INTRODUCTION
Carbapenemase-producing Klebsiella pneumoniae (CPKP) is an ever-increasing clinical problem in hospitals which significantly limits treatment options for infections with these organisms. The most clinically significant carabapenemases are the KPC-type (Ambler class A), IMP-, VIM- and NDM-types (class B) and OXA-48 (class D), which have mostly been identified in K. pneumoniae isolates as sources of nosocomial outbreaks [Reference Dortet1]. In Chinese hospitals there is an ongoing epidemic of K. pneumoniae clonal strains, predominantly sequence type (ST)11, harbouring class A (KPC) and/or class B (MBLs) carbapenemases [Reference Chen2]. The carbapenemases are often encoded by genes located on large plasmids which also carry genes for resistance to other antimicrobial agents such as aminoglycosides and fluoroquinolones [Reference Sheng3]. The carbapenemase genes are associated with a variety of plasmid types, although commonly with broad range IncA/C elements [Reference Kumarasamy4]. As plasmids are the principal vehicles for the dissemination of a great variety of resistance genes, their study and understanding is critical for reversing the increasing trend in antibiotic resistance rates worldwide [Reference Mata5].
Until now, there have been no attempts to analyse potential connections between STs and plasmid replicon types of K. pneumoniae isolates producing carbapenemases. Moreover, there is little information regarding the association of carbapenemase genes with specific plasmid families. Such linkages are potentially important owing to the spread of the KPC-producing lineage of K. pneumoniae ST11 in China [Reference Liu6]. The aim of this study was therefore to determine the plasmid families and ST diversity of a collection of K. pneumoniae isolates producing carbapenemases in a large teaching hospital in China.
MATERIALS AND METHODS
Isolation and identification of bacterial strains
This study was conducted at the First Affiliated Hospital of Nanchang University, a 2900-bed teaching hospital with 400 adult intensive care unit beds and 3 650 000 annual inpatient discharges. Over a 13-month study period (December 2010 to January 2012), initiated with the identification of the first isolate under investigation, a total of 180 carbapenem-resistant K. pneumoniae isolates (imipenem, meropenem, or ertapenem resistant) were recovered from clinical specimens. Of these isolates, 18 were shown to be carbapenemase producers which were randomly selected for a battery of phenotypic tests and the molecular study. Identification of isolates was performed using an automated microbiology analyser (bioMérieux, France) according to the manufacturer's instructions. Semi-quantitative counts of isolates of >107 c.f.u./ml were considered indicative of pulmonary infection.
Antimicrobial susceptibilities
Minimum inhibitory concentration (MICs) for carbapenems (imipenem and meropenem) and other antimicrobial agents (amoxicillin/clavulanic acid, cefotaxime, ceftazidime, cefepime, aztreonam, cefoxitin, amikacin, gentamicin, tobramycin, ciprofloxacin, trimethoprim-sulfamethoxazole) were determined by a broth microdilution method (bioMérieux). Additional susceptibilities for ertapenem were performed using E-tests according to the manufacturer's instructions (AB BioDisk, Sweden). The Clinical and Laboratory Standards Institute (CLSI) M100-S22 interpretive breakpoints were used to interpret the MIC results for all antimicrobial agents studied [7]. Carbapenemase production was detected using the modified Hodge test (MHT) and a combined disc test using meropenem plus boronic acid or EDTA [Reference Ruiz-Garbajosa8].
Detection of antimicrobial resistance determinants
KPC, OXA-48-like, VIM, IMP, and NDM carbapenemases were identified using polymerase chain reaction (PCR) amplification and sequencing as described previously and a microarray assay (Check-MDR CT101; Check-Points, The Netherlands) was used to detect additional β-lactamases. The amplification of the qnrA, qnrS, and qnrB genes was undertaken in all isolates with multiplex PCR [Reference Lascols9]. Genes aac(6′)-Ib and qepA were amplified in separate PCRs using published primers and conditions [Reference Ahmed-Bentley10]. The variant aac(6′)-Ib-cr was further identified by digestion with BstF5I and sequencing [Reference Kim11] (New England Biolabs, USA). The amplification of genes encoding 16S rRNA methylases was determined using a multiplex PCR. Detection of intl1, ISCR1 and complex class 1 integron was accomplished using PCR and nucleotide sequencing with previously published primers [Reference Liu12, Reference Liu13].
Transferability of carbapenemase genes and plasmid characterization
Plasmids were isolated using the QIAprep spin miniprep kit (Qiagen GmbH, Germany) and the incompatibility group was determined according to the PCR-based replicon typing scheme [Reference Pérez-Moreno14]. The size of the carbapenemase plasmids and association with replicon type were confirmed by hybridization of S1 nuclease-digested genomic DNA from Escherichia coli transconjugants (or wild-type strains in the absence of transfer) with appropriate probes. The potential for conjugational transfer of carbapenem resistance was examined using representative isolates and E. coli J53AzR as the recipient strain. Transconjugants were selected on Luria–Bertani (LB) agar containing sodium azide (50 mg/l) supplemented with ceftazidime (50 mg/l) or imipenem (1 mg/l). In isolates unable to transfer carbapenemase by conjugation the gene was transferred by transformation using plasmid extracts purified using a Gene Pulser Xcell (Bio-Rad, USA) with E. coli DH5α as a recipient. Transformants were selected on LB agar plates with 100 mg/l ampicillin. Similarity between the carbapenemase plasmids was assessed by comparing EcoRI-, PstI- and HpaI-digested plasmid DNA profiles in all transconjugants.
Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)
PFGE of XbaI-digested genomic DNA of the isolates under investigation was performed with a CHEF-DR-III system (Bio-Rad, UK), with a running time of 23 h and pulse times ranging from 3 s to 20 s. MLST was performed using seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, tonB) (http://www.pasteur.fr/recherche/genopole/PF8/mlst/K.pneumoniae.html).
RESULTS
Patients' characteristics
Eighteen patients had proven or suspected acquisition of CPKP, including eight with invasive infection. The patients were mostly elderly (median age 64 years, range 26–85 years) with multiple underlying illnesses (Table 1). Infection was documented in all cases, the most prevalent being respiratory tract infection. All patients had a prolonged hospital stay (median 41 days, range 9–263 days) and the median number of days between admission and the first positive culture with CPKP was 21 days (range 8–86). All patients had a history of recent hospitalization in our institution and two patients had travelled abroad or been transferred from another hospital.
Clinical features of the carbapenemase-producing Klebsiella pneumoniae strains and patients' characteristics
LOS, Length of stay; M, male; F, female; RU, respiratory unit; ICU, intensive care unit; BU, burns unit; CCU, coronary care unit; TCU, transplantation care unit; OD, orthopaedics department; ED, emergency department; DM, diabetes mellitus; UTI, urinary tract infection; COPD, chronic obstructive pulmonary disease; ARDS, acute respiratory distress syndrome; VAP, ventilator-associated pneumonia; IPM, imipenem; FEP, cefepime; SCF, cefoperazone/sulbactam; MEM, meropenem; AMK, amikacin; TGC, tigecycline; SXT, trimethoprim-sulfamethoxazole; TZP, piperacillin/tazobactam.
Antimicrobial susceptibility
Based on the results of initial antibiotic susceptibility tests, all 18 CPKP strains were highly resistant to gentamicin, tobramycin, ciprofloxacin, cefepime, cefotaxime and carbapenems. The majority (83·3%) proved resistant to aztreonam, 72·2% to amoxicillin/clavulanic acid, 66·7% to trimethoprim-sulfamethoxazole, 61·1% to levofloxacin, 61·1% to amikacin, and 55·6% to ceftazidime. The MHT and the carbapenem/boronic acid combination disc test were positive for all strains.
β-Lactamase identification
The carbapenemase genes bla KPC-2, bla IMP-4, bla VIM-2, and bla NDM-1 were identified by PCR and subsequent sequencing and all of the 18 patients' representative carbapenemase-producing strains tested positive for at least one of the β-lactamase-encoding genes by PCR with bla KPC-2 being the most frequent (Table 2). However, extended-spectrum β-lactamase (ESBL) genes could be sequenced in only 13/18 strains and the remainder contained only bla TEM-1 (n = 4) or bla SHV-11 (n = 1), which are not ESBL enzymes (Table 2). The dominant ESBL types detected were bla SHV-12 (n = 6), bla CTX–M-14 (n = 4) and bla CTX-M-15 (n = 4).
Characteristics of the carbapenemase-producing Klebsiella pneumoniae clones
PFGE, Pulsed-field gel electrophoresis; ST, sequence type; MIC, minimum inhibitory concentration; MEM, meropenem; IPM, imipenem; ERT, ertapenem; n.t., non-typable.
* Plasmids from Kpn-A1/395, Kpn-A2/134, Kpn-A3/11, and Kpn-D/39 clones were transferred by conjugation and showed a similar RFLP pattern.
† Resistance markers were co-harboured by the carbapenemase gene- (bla KPC-2, bla VIM-2 or bla NDM-1) carrying plasmids.
Association of other resistance genes with carbapenemase production
All 18 CPKP strains harboured at least one of the five plasmid-mediated quinolone resistance (PMQR) genes tested; qnrA, qnrB, qnrS and aac(6′)-Ib-cr genes were detected in eight, nine, 11 and 10 of the strains, respectively. The qnr genes included eight qnrA1, eight qnrB4, one qnrB6 and 11 qnrS1 genes; all were negative for qepA. Three KPC-producing strains were positive for qnrS1, three for acc(6′)-Ib-cr with qnrA1 and qnrS1, one each for qnrA1 and acc(6′)-Ib-cr with qnrB4. Four NDM producers were positive for qnrB4 with qnrS1. Two each of strains that co-produced KPC and IMP were positive for acc(6′)-Ib-cr with qnrB4 and acc(6′)-Ib-cr with qnrA1 and qnrS1, one was positive for acc(6′)-Ib-cr with qnrA1 and qnrB4. Only one VIM producer was positive for acc(6′)-Ib-cr with qnrA1 and qnrB6 (Table 2).
All 18 strains had at least one of the three aminoglycoside resistance determinant (ARD) genes tested. The aac(6′)-Ib, armA and rmtB genes were detected in eight, three, and 13 of the strains, respectively. Six KPC strains were positive for rmtB, three were positive for armA. Two each of NDM producers were positive for acc(6′)-Ib and rmtB and five co-producers of KPC and IMP were positive for rmtB with acc(6′)-Ib. Only one VIM producer was positive for armA with acc(6′)-Ib. Mobile elements ISCR1, class 1 integron and the complex class 1 integron were detected in 12, three, and four strains, respectively (Table 2); four KPC producers harboured the complex class 1 integron; two with ISCR1 and two with class 1 integron. All NDM producers and co-producers of KPC and IMP were positive for ISCR1. Only one VIM producer was positive for class 1 integron.
Genetic context of carbapenemase and plasmid analysis
The carbapenemase genes were successfully transferred from all strains either by conjugation or transformation into recipient E. coli, suggesting plasmid localization (Table 2). S1 nuclease PFGE and in-gel hybridization of all isolates (data not shown) showed that diverse carbapenemase genes were located on plasmids ranging from ~20 to ~100 kb. Co-transfer of several carbapenemase genes with other resistance determinants and mobile elements was observed. The co-transfer of bla KPC-2, bla CTX-M-15 and rmtB as observed in isolate Kpn-A/ST395, resulting in resistance to all β-lactams and aminoglycosides, is notable as dissemination of such multidrug resistance plasmids could have serious consequences for treatment options.
The bla KPC-2 gene was transferable for strains of six genotypes and this gene was located either on transferable ~65 kb IncL/M and ~100 kb IncA/C plasmids (Table 2). On the other hand, bla NDM-1 was located on a ~70 kb plasmid showing a highly similar restriction fragment length polymorphism (RFLP) pattern in all transconjugants. The nucleotide sequence of these replicons was 99% identical to those of the pMR0211 plasmid (GenBank accession nos. JF826284·1), which belongs to the A/C group. The bla VIM-2 gene was associated with a non-typable and non-transferable ~20 kb plasmid (Table 2).
Molecular epidemiology
Based on an 80% similarity as the cut-off to discriminate between DNA profile clusters, seven PFGE types were evident in the 18 strains; the most common type A was further discriminated into three subtypes. The linkage between PFGE type and MLST type is shown in Table 3. Three genotypes, Kpn-A1/ST395, Kpn-B/ST263 and Kpn-D/ST39 accounted for five, four and two strains respectively and the remaining strains were characterized by unique genotypes. With the exception of ST11, a single-locus variant of ST258, none of the strains belonged to the CG258 group.
Genotypic characteristics, PFGE patterns and their correspondence with MLST profiles of the 18 CPKP strains
PFGE, Pulsed-field gel electrophoresis; MLST, multilocus sequence type; PMQR, plasmid-mediated quinolone resistance; ARD, aminoglycoside resistance determinant.
Table 3 also shows that regarding carbapenemase types and resistance gene content, four NDM-1 producers fell in genotype Kpn-B/ST263, which is the first identification of this ST in China having been originally reported in Korea [Reference Ko15]. Thirteen strains in seven different STs (11, 37, 39, 134, 395, 890, 1153) harboured the bla KPC-2 gene. Interestingly, five KPC-2 strains were additionally positive for the metallo-β-lactamase bla IMP-4, an association recently reported in China [Reference Wei16, Reference Wang17]. MLST assigned the VIM-2-producing isolates to ST1151. Only four K. pneumoniae genotypes (Kpn-A1/ST395, Kpn-A2/ST134, Kpn-B/ST263, Kpn-D/ST39) were isolated for more than a 1-month period in the hospital, while others were more sporadic in occurrence.
DISCUSSION
Of 180 K. pneumoniae isolates with decreased susceptibility to carbapenems, 18 were shown to be carbapenemase producers. For the other isolates, no carbapenemase activity or carbapenemase genes were identified, suggesting a non-carbapenemase-related resistance mechanism. Reduced susceptibility to carbapenems may have been due to combined mechanisms such as overproduction of the chromosomal cephalosporinase or ESBL associated with decreased permeability of the outer membrane. In this scenario, PCR and sequencing analysis of the 18 strains revealed four diverse carbapenemase genes (bla NDM-1, bla KPC-2, bla IMP-4, bla VIM-2), with the bla KPC-2 genes being the most common. The strain co-producing IMP-4 and KPC-2 enzymes was fully resistant to all carbapenems, whereas all other KPC-2-producing strains exhibited variable susceptibility to carbapenems, with only a slight increase in carbapenem MICs (Table 2). NDM-1-producing and VIM-2-producing strains also exhibited variable susceptibility to carbapenems. As frequently observed for carbapenemase producers, all of the 18 strains studied exhibited a multidrug resistance phenotype. All types of carbapenemase were highly associated with other resistance genes (PMQR, ESBLs, quinolone resistance determinant genes) and various highly efficient mobile elements (ISCR1 and class 1 integron), which is in keeping with the finding of Huang et al. [Reference Huang18].
Considerable genetic diversity was evident in the characterized strains with seven clusters defined by PFGE and nine STs. Of the KPC-2-producing K. pneumoniae, five different STs were identified, underlining the genetic diversity of the KPC-2-positive backgrounds circulating in the same hospital (Table 3). Kpn-A/ST395 was the most common genotype with six representatives. Thus far, reports of STs of outbreak or nosocomial dissemination of NDM-1-producing K. pneumoniae around the world have involved ST231 and ST340 [Reference Mi19]. However, the ST of the NDM-1 clone described herein is ST263, which has not yet been reported to harbour NDM-1. This finding supports the conclusion that bla NDM-1 occurs in K. pneumoniae belonging to diverse phylogenetic lineages and also emphasizes the need to study the plasmids carrying this gene in the species. Two novel sequence types (ST1151 and ST1153) were first described in our study and have been deposited in the K. pneumoniae MLST database.
In addition to the successful clones, previous studies have demonstrated that the spread of diverse carbapenemase genes is also linked to different types of transferable IncA/C, IncL/M and IncN plasmids [Reference Andrade20]. The bla KPC-2 gene was transferred from K. pneumoniae isolates of STs 11, 37, 39, 134, 395 and 1153, albeit at relatively low frequencies (1·8 × 10−6 to 5·2 × 10−7 transconjugants per donor cell), and fell into incompatibility groups, IncL/M and IncA/C. Such bla KPC-2-harbouring plasmids have been mainly described in K. pneumoniae ST11 in different countries including China, Greece and the USA [Reference Baraniak21–Reference Giakkoupi23]. Our work therefore underscores the fact that, intraclonal spread of bla KPC-2-containing plasmids and STs occurs as previously reported by Huang et al. [Reference Huang18]. Notably, determination of the sequences flanking bla KPC-2 revealed that the genetic environments of the gene for most strains were consistent with the genetic structure of bla KPC-2 on the plasmid pKP048 [Reference Giakkoupi23]. In four CPKP strains, the bla NDM-1 gene was carried on a 70 kb self-conjugative plasmid with an IncA/C-type backbone (Table 3). In addition, we identified the same plasmid with the bla NDM-1 gene in both K. pneumoniae and E. coli isolates recovered sequentially from the same patient (data not shown), which is suggestive of in vivo plasmid transfer. Such transfer of a NDM-1-encoding IncA/C plasmid from K. pneumoniae ST263 to E. coli, which might act as an accidental recipient present in the patients' microbiota has previously been documented [Reference Carattoli24]. The bla VIM-2 gene was carried on an untypable and non-conjugative plasmid. Most often, several resistance markers and mobile elements were co-harboured by the plasmids that carried the carbapenemase genes.
To our knowledge, this is the first systematic molecular survey reporting prevalence and characteristics of four diverse types of carbapenemase in a single Chinese hospital. Although the number of isolates studied was small the findings are disturbing as 18 distinct CPKP strains were recovered from different patients over 13 months. Such patterns imply wider persistence, although it is impossible to be certain of this without active surveillance. In addition, we have described the rapid penetration of four types of carbapenemase genes into different unrelated K. pneumoniae clones and highlighted the importance of horizontal gene transfer in the dissemination of these genes and the role of the local clonal pool as a potential substrate for their acquisition. Microbiologists and clinicians need to be made aware of this threat and implement the necessary control measures to prevent further spread in the wider population. Finally our study underlines the importance of surveillance programmes supported by powerful molecular epidemiological techniques to identify the transmission of STs and their respective plasmids.
ACKNOWLEDGEMENTS
This study was supported by grants from the Education Department of Jiangxi Province, China (GJJ13200) and the Health and Family Planning Commission of Jiangxi Province (2013A035).
DECLARATION OF INTEREST
None.
