First Report of Klebsiella oxytoca Strain Coproducing KPC-2 and IMP-8 Carbapenemases

Abstract

The study shows for the first time the presence of the Klebsiella oxytoca strain fp10 coproducing plasmid-mediated KPC-2 and IMP-8 carbapenemases. The strain was obtained from the fecal sample of an inpatient and showed high-level resistance to imipenem and ertapenem (MICs > 32 μg/ml). Conjugation experiments demonstrated the transferability of the carbapenem-resistant determinants. The results of plasmid analysis and Southern hybridization revealed that the bla_KPC-2 gene was located on transferable plasmid pFP10-1 (∼54 kb), whereas the bla_IMP-8 gene was on transferable plasmid pFP10-2 (∼180 kb). Analysis of the genetic environment of these two genes has demonstrated that ISKpn6 and ISKpn8 are involved in the spread of the bla_KPC-2 gene, while the transposable elements IS26, intI1, and tniC might contribute to the dissemination of the bla_IMP-8 gene. The chimera of several transposon-associated elements indicated a novel genetic environment of IMP-type metallo-β-lactamase gene in Enterobacteriaceae from China.

INTRODUCTION

The carbapenems, such as imipenem and meropenem, are used widely during the treatment of serious infections caused by multiresistant Gram-negative bacteria, especially strains producing high levels of AmpC cephalosporinases or extended-spectrum β-lactamase (ESBLs). Recently, however, carbapenem resistance has increasingly reported in Enterobacteriaceae, and the carbapenem resistance can mostly be mediated by carbapenem-hydrolyzing enzymes, such as metallo-β-lactamases (MBLs) and Klebsiella pneumoniae carbapenemases (KPCs) (12, 18). The emergence and spread of carbapenemases has caused problems regarding therapy and control.

Carbapenem-hydrolyzing KPC β-lactamases are classified into subgroup 2f serine β-lactamases, which are capable of hydrolyzing carbapenems, penicillins, cephalosporins, and aztreonam. These enzymes can be inhibited by clavulanic acid and tazobactam (2). KPCs in particular have recently been associated with major outbreaks of multidrug-resistant Gram-negative bacterial infections (2). KPC-producing bacteria were first reported in North Carolina (29). bla_KPC is now present among Enterobacteriaceae isolates worldwide (12, 18) and can even be detected among nonfermentative bacilli, such as Pseudomonas aeruginosa and Acinetobacter species (16, 19, 26). The dissemination of KPC producers poses a serious threat to public health.

MBLs belong to functional subgroup 3a β-lactamases, which can hydrolyze all β-lactam antibiotics except monobactams (2) and are not inhibited by β-lactamases inhibitors. However, the activity of these enzymes can be inhibited by metal ion chelators such as EDTA (2). MBLs, especially IMP and VIM, have emerged in multiple species and recently disseminated into several members of the Enterobacteriaceae family (1, 5, 8). In China, there have been several reports of IMP in Enterobacteriaceae (3, 6, 10). However, there has been no report of coproduced KPC and IMP carbapenemases in Enterobacteriaceae thus far in the world. In the present study, we describe a commensal carbapenem-resistant Klebsiella oxytoca strain that produces both KPC-2- and IMP-8-type carbapenemases, and we characterize the genetic environment of both resistance genes. This is the first report, to our knowledge, of detection of the plasmid-mediated carbapenem-hydrolyzing KPC-2 and IMP-8 simultaneously in one Enterobacteriaceae.

MATERIALS AND METHODS

Bacterial strain.

During January to May 2009, an investigation was carried out in one region of China to study the prevalence of fecal carriage with ESBL-producing Enterobacteriaceae in healthy persons and inpatients. All of the fecal samples were seeded onto MacConkey agar plates, which were supplemented with 1 μg of cefotaxime/ml. Also, one ESBL-producing K. oxytoca strain (fp10) was isolated from the fecal sample of a randomly selected patient, who was a 5-year-old patient hospitalized in the pediatric ward in the Union Hospital of Fujian Medical University. The strain showed resistant to imipenem and ertapenem. The patient suffered from acute leukemia and had some clinical infections, including bacteremia and perianal abscess. During his hospitalization, imipenem was repeatedly administered. Identification of the strain was determined by using the Vitek system.

Antimicrobial susceptibility testing and phenotypic screening.

The MICs of imipenem, ertapenem, cefepime, ceftazidime, cefotaxime, ceftriaxone, cefoxitin, aztreonam, cefoperazone-sulbactam, piperacillin-tazobactam, ciprofloxacin, gentamicin, and amikacin were determined on strain fp10 by the Etest technique (AB Biodisk, Sweden). Antibiotic susceptibility testing of ertapenem and aztreonam was performed on the transconjugant strains by the disc diffusion method. The susceptibility breakpoints were interpreted as recommended by the Clinical and Laboratory Standards Institute (4). Escherichia coli ATCC 25922 was used as a quality control. A phenotypic detection method using combined disc tests of meropenem alone and with phenylboronic acid (PBA) or EDTA or both PBA and EDTA was evaluated for the detection of carbapenemase (25).

PCR detection and DNA sequencing.

Plasmid DNAs isolated from K. oxytoca fp10 and E. coli transconjugant were obtained by the alkaline lysis method (20) and were used as a template in PCR analyses with primers that are specific for bla_KPC, bla_IMP, bla_VIM, and OXA-type carbapenemases as described previously (14, 15, 23). The bla_TEM, bla_SHV, bla_CTX-M, and plasmidic AmpC genes were also amplified by PCR (13, 28). The PCR products were purified and sequenced twice on both strands by Invitrogen (Invitrogen, Shanghai).

Conjugation experiment and plasmid analysis.

Conjugation transfer assay was performed in broth culture with E. coli J53 Az^r as the recipient (28). Donor and recipient cells were mixed at a ratio of 1:1. Transconjugants were selected on MacConkey agar containing ampicillin (100 mg/liter) supplemented with sodium azide (100 mg/liter; Sigma Chemical Co.). The colonies grown on the selecting medium were selected and identified by the Vitek system. Plasmid DNA was isolated by alkaline lysis method and examined by agarose gel electrophoresis. The plasmid sizes were estimated by comparison to the plasmids from E. coli V517 (54.2, 7.3, 5.6, 5.2, 3.9, 3.1, 2.7, and 2.1 kb) (9) and E. coli R27 (182 kb) (24) as the standard markers. Primers targeting bla_KPC and bla_IMP genes were as described previously.

Southern hybridization.

Plasmid DNA was extracted by alkaline lysis method (20). Purified plasmids were electrophoresed in a 1.0% agarose gel and transferred to a positively charged nylon membrane (Roche Applied Science, Mannheim, Germany). The membrane was probed with PCR-amplified KPC-2 and IMP-8 gene probes, respectively. The probes were prepared by using a DIG High-Prime DNA labeling and detection kit (Roche Applied Science).

Genetic environment of carbapenem-resistant determinants.

The genetic organization of carbapenem-resistant determinants was investigated by gene cloning with plasmid DNA digested and ligated in the EcoRI and BamHI sites of the pACYC 184 vector (28). All enzymes for DNA manipulations were used according to the recommendations of the supplier (Amersham Biosciences). Recombinant plasmids were transformed into E. coli DH5α. The transformants harboring the recombinant KPC- or IMP-encoding plasmids were selected on LB agar plates supplemented with ampicillin (100 mg/liter). The molecular sizes of the inserts were estimated from the results of restriction digestion and electrophoresis in 1% agarose gel in TAE buffer. Finally, inserted fragments were sequenced on both strands on an ABI Prism 3730XL DNA analyzer (Applied Biosystems).

Nucleotide sequence accession numbers.

The nucleotide sequences reported here have been assigned to the EMBL/GenBank nucleotide database under the accession numbers HQ651092 and HQ651093.

RESULTS

Antimicrobial susceptibility testing and phenotypic screening.

The MICs of a variety of antimicrobial agents against strain fp10 are shown in Table 1. The results showed that fp10 strain was a multidrug-resistant isolate, and it also exhibited high-level resistance to imipenem and ertapenem (MICs ≥ 32 μg/ml). Of note, the strain was only susceptible to ciprofloxacin (MIC = 0.75 μg/ml). The zone diameters of meropenem alone, with PBA or EDTA, or with both PBA and EDTA were 6, 6,10, and 16 mm, respectively. The simultaneous presence of both MBLs and KPCs was indicated by a ≥5-mm increase in the zone diameter of the combined disc test using meropenem with or without both PBA and EDTA.

Table 1.

MICs of antibiotics for K. oxytoca fp10, various E. coli transconjugants, E. coli DH5α, and the E. coli J53 clone

Antimicrobial agent	MIC (μg/ml) for:
	K. oxytoca fp10	E. coli DH5α (pACYC184-blaKPC-2)	E. coli DH5α (pACYC184-blaIMP-8)	E. coli J53 (blaKPC-2 + blaIMP-8)	E. coli J53 (blaIMP-8)	E. coli DH5α	E. coli J53
Imipenem	>32	32	8	>32	>32	0.19	0.19
Ertapenem	>32	16	1.5	>32	>32	0.006	0.016
Cefepime	>256	1	6	128	16	0.032	0.016
Cefotaxime	>256	6	6	128	32	0.032	0.016
Ceftazidime	>256	6	12	>256	32	0.064	0.016
Ceftriaxone	>256	16	12	128	16	0.032	0.023
Cefoxitin	>256	24	24	64	24	0.032	0.016
Piperacillin-tazobactam	>256	32	12	128	32	0.032	0.032
Cefoperazone-sulbactam	>256	24	16	256	16	0.032	0.016
Aztreonam	>256	24	0.064	64	0.064	0.016	0.032
Gentamicin	>256	0.5	0.25	>256	>256	0.094	0.125
Amikacin	>256	0.75	1.5	>256	>256	0.75	0.75
Ciprofloxacin	0.75	<0.002	<0.002	0.032	0.006	0.008	0.006

PCR detection and DNA sequencing.

PCR showed that strain fp10 was positive for bla_TEM, bla_KPC, and bla_IMP genes and negative for the remaining genes tested, including all bla_SHV and bla_CTX-M variants. Sequencing analysis identified bla_TEM-1, bla_KPC-2, and bla_IMP-8 alleles.

Conjugation experiment and plasmid analysis.

Plasmid in strain fp10 successfully transferred carbapenem resistance into E. coli J53 by conjugation. Transconjugants exhibited a phenotype of resistance or reduced susceptibility to ertapenem. Phenotypic testing of many transconjugant colonies showed that there was some different resistance to aztreonam. PCR assays showed that all aztreonam-resistant transconjugant colonies tested were positive for bla_KPC-2 and bla_IMP-8 genes, while the remaining aztreonam-susceptibility colonies tested were positive for bla_IMP-8 gene. The plasmid analysis of the parental isolate showed that the strain harbored four plasmids of ca. 3.8, 4.1, 54,and 180 kb (data not shown). The transconjugants bearing bla_KPC and bla_IMP had two plasmids of ∼54 (pFP10-1) and ∼180 kb (pFP10-2), while transconjugants bearing bla_IMP but not bla_KPC had a single plasmid of ∼180 kb (pFP10-2). PCR analysis, using as a template the gel-extracted plasmid DNA band of the transconjugants, confirmed the carriage of the bla_KPC gene on the plasmid pFP10-1, and the bla_IMP gene was on plasmid pFP10-2. Hybridization analysis with the probe specific for the bla_KPC-2 and bla_IMP-8 genes revealed that bla_KPC-2 was located on the plasmid of ∼54 kb, while bla_IMP-8 gene was on the ∼180-kb plasmid (Fig. 1). Repeated mating experiments were carried out but failed to isolate any transconjugants with a single plasmid pFP10-1. Electrophoretic profiles of K. oxytoca strain fp10 and hybridization analysis results are shown in Fig. 1. The MICs for the transconjugant colonies (i.e., either bearing both bla_KPC and bla_IMP or bearing bla_IMP but not bla_KPC) of the antibiotics are given in Table 1.

Fig. 1.

Electrophoretic profiles of plasmids (A) and hybridization with a bla_KPC-2-specific (B) or a bla_IMP-8-specific (C) probe. Lanes 1, 6, and 11, λ-HindIII-digested DNA (New England Biolabs, Beverly, MA); lanes 5, 10, and 15, marker (E. coli V517); lane 2, transconjugant harboring plasmid pFP10-2; lane 3, transconjugant harboring plasmids pFP10-1 and pFP10-2; lane 4, K. oxytoca strain fp10.

Characterization of the genetic environment of the bla_KPC-2 and bla_IMP-8 genes.

For the bla_KPC-2 gene, cloning isolates were found to have recombinant plasmids carrying a 6,964-bp inserted fragment. The nucleotide sequence of the inserted fragment was determined and revealed several open reading frames (ORFs) (Fig. 2). Upstream of the bla_KPC-2 gene was located a putative ISKpn8 element, and a truncated bla_TEM gene was inserted between them. An ISKpn6-like element was located downstream of the carbapenemase gene, followed by the pKP048-08, the klcA, the pKP048-09, and the korC genes. pKP048-08 and pKP048-09 genes encode hypothetical proteins. The klcA gene encodes the antirestriction protein KlcA, while the korC gene encodes the transcriptional repressor protein KorC. All four of these genes shared 100% nucleotide identity with the corresponding sequence on plasmid pKP048 (GenBank accession no. FJ628167).

Fig. 2.

(a) Schematic representation of the genetic structure involved in the bla_IMP-8 gene in plasmid pFP10-2. (b) Schematic representation of the genetic environment of the bla_KPC-2 gene in plasmid pFP10-1 compared to that found around the bla_KPC-2 gene in plasmid pKP048. The diagram of pKP048 is drawn based on the nucleotide sequences available in GenBank under accession number FJ628167.

For bla_IMP-8, a nucleotide sequence of 5,201 bp surrounding the gene was obtained. The annotation of this sequence revealed several ORFs, and some of these have been associated with the bla_IMP-8 gene (Fig. 2). The bla_IMP-8 genes cassette is located downstream of the attI1 recombination site, followed by an aacA4 cassette and a tniC gene cassette. TniC is a putative resolvase, which involves in transposition. Upstream of the bla_IMP-8 gene are located a class 1 integron, orf44, and one short gene fragment. The orf44 gene shares 100% identical to the corresponding sequence on plasmid pCTX-M3 (GenBank accession no. AF550415) and encodes the hypothetical protein Orf44. One replication initiator protein is encoded by the short gene fragment, which shares 99% nucleotide identity with the Rep_3 gene on plasmid pKPN5 (GenBank accession no. CP000650). However, the intI1 gene in the 5′-conserved region is disrupted and flanked by a putative IS26 insertion.

DISCUSSION

In 2009, 0.4% of Enterobacteriaceae isolates showed resistance to imipenem in the hospital where this study was conducted. Among the nonfermentative bacteria, the resistances of P. aeruginosa and Acinetobacter baumannii to imipenem were 19.8 and 17.9%, respectively (data not published). The data suggested that carbapenem resistance in Enterobacteriaceae isolates emerged in the area, while the carbapenem resistance status in P. aeruginosa and A. baumannii to imipenem remained at a high level. However, no molecular epidemiological data are available on the carbapenemase-producing Gram-negative bacteria in this hospital up to now. In the present study, we reported a fecal K. oxytoca strain that coproduced bla_KPC-2 and bla_IMP-8. The strain was highly resistant to many antibiotics, including imipenem and ertapenem. Interestingly, the strain was only susceptible to ciprofloxacin. Conjugation assay and Southern hybridization results showed that both of these two resistance genes were located on different transferable plasmids. In addition, the results of the conjugation assay in the present study demonstrated that plasmid pFP10-1 could not transfer from parental strain to the recipient strain without plasmid pFP10-2. Further experiments are needed to investigate the relationship between these two plasmids.

In recent years, many reports have described the presence of KPC β-lactamases (especially KPC-2) in strains of the family Enterobacteriaceae with carbapenem resistance or reduced carbapenem susceptibility from different areas in China (10, 12, 18). It seems that KPC enzymes are becoming widely disseminated in this region, and plasmids of different sizes could harbor the bla_KPC-2 gene in China (22).

The genetic environments of the bla_KPC gene were characterized in some studies, and various transposon elements seem to be responsible for the rapid spread of bla_KPC (11, 22, 28). Tn4401 was considered to be at the origin of bla_KPC-like gene acquisition and dissemination (11), while Shen et al. revealed a distinct genetic environment with the chimera of several transposon-associated elements in China, such as Tn3, ISKpn8, and an ISKpn6-like element on plasmid pKP048 (22). In the present study, the genetic environment of the bla_KPC-2 gene from a fecal strain showed a structure and context similar to those found in plasmid pKP048, except that a truncated bla_TEM gene was inserted and located between ISKpn8 and the bla_KPC-2 gene (Fig. 2b). Of note, the similar gene structure (ISKpn8, bla_KPC-like gene, and ISKpn6-like element) found in different plasmids in China probably suggests a common origin in this region. The results found in these studies imply that the diversity of genetic environment harboring bla_KPC-2 contributes to the wide dissemination of this carbapenem-hydrolyzing β-lactamase in China.

Several studies have shown that bla_IMP has emerged in some members of the Enterobacteriaceae in China (3, 6, 10). However, the genetic elements around this MBL gene are still unclear in this region. Most of the IMP-type MBLs are commonly located in class 1 integrons with a site-specific recombination site (attI), which is able to capture genes (18, 27). The bla_IMP genes inserted into class 1 integrons might be transferable horizontally because some of them are found on self-transmissible plasmids. However, in the present study, the genetic environment of the bla_IMP-8 gene is different. The bla_IMP-8 gene is located immediately downstream of the intI1 gene, which is disrupted by IS26 and results in the formation of a composite transposon. This structure is similar to that found in P. aeruginosa (7). As described before, this novel element may facilitate IS26-mediated mobilization of the bla_IMP gene (7).

It is also interesting that the tniC gene is located downstream of bla_IMP-8. The product of the gene is a resolvase, TniC, which is involved in transposition and plays an important role in spreading MBL genes (21). It seems that the cooperative action of several transposon elements contributes to the dissemination of bla_IMP gene among different species of Enterobacteriaceae. Moreover, it is worth noting that bla_VIM genes, along with the bla_KPC-2 gene, have emerged in K. pneumoniae (17, 30).

In conclusion, our study has demonstrated for the first time the coproduction of plasmid-mediated bla_KPC-2 and bla_IMP-8 genes in a K. oxytoca strain, and the genetic elements contributing to the dissemination of the two resistance genes were also characterized. The prevalence of the Enterobacteriaceae strains coproducing bla_KPC-2 and bla_IMP-8 may have serious consequences for clinical therapy, and we suggest that an effective surveillance and strict infection control strategies should be implemented soon to prevent potential outbreaks of nosocomial infections by such pathogens in China.