Scholarly article on topic 'Epidemic Plasmid Carrying blaCTX-M-15 in Klebsiella penumoniae in China'

Epidemic Plasmid Carrying blaCTX-M-15 in Klebsiella penumoniae in China Academic research paper on "Biological sciences"

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Academic research paper on topic "Epidemic Plasmid Carrying blaCTX-M-15 in Klebsiella penumoniae in China"

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Epidemic Plasmid Carrying biaCTX.M.15 in Klebsiella penumoniae in China

Chao Zhuo1*, Xiao-qiang Li1, Zhi-yong Zong2, Nan-Shan Zhong1

1 State Key Laboratory of Respiratory Diseases, Guangzhou Medical College, Guangzhou, China, 2 Department of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China

Abstract

Objective: To investigate the local epidemiology of Klebsiella penumoniae carrying blaCTX-M-15 in southern China and to characterize the genetic environment of blaCTX-M-15.

Methods: PCR and DNA sequencing were used to detect and characterize the genetic contexts of blaCTX-M-15. The clonal relatedness of isolates carrying blaCTX-M-15 was determined by pulse-field gel electrophoresis. Conjugative plasmids carrying blaCTX-M-15 were obtained by mating and were further subject to restriction analysis and replicon typing.

Results: A total of 47 CTX-M-15 ESBL-producing isolates of K. pneumoniae were collected from nine hospitals in China from October 2007 to October 2008. Isolates were clustered into various clonal groups. The local spread of blaCTX-M-15 was mainly mediated by one major conjugative plasmid as determined by S1-PFGE and restriction analysis. A 90-kb plasmid belonging to incompatible group FII was the major carrier of blaCTX-M-15 in K. pneumoniae. Except blaTEM-1, the resistance genes such as blaSHV, blaDHA-1, blaOXA-1, qnrB, qnrS, aac(3)-II, and aac(6')-Ib were not found in the plasmid. In the comparing of conjugative gene sequence, it is 100% identical with the plasmid pKF3-94, which was found in K. pneumonia from Zhejiang province of china previously.

Conclusions: blaCTX-M-15 was prevalent in K. pneumonia of southern China. The dissemination of blaCTX-M-15 appeared to be due to the horizontal transfer of a 90-kb epidemic plasmid.

Citation: Zhuo C, Li X-q, Zong Z-y, Zhong N-S (2013) Epidemic Plasmid Carrying bloCTX-M-15 in Klebsiella penumoniae in China. PLoS ONE 8(1): e52222. doi:10.1371/ journal.pone.0052222

Editor: Holger Rohde, Universitatsklinikum Hamburg-Eppendorf, Germany Received July 9, 2012; Accepted November 16, 2012; Published January 29, 2013

Copyright: © 2013 Zhuo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by a grant (no.201002021) from Special Fund for Health-Scientific Research in the Public Interest: Research & application for the prevention & control of nosocomial infections coursed by multi-drug resistant bacteria, and a grant (no. 2009A030301011) from the Key Foundation of Guangdong province Bureau of Science and Technology. Grant from the Natural Science Foundation of China (No.81271881). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: chao_sheep@263.net

Introduction

CTX-M enzymes have emerged as the predominant type of extended-spectrum b-lactamases (ESBL) produced by clinical isolates of Enterobacteriaceae in the world [1]. At present, more than 90 CTX-M variants have been designated (http://www.lahey. org/Studies/other.asp), of which CTX-M-15 is the most prevalent variant globally [2]. The global spread of b/aCTX-M-i5 is largely due to Escherichia coli of sequence type (ST) 131 and IncFII plasmids [3,4]. In China, blaCTX-M-14 has been the dominant blaCTX-M variant, especially in E. coli [5,6]. A recent small-scale investigation shown that 64.7% (11/17) E. coli isolates and 27.3% (3/11) K pneumoniae isolates carried blocTx-M-14 [7]. Interestingly, 64.3% of the blaCTX-M-14 genes were mainly located on an IncF plasmid in the 14 blaCTX-M-14-positive isolates, which indicate the IncF

plasmids plays a main role in dissemination of blaCTX-M-14 among

the E. coli isolates in China [7].

The resistant rate of ceftazidime has increased sharply in both E. coli and K. pneumoniae recently in China [8]. However, the data about blaCTX-M-15 in Klebsiella pneumoniae is scarce, especially the clonal relatedness of the plasmids in K. pneumoniae

isolates carrying blaCTX-M-15 in China. Owing to CTX-M-15 enzyme hydrolyse ceftazidime at a higher rate than cefotaxime, it was speculated that blaCTX-M-15 gene may be emerging and spreading in China. Our previous study has also shown that blaCTX-M-15 had emerged as the more common type of blaCTX-M genes in K. pneumoniae in Guangzhou during 2007 to 2008, and revealed that 28.3% and 26.1% ESBL-producing K. pneumoniae isolates carried blaCTX-M-14 and blaCTX-M-15, respectively [9]. The mechanisms responsible for the high prevalence of blaCTX-M-15 in K. pneumoniae occurred in the period remained undetermined. Therefore, all of the 47 K. pneumoniae isolates carrying blaCTX-M-15 from our previous study were studied for clonal relatedness and were also subjected to plasmid analysis including replicon typing and restriction analysis. This study show that blaCTX-M-15 was prevalent in K. pneumoniae of southern China and the dissemination of blaCTX-M-15 appeared to be due to the spread of a 90-kb epidemic plasmid.

Materials and Methods

Clinical Isolates

Clinical K. pneumoniae isolates carrying b/aCTX-M-i5 identified in our pervious study [9] were collected from nine hospitals, as part of the MOH National Antimicrobial Resistant Investigation net (Mohnarin) Program from October 2007 to October 2008. Briefly, Nine hospitals are scattered geographically over the three cities in southern China, Hospital A, B, C, D, E are located in the city I, and hospital F, G, H in the city II, and hospital K in the city III. Hospital A, B, C are close to each other, and the nearest distance between two hospitals is about 1 km, the city II and city III are far from the city I, and the distance is about 100 km and 300 km, respectively. Sometimes, the same patients may walk through the hospitals in one city for treatment. All the 181 isolates K. pneumoniae producing ESBLs were screened by PCR with special primers for all known CTX-M type encoding gene [9]. Purified PCR products were directly sequenced from both ends or cloned in pMD18-T and then sequenced. The DNA sequences and deduced amino acid sequences were compared to genes in GenBank or the b-lactamases classification system (www.lahey.org/studies/webt. html) to conform the subtypes of b-lactamase genes. The species identification for all isolates was performed using the VITEK-2 (bioMerieux, Marcy l'Etoile, France) automated microbiological analyzing system.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing of isolates and their trans-conjugants carrying blaCTX-M-15 was performed using the microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. Antimicrobial agents tested were ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobac-tam, cefoperazone-sulbactam, cefoxitin, cefotaxime, ceftazidime, cefepime, imipenem, aztreonam, amikacin and ciprofloxacin. All of the agents were provided by the Chinese National Institute for the Control of Pharmaceutical and Biological Products, except for cefoperazone-sulbactam, which was obtained from Pfizer (New York, NY). E. coli ATCC 25922 was used as the control strain. Isolates were classified as susceptible or resistant according to the interpretative standards recommended by CLSI.

Clonal Relatedness of Isolates Carrying ba/CTX-M-15

Pulsed-field gel electrophoresis (PFGE) analysis of XbaI-digested genomic DNA was performed to determine the genetic relatedness of CTX-M-producing K. pneumoniae isolates using a CHEF-Mapper XA System (Bio-Rad Laboratories, Hercules, CA, USA) as described by Seifert [11]. The interpreting criteria were described by Tenover [12] combining UPGMA (unweighted pair group method with hierarchic averages) method, Isolates were assigned the same pulsetype if the value of Dice coefficient of similarity was >80% [13].

Conjugation

Conjugation experiments were performed in broth as described previously [14]. K. pneumoniae strains of different clones carrying blaCTX-M-15 were used as donor strains, while a rifampicin-resistant variant ofE. coli strain C600, C600 (Rif), was used as the recipient. Transconjugants were selected on MH agar (Oxoid, Basingstoke, UK) supplemented with ceftazidime (2 mg ml"1) plus rifampicin (300 mg ml"1).

Plasmid Analysis

Plasmids from transconjugants were prepared using a modified alkaline lysis method [15]. Plasmid DNA was linearized with the

S1 enzyme followed by PFGE [16]. The estimation of the plasmid size was compared with the molecular weight marker, Salmonella braenderup H9812. Plasmids were restricted with EcoRI. Plasmid replicons were determined using the PCR-based replicon typing scheme(PBRT) by using eighteen pairs ofprimers to perform PCR which recognized F,FIA, FIB, FIC, HI1, HI2, I1-Ic, L/M, N, P, W, T, A/C, K, B/O, X, Y and FII replicons as described by Carattoli [17]. All the primers and targets were listed in Table S2. To further determine the backbone of the plasmid, the genes traF, traH, traN, traU, traW and trbC, specific to the backbone of F system and ought to demonstrate the relationship of plasmid [18], were employed to analyze the backbone of the 90-kb plasmid by PCR and sequenced. (Primers listed in Table 1).

To determine whether some other resistance genes and insertion sequences were co-transferred with blaCTX-M-15, trans-conjugants obtained were screened for ISEcp1, IS26, blaTEM, blaSHV, blaDHA-1, blaOXA-1, qnrB, qnrS, aac(3)-II, and aac(6')-Ib by PCR (primers listed in Table 1).

Results

Antimicrobial Susceptibility Testing

A total of 47 out of 125 blaCTX-M postive strains of K. pneumoniae carrying blocTX-M-15, were originated from sputum (n = 38), blood (n = 6), abscess (n = 3). Results of the microdilution method showed that both types of ESBL-producing strains had low resistance to imipenem, piperacillin/Tazobactam and cefopera-zone/sulbactam, with the resistance rate of 2.1%, 10.6% and 14.9%, respectively. The third-generation cephalosporin and cefepime had an antimicrobial resistance rate ranging from 57.4% to 89.5% (Table S1).

Clonal Relatedness of Isolates Carrying b/aCTX-M-15

According to the patterns of PFGE isolates, 30 pulsotypes were designated for 47 blaCTX-M-15-producing K. pneumoniae isolates (Fig. S4). Besides 25 individual pulsotypes, the remaining 22 isolates collected from three hospitals were classified into 5 pulsotypes, designated types A, B, C, D and E (Fig. 1). Five isolates from hospital A belonged to type A, 3 isolates from hospital A and 3 isolates from hospital B belonged to type B, 4 isolates from hospital B belonged to type C, 2 isolates from hospital B and 2 isolates from hospital C belonged to type D, 3 isolates from hospital C belonged to type E. In summary, the small clonal dissemination just occurred in each hospital or near hospital. There is no a predominant clone carrying blaCTX-M-15 found in this study.

Antibiotic Resistance Profile of 30 K. pneumoniae Isolates and the Transconjugants

For isolates of the same pulsotype, only one was chosen as a representative for further study. Therefore, a total of 30 non-clonal isolates carrying blaCTX-M-15 were subject to plasmid analyzing. The conjugative plasmids were obtained in all the 30 isolates, and 21 transconjugants containing a single 90-kb conjugative plasmid carrying blaCTX-M-15, while 9 transconjugants containing three different size ofplasmid (60-kb,90-kb,160-kb) (Fig. S1). The susceptibility characteristics of all transconjugants are shown in Table 2. All the transconjugants were susceptible to imipenem and ciprofloxacin, while almost of them were resistant to ampicillin, piperacillin, aztreonam and cefotaxime. For these above antimicrobial agents, transconjugants shared similar MICs with their corresponding parent strains. In addition, the MIC of ceftazidime for transconjugants, were ranged from 4 to 64 mg/l, with 2-8 folds decreased than the parent strains, respectively. We also observed that 30.0% (9/30) isolates and 83.3% (25/30) transcon-

Table 1. Primers used in this study.

Primer sequence (5 r3 ) GenBank accession no

blaCTX-M group F:ATGTGCAGYACCAGTAARGTKATGGC AY458016

R:TGGGTRAARTARGTSACCAGAAYCAGCGG

blaCTX-M-1 group F:CAGCGCTTTTGCCGTCTAAG AY458016

R:GGCCCATGGTTAAAAAATCACTGC

blaTEM F: GAGTATTCAACATTTTCGT AY458016

R: ACCAATGCTTAATCAGTGA

blaSHV F:CGCCGGGTTATTCTTATTTGTCGCG X98101

R:TCTTTCCGATGCCGCCGCCAGTCA

blaOXA F:CCAAAGACGTGGATG FJ594766

R:GTTAAATTCG ACCCCAAGTT

blaDHA-1 F:CTCATCCTCCATAAAACAGC FJ715937

R:TTATCTCACACCTTTATTACT

qnrA F:AAGGAAGCCGTATGGATATT AB469045

R:AGCTAATCCGGCAGCACTAT

qnrB F:CGACCTGAGCGGCACTGAAT FJ233873

R:TGAGCAACGATGCCTGGTAG

qnrS F:ACCTTCACCGCTTGCACATT FJ418153

R:CCAGTGCTTCGAGAATCAGT

aac-(6')-Ib-cr F:ATGACTGAGCATGACCTTGC FJ790516

R:TTAGGCATCACTGCGTGTTC

aac(3')-II

R:CTCGAATGCCTGGCGTGTTT

F:ATATCGCGATGCATACGCGG

R:GACGGCCTCTAACCGGAAGG

F:TGGCAGTGGTATAACGAGA

R:CCATAGGTATCCCTGAAGC

F:CTATGGTGGCTCCCTGTAT

R:TGTTCTGGTAACGGCTGA

F:TGTGGTGGTGATGTCTTCTG

R:CAAACCCGATACGCAACT

F:CCATTGGTTACTGGGAGC

R:GCGTTCTTTAGGCAGGATT

F:GTATCGGACGCACGGAGA

R:AGTAAACACGGCTGTCCAGAG

F:AGGGTGCCCTGTATTTTGTGTCGTT

R:TGGCGGCCACTTTCTCCACG

GQ343184.1

NC005327

NC005327

NC005327

NC005327

NC005327

NC005327

K=G or T; R = A or G; S = C or G; Y = C or T. doi:10.1371/journal.pone.0052222.t001

jugants were susceptible to ceftazidime, according to CLSI breakpoint.

Plasmids Carrying b/aCTX-M-15

To determine the role of plasmid in dissemination of blaCTX-M-15, the single 90-kb conjugative plasmids were subject to analyzed. Firstly, all the 90-kb plasmids were confirmed to be the IncFII group by PCR-based replicon typing (PBRT). Restriction analysis revealed that the twenty-one 90-kb plasmids restricted with EcoRI had highly similar patterns (Figure 2). The sequences of traF, traH, traN, traU, traW and trbC in all the 90-kb plasmids were 100% identical to the plasmid pKF3-94 which is an epidemic plasmid carried blaCTX-M-15 in Zhejiang province of China [18]. Meanwhile, these sequences were found to have low identities to those of

plasmid pC15-1a though both were in similar size. Besides, we also found that the length of EcoRI-EcoRI fragment of pKF3-94 mated with some restriction fingerprints of the epidemic plasmid theoretically, as shown in Table S3, there are 13 restriction fragments digested by EcoRI in pKF3-94 with the similar length of fragments of the restricted plasmids.

To demonstrate that the blaCTX-M-15 is located on the 90-kb plasmid of the remaining 9 transconjugants which contains multi-plasmid (60-kb, 90-kb, 160-kb) (Fig. S2), the 90-kb plasmid DNA was acquired by plasmid electrophoresis and gel extraction, and then it was used as a template, blaCTX-M-15 was amplified by PCR and sequenced. Together, these results indicated that all the 90-kb plasmid harbored blaCTX-M-15.

Figure 1. Dendrogram to illustrate the relatedness of 47 CTX-M-15 producing K. pneumoniae isolates. The same pulsetype defined by PFGE profiles with $80% similarity (UPGMA, Dice; black vertical line) are indicated. 30 pulsotypes were designated. doi:10.1371/journal.pone.0052222.g001

Genetic Environment of b/aCTX-M-15

To compare the resistance region between the 90-kb plasmid and other epidemic plasmids carrying blaCTX-M-15, the common elements involved in the mobilization and expression of blaCTX-M-15 gene were analyzed. The insertion of ISEcpl was observed at

48 bp upstream of the start codon of all the 47 CTX-M-15 group genes by DNA sequence analysis. IS26, related to the transmission of ß-lactamase genes such as DHA-1, CFE-1, ACC-1 and SHV-2a, was usually found in the IncFII plasmid [19-20]. However, PCR amplification with primers specific for the tnpA genes of IS26

Table 2. MICs of antimicrobial agents for K. pneumoniae clinical isolates and the transconjugants.

strain AMP SAM PIP FOX CTX CAZ FEP CSL TZP AZM IPM CIP AMK

K7 $256 32 $256 4 $256 32 64 16 16 64 0.5 1 32

Tr7 $128 8 $256 2 32 8 32 2 2 $128 #0.25 0.5 0.5

K13 8 4 16 32 8 16 4 16 8 8 0.5 0.5 1

Tri 3 8 2 8 2 8 4 2 4 4 8 #0.25 0.5 #0.25

K15 $256 128 $256 64 $256 64 64 $128 16 $256 0.5 4 64

Tr15* $128 8 $256 32 64 32 32 16 8 64 #0.25 0.5 32

K20 $256 64 $256 4 $256 16 64 16 16 64 #0.25 1 8

Tr20 $128 4 $256 0.5 16 8 32 4 8 64 #0.25 0.5 #0.25

K24 $256 64 $256 4 $256 32 64 32 16 128 ##0.25 0.5 8

Tr24 $128 4 $256 0.5 64 4 32 8 4 $128 #0.25 0.5 1

K140 16 8 16 8 4 16 8 2 2 16 0.5 2 16

Tr140 16 2 16 1 4 2 4 1 1 16 #0.25 0.5 1

K251 $256 64 $256 32 $256 8 64 16 8 64 #0.25 1 4

Tr251 $128 8 $256 2 $128 4 32 4 4 64 #0.25 0.5 0.5

K535 $256 64 $256 4 $256 16 64 16 16 $256 0.5 0.5 4

Tr535 $128 16 $256 0.5 32 4 32 4 1 $128 #0.25 0.5 0.5

K579 $256 8 $256 $128 $256 32 $128 32 16 $256 0.5 2 64

Tr579* $128 2 $256 64 $128 32 64 4 4 $128 #0.25 0.5 32

K714-1 $256 8 $256 4 $256 8 64 16 16 64 #0.25 1 8

Tr714-1 $128 2 $256 #0.25 $128 8 32 2 4 64 #0.25 0.5 1

K732-1 $256 8 $256 4 $256 $128 64 8 4 64 0.5 2 8

Tr732-1 $128 1 $256 1 $128 32 16 4 1 32 0.5 0.5 2

K948 $256 32 $256 8 $256 4 8 8 8 128 0.5 1 16

Tr948 $128 4 $256 1 $128 4 4 4 4 $128 #0.25 0.5 2

K997-2 $256 8 $256 4 $256 32 32 4 8 64 #0.25 0.5 16

Tr997-2 $128 1 $256 1 $128 4 8 2 2 64 #0.25 0.5 0.5

K1147 $256 32 $256 4 $256 8 $128 16 16 128 #0.25 1 8

Tr1147 $128 8 $256 0.5 $128 4 64 16 4 64 #0.25 0.5 1

K1150 $256 64 $256 4 $256 32 64 64 $128 64 0.5 2 32

Tr1150 $128 8 $256 1 64 8 64 16 16 32 0.5 0.5 0.5

K1162 $256 4 $256 4 $256 4 8 4 2 64 #0.25 1 64

Tr1162 $128 0.5 $256 2 64 4 4 2 0.5 64 #0.25 0.5 0.5

K1452 $256 128 $256 8 $256 $128 64 $128 64 $256 0.5 $8 64

Tr1452 $128 64 $256 2 $128 32 16 16 16 $128 #0.25 0.5 16

K1474 $256 64 $256 2 $256 8 64 64 $128 $256 #0.25 0.5 8

Tr1474 $128 2 $256 0.5 $128 4 8 16 32 $128 #0.25 0.5 0.5

K1479 $256 64 $256 8 $256 16 16 32 64 64 #.25 2 2

Tr1479 $128 4 $256 0.5 32 8 4 8 8 64 #0.25 0.5 0.5

K1562 $256 64 $256 4 $256 16 64 32 32 $256 #0.25 0.5 8

Tr1562 $128 2 $256 1 64 8 64 8 4 $128 #0.25 0.5 0.5

K2014 $256 64 $256 4 $256 $128 64 32 32 $256 #0.25 4 32

Tr2014 $128 2 $256 0.5 64 16 16 4 4 64 #0.25 0.5 0.5

K2251 $256 128 $256 4 $256 8 64 16 16 64 #0.25 0.5 2

Tr2251 $128 64 $256 1 $128 4 16 16 8 64 #0.25 0.5 0.5

K2301 $256 8 $256 8 $256 16 8 8 4 $256 0.5 $8 16

Tr2301 $128 4 $256 2 $128 2 2 4 2 $128 #0.25 0.5 0.5

k3272 $256 8 $256 64 $256 64 64 4 16 64 #0.25 0.5 0.5

Tr3272 64 4 $256 4 64 8 16 4 4 64 #0.25 0.5 0.5

K4294 $256 64 $256 8 $256 8 8 4 4 64 1 4 64

Table 2. Cont.

strain AMP SAM PIP FOX CTX CAZ FEP CSL TZP AZM IPM CIP AMK

Tr4294 64 8 $256 2 $128 4 4 4 1 64 #0.25 0.5 0.5

K6180 $256 64 $256 4 $256 32 16 16 16 $256 #0.25 0.5 1

Tr6180 $128 16 $256 0.5 64 8 4 8 4 $128 #0.25 0.5 0.5

K6259 $256 8 $256 32 $256 32 64 8 8 64 1 4 64

Tr6259* $128 2 $256 4 $128 8 8 2 2 64 0.5 0.5 0.5

K701144 $256 16 $256 8 $256 64 64 8 4 $256 #0.25 0.5 4

Tr701144 $128 2 $256 1 $128 8 64 4 4 64 #0.25 0.5 0.5

K707344 $256 16 $256 4 $256 $128 $128 16 32 $256 1 4 16

Tr707344 $128 4 $256 0.5 64 8 64 4 4 64 #0.25 0.5 0.5

K709305 $256 128 $256 4 $256 8 4 64 64 128 0.5 $8 8

Tr709305 $128 16 $256 0.5 64 4 2 8 4 $128 #0.25 0.5 1

Recipient #2 #2 #0.25 0.5 0.5 #0.25 #0.25 2 0.25/4 #0.25 #0.25 #0.25 #0.25

C600 (Rif)

Note: K type: clinical isolates of K. pneumoniae. Tr type: transconjugants.

*the transconjugants containing three different size of plasmid (60 kb,90 kb,160 kb) AMP:Ampicillin SAM:Ampicillin/Sulbactam PIP:Piperacillin FOX:Cefoxitin CTX:Cefotaxime CAZ:Ceftazidime FEP:Cefepime CSL:Cefoperazone/Sulbactam TZP:Piperacillin/Tazobactam AZM:Aztreonam IPM:Imipenem CIP:Ciprofloxacin AMK:Amikacin.

doi:10.1371/journal.pone.0052222.t002

Figure 2. Dendrogram to illustrate the similarity of 90-kb plasmid digested by the EcoRI. The similar patterns were defined with $80% similarity (UPGMA, Dice; black vertical line). 21 transconjugants containing a single 90-kb conjugative plasmid carrying b/aCTX-M-15 were analyzed. doi:10.1371/journal.pone.0052222.g002

was negative for all 47 CTX-M-15 groups. In addition, blaTEM-1 was detected in all the 90-kb plasmid of transconjugants by sequenced. Other resistance genes such as blaSHV, blaDHA-1, blaOXA-1, qnrB, qnrS, aac(3)-II, and aac(6')-Ib were not found in the 90-kb plasmids consistent with the MIC data.

Discussion

Here, we show that K. pneumoniae carrying blacT~x-M-i5 have emerged in southern China. The local spread of blaCTX-M-15 in K. pneumoniae was not mainly due to one or several dominant clones as various pulsotypes were identified in this study, though there were some medical tourism might occurred among the nine hospitals in the study period, unlike the situation seen in E. coli ST131[21].

In contrast to the diversity of clonal relationships, many local isolates harbored a 90-kb IncFII plasmid carrying blaCTX-M-15, suggesting that this plasmid appeared to be a major vehicle mediating the local dissemination of blaCTX-M-15 in K. pneumoniae. Indeed, previous reports [1] suggested that plasmid is one major factor responsible for the worldwide spread of blaCTX-M-15. For example, in E. coli, many plasmids carrying blaCTX-M-15 found in France, Tunis, Bangui and India [22-23], shared common features with pC15-1a from Canada [3]. Furthermore, emergence of K. pneumoniae isolates producing CTX-M-15 were also found in European countries and blaCTX-M-15 transfer were mediated by IncFII-related plasmids with different sizes among part of them [24]. In this study, non-clonal isolates of K. pneumoniae from different hospitals had the same plasmid carrying blaCTX-M-15. To our best knowledge, this is the first evidence of an epidemic plasmid carrying blaCTX-M-15 in K. pneumoniae.

IncFII-related plasmids are narrow-host range plasmids that are frequently involved in the worldwide dissemination of the blaCTX-M-i5 gene [1]. However, unlike plasmids belonging to other incompatibilty groups, IncFII-related plasmids possess a great versatility of intracellular adaptation by the rapid evolution of the regulatory sequences of the replicons. Therefore, the backbones of IncFII-related plasmids exhibit a significant heterogeneity in terms of the size and number of replicons [25]. A genetic comparison of two widely distributed blaCTX-M-15-carrying IncFII-related plas-mids pC15-1a and pEK516 revealed three genetic events potentially accounted for all of the differences [26] though a 60kb region is higher homologous between the two plasmid which is originated from the non-R-determinant region of plasmid R100 [4]. For the 90-kb IncFII-related plasmid identified in this study, it appeared to have genetic components in both the backbone and the resistance region with different origins from those of plasmid R100. In contrast, this 90-kb plasmid appears to be closer to pKF3-94 as both plasmids originated from K. pneumoniae, belonged to the IncFII group, carried blaTEM and blaCTX-M-15, and had the same traF, traH, traN, traU, traW and trbC sequences. The close relatedness between the 90-kb plasmid and pKF3-94 was also evidenced by their almost identical EcoRI restriction patterns, suggesting that the two plasmids may contain a common backbone. Interestingly, this 90-kb plasmid was not detected to harbor certain resistance genes such as blaOXA-1, qnrB, qnrS, and aac(6 )-Ib, which were usually found in other IncFII-related plasmid carrying blaCTX-M-15. The phenomenon is also found in the resistance region of pKF3-94 as only the ISEcp1-blaCTX-M-15 genetic stucture (positions 88110-88985) and blaTEM-1 (positions 89767-90627) but no IS26 found on pKF3-94.

IncFII-related plasmids are complex and sophisticated vehicles mediating the dissemination of antimicrobial resistance genes.

IncFII-related plasmids may co-exist with other Inc type plasmids and even are able to co-exist each other due to the differences on the inc sequence, which controls incompatibility in the same hosts [27]. The co-existence of plasmids were evidenced that three different sizes of plasmids existed in 9 out of 30 transconjugants containing 90-kb IncFII plasmid. The extensive recombination between different plasmids or plasmid fusions could facilitate IncFII-related plasmids to acquire various resistance genes and therefore generate new vehicles encoding multiple resistances. It could be predicted that the local 90-kb IncFII-related plasmid or pKF3-94 plasmid may evolve into plasmids with new phenotypes by acquiring multiple resistant genes during their further spread.

In summary, this study reveals that a high prevalence of blaCTX-M-15 genes in K. pneumoniae was contributed to a 90-kb IncFII-type plasmid which has different backbone structure with the epidemic plasmid found in Europe and other countries. It looks like that the epidemic IncFII plasmid carrying blaCTX-M-15 is not restricted in southern China but might have been spread the whole country.

Supporting Information

Figure S1 Fingerprints of transconjugant containing three conjugative plasmids.

Figure S2 Restriction enzyme fingerprints of 90-kb conjugative plasmid digested by EcoRI.

Figure S3 Restriction enzyme fingerprints of 90-kb conjugative plasmid digested by EcoRI+Hindlll.

Figure S4 PFGE patterns of 47 isolates of K. pneumonia.

Table S1 Antimicrobial susceptibility of K.pneumoniae isolates carrying b1aCTX-M-15.

Table S2 Primers used in the PCR-based replicon typing scheme.

Table S3 Comparison the length of restriction between the 90-kb plasmid and pKF3-94.

Table S4 Distribution of the CTX-M genotype in test strains.

Acknowledgments

We thank the members of 9 medical centers participating in MOH National Antimicrobial Resistant Investigation net (Mohnarin) Program, including Dan-hong Su, Cha Chen, Kang Liang, Lu-xia Wang, Hong-yu Li, Mei Wang, Zhi-quan Zhi, Zhong-hui Guo, Sui-na Geng for sending the clinical isolates.

Author Contributions

Conceived and designed the experiments: CZ NSZ. Performed the experiments: XQL. Analyzed the data: XQL ZYZ. Contributed reagents/ materials/analysis tools: XQL. Wrote the paper: CZ.

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