-

8 Figure 1. Structure of pkpc-dk05 plasmid. (A) Major structural features of the pkpc- DK05 plasmid in comparison with those of the IncN3-type plasmid pn-cit of Citrobacter freundii (GenBank accession No. JQ ) and of the IncN1 prototype backbone pr46 (GenBank accession No. AY046276). Open reading frames are indicated by black arrows and are coloured according to predicted gene functions. White dotted bars denote replicationassociated genes; plasmid transfer-associated tra-loci are indicated by black bars; stabilityassociated genes are indicated by grey bars. The regions of the bla KPC-2 -carrying transposon Tn4401b are indicated by the diagonal bar. (B) Comparison of the parts of the bla KPC-2 - carrying transposon Tn4401b in the pkpc-dk05 plasmid that was found in this study with those from the known IncN-type plasmids pbk31551, pkpc_fcf13/05, pkox105, pkpc- LKEc, and pecn580. Light grey shading denotes regions of homology. Open reading frames are shown as large arrows. Inverted repeats (IR) of the respective mobile elements are shown as small grey arrows; small black arrows represent target site duplications (TSD). Downloaded from on September 6, 2018 by guest -8-

9 Fig. 1A kika tral orf 6 0 pkpc-dk05 (56,728 bp) bla KPC-2 30,000 mpr Replication genes Stability genes Conjugation genes Tn4401b with bla KPC-2 Citrobacter freundii pn-cit Salmonella enterica pr46 (IncN prototype backbone) K. pneumoniae ppmk1-b Other backbone genes

10 Fig. 1B GCGCT 5 bp TSD Tn4401b Tn6901 tnpr tnpa ISKpn7 ISKpn7 ISKpn7 bla KPC-4 ISkpn6 Δ ista Δ ista Δ istb tnpr tnpa IRL ISKpn7 ISKpn7 IRR bla KPC-2 IRL ISkpn6 256 bp Insertion ista istb Δ tnpr tnpa ISKpn7 ISKpn7 bla KPC-2 ISkpn6 ista istb tnpr tnpa ISKpn7 ISKpn7 IRR bla KPC-2 ISkpn6 ista istb IRR GCGCT 5 bp TSD uvp1 IS26-u ΔtnpR ISKpn8 bla KPC-2 Δ ISKpn6 IS26-d traf uvp1 IS26-u ΔtnpR ISKpn8 bla KPC-2 Δ ISKpn6 IS26-d Intl1 1 kb pkpc-dk05 K. Pneumoniae ST1612 (IncN, Korea) pkpc_fcf13/05 K. pneumoniae ST442 (IncN7, Brazil) pkox105 K. oxytoca (IncN3, Italy) pkpc-lkec E. coli ST410 (IncN3, Taiwan) pecn580 E.coli ST131 (IncN3, China) pbk31551 K. pneumoniae ST834 (IncN2, USA) Figure 1. Structure of pkpc-dk05 plasmid. (A) Major structural features of the pkpc-dk05 plasmid in comparison with those of the IncN3-type plasmid pn-cit of Citrobacter freundii (GenBank accession No. JQ ) and of the IncN1 prototype backbone pr46 (GenBank accession No. AY046276). Open reading frames are indicated by black arrows and are coloured according to predicted gene functions. White dotted bars denote replication-associated genes; plasmid transfer-associated tra-loci are indicated by black bars; stability-associated genes are indicated by grey bars. The regions of the bla KPC-2 -carrying transposon Tn4401b are indicated by the diagonal bar. (B) Comparison of the parts of the bla KPC-2 -carrying transposon Tn4401b in the pkpc-dk05 plasmid that was found in this study with those from the known IncN-type plasmids pbk31551, pkpc_fcf13/05, pkox105, pkpc-lkec, and pecn580. Light grey shading denotes regions of homology. Open reading frames are shown as large arrows. Inverted repeats (IR) of the respective mobile elements are shown as small grey arrows; small black arrows represent target site duplications (TSD).