Detection of plasmid-mediated IMP-1 metallo-β-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate

Detection of plasmid-mediated IMP-1 metallo-β-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate 1 2 first-author 1 2 1 1 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chengong218@163.com †E-mail:chengong218@163.com chengong218@163.com Clinical Laboratory Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Clinical Laboratory Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Cancer Institute, Zhejiang University, Hangzhou 310009, China Cancer Institute, Zhejiang University, Hangzhou 310009, China Objective: 2 To investigate the mechanism of carbapenem resistance and the occurrence of plasmid-mediated quinolone resistance determinants qnr and aac(6 ′ )-Ib-cr in a clinical isolate of Enterobacter cloacae . Methods: 2 An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction (PCR), and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Urea-SDS-PAGE). Results: 2 Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem for ZY106 were 2, 4, and 16 μg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-β-lactamase and CTX-M-3 extended-spectrum β-lactamase, and E. coli transconjugant produced IMP-1. Plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1 -encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa. Conclusion: 2 It is the first IMP-1-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blaIMP and qnrS genes as well. The blaIMP-1 , blaCTX-M-3 , and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and meropenem susceptibility in E. cloacae . Antibiotic resistance Carbapenem Enterobacteriaceae Outer membrane proteins (OMPs) INTRODUCTION 1 Carbapenems are commonly used to treat serious infections caused by multi-resistant gram-negative bacilli, especially strains producing high-level of AmpC cephalosporinases or extended-spectrum β-lactamases (ESBLs) (Baldwin et al., 2008 2008 ). However, the emergence of carbapenemase is becoming a therapeutic challenge. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D (Walsh, 2008 2008 ). The class B enzymes, metallo-β-lactamases (MBLs), are the most clinically threatening carbapenemases, as these enzymes are capable of hydrolyzing all β-lactams except monobactams, and are not susceptible to therapeutic β-lactamase inhibitors such as clavulanate, sulbactam, and tazobactam. The most commonly acquired MBL families include the IMP, Verona integron-encoded metallo-β-lactamase (VIM), Sao Paulo metallo-β-lactamase (SPM), German imipenemase (GIM), and Seoul imipenemase (SIM). SPM, GIM, and SIM MBLs were detected in some specific regions of origins, while IMP and VIM, which are becoming common in Pseudomonas aeruginosa and other nonfermenting gram-negative bacteria, spread worldwide. IMP-1 was first detected in a P. aeruginosa isolate in Japan in 1990 (Watanabe et al., 1991 1991 ). Soon afterward, IMP-1 was prevalent in Serratia marcescens and spread into several members of the family Enterobacteriaceae in Japan (Walsh et al., 2005 2005 ; Queenan and Bush, 2007 2007 ). There has been an increase in the detection and spread of the acquired MBLs in Enterobacteriaceae from Australia and Taiwan region of China, and IMP-4 and IMP-8 were the predominant MBLs (Walsh et al., 2005 2005 ; Queenan and Bush, 2007 2007 ; Peleg et al., 2005 2005 ; Herbert et al., 2007 2007 ; Wu et al., 2007 2007 ; 2008 2008 ; Liu et al., 2008 2008 ). MBLs in Enterobacteriaceae have occurred sporadically in other countries. IMP-1 has been reported in Klebsiella pneumoniae from Singapore (Koh et al., 1999 1999 ), Brazil (Lincopan et al., 2005 2005 ; 2006 2006 ), Turkey (Aktas et al., 2006 2006 ), and the UK (Woodford et al., 2007 2007 ), in Enterobacter cloacae from Turkey (Deshpande et al., 2006 2006 ), and in Enterobacter aerogenes from France (Biendo et al., 2008 2008 ). In China, there were only two reports on the production of IMP in Enterobacteriaceae. An IMP-4-producing Citrobacter youngae was isolated from Guangzhou in 2001 (Hawkey et al., 2001 2001 ) and an IMP-4-producing K. pneumoniae from Wuhan in 2008 (Mendes et al., 2008 2008 ). In this study, an ertapenem-resistant E. cloacae strain was collected from our hospital and its mechanisms of carbapenem resistance were analyzed. It is the first detection of IMP-1 in Enterobacteriaceae from China. A further study revealed that this E. cloacae strain lacked an outer membrane protein (OMP) and harbored a plasmid-mediated quinolone resistance determinant qnrS1 . MATERIALS AND METHODS 1 Bacterial strains 2 In August 2007, an E. cloacae strain (ZY106) was isolated from a purulent exudate sample from abdominal cavity of a patient in oncology ward in the Second Affiliated Hospital of Zhejiang University, China. The patient was diagnosed as gastric cancer and received surgery. During hospitalization, the patient was treated with cefoperazone/sulbactam and ciprofloxacin before the isolation of the ertapenem-resistant E. cloacae strain. Antimicrobial susceptibility testing 2 Minimum inhibitory concentrations (MICs) were determined using the agar dilution method according to Clinical and Laboratory Standards Institute ( 2007 2007 ) recommendations. Conjugation experiments 2 Conjugal transfer experiments were carried out in mixed broth cultures (Zhang et al., 2008 2008 ). Rifampin-resistant Escherichia coli EC600 and streptomycin-resistant E. coli C600 were used as recipients. IMP-producing E. coli transconjugants were selected on Muller-Hinton agar plates containing 400 μg/ml rifampin (or 400 μg/ml streptomycin) and 8 μg/ml ceftazidime, and qnr positive E. coli transconjugants were selected on plates containing 400 μg/ml rifampin (or 400 μg/ml streptomycin) and 0.25 μg/ml ciprofloxacin. The selected colonies were picked up and identified by Vitek system (bioMérieux, Hazelwood, MO, USA). Plasmids from E. cloacae ZY106 and E. coli transconjugants were extracted using AxyPrep Plasmid Miniprep Kit (Axygen Scientific, Union City, CA, USA) and examined by electrophoresis. Isoelectric focusing (IEF) of β-lactamase 2 Crude β-lactamase preparations were obtained by ultrasonic method. IEF was carried out on PhastGel polyacrylamide gel (pH 3 to 9, Amersham Biosciences, Uppsala, Sweden) using the PhastSystem (Pharmacia Biotech, Uppsala, Sweden) by the method of Mathew et al.( 1975 1975 ). β-lactamase activity was visualized by staining the gel with Nitrocefin (Oxoid, Basingstoke, Hampshire, England). The isoelectric points (pIs) were determined as described previously (Zhang et al., 2008 2008 ). Polymerase chain reaction (PCR) amplification and DNA sequence analysis of bla and plasmid-mediated quinolone resistance genes 2 Plasmids from E. cloacae ZY106 and E. coli transconjugants were used as template. The primers used to amplify bla TEM , bla SHV , bla CTX-M (Yu et al., 2007 2007 ), bla IMP-1 , bla IMP-2 (Shibata et al., 2003 2003 ), qnrA , qnrB , qnrS (Robicsek et al., 2006 2006 ), and aac(6 ′ )-Ib (Jiang et al., 2008 2008 ) were described previously. Prepared bacterial DNA of the ZY106 was used as the template for PCR amplifications of gyrA , parC , and gene of chromosomal cephalosporinase (Deguchi et al., 1997 1997 ; Conceição et al., 2004 2004 ). The reaction was conducted in a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR amplification products were purified and sequenced using an ABI3730 Sequencer (Applied Biosystems). We determined partial sequences of the gyrA and parC genes of the ZY106, including the regions analogous to the quinolone resistance-determining region of the E. cloacae ATCC13047 gyrA and parC genes and the deduced amino acid sequences of the ZY106 gyrA and parC genes were compared with the corresponding regions of the E. cloacae ATCC13047 GyrA and ParC proteins. Detection of KPC-2 of the ZY106 was conducted according to our previous report (Zhang et al., 2008 2008 ). Analysis of outer membrane proteins (OMPs) 2 OMPs of E. cloacae ZY106 and E. cloacae ATCC13047 were isolated and separated on 11.6%(w/v) acrylamide-0.4% (w/v) bisacrylamide-0.1% (w/v) sodium dodecyl sulfate gel containing 20% (w/v) urea (urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Urea-SDS-PAGE) as described previously (Zhang et al., 2008 2008 ). RESULTS 1 Antibiotics susceptibility 2 E. cloacae ZY106 showed reduced susceptibility to imipenem and meropenem with MICs of 2 and 4 μg/ml, and resistance to ertapenem with MIC of 16 μg/ml. The isolate was highly resistant to β-lactams except piperacillin/tazobactam and aztreonam. MICs of carbapenems significantly decreased (≥4-fold) in the presence of ethylenediaminetetraacetic acid (EDTA) (a constant concentration of 320 μg/ml) (Table 1 1 ). These results suggest the production of MBL. Similarly, MIC of ceftazidime was reduced from > 256 μg/ml to 1 μg/ml, and that of cefotaxime was reduced from 256 μg/ml to 64 μg/ml, suggesting the production of CTX-M ESBL. E. cloacae ZY106 was also resistant to quinolones and aminoglycosides. The final concentration of EDTA was fixed at 320 μg/ml 2 Antimicrobial agents 6 MIC (μg/ml) E. cloacae ZY106 E. coli C600 Transconjugant A E. coli EC600 Transconjugant B 1 Transconjugant B 2 Imipenem 2 ≤0.0625 0.5 0.125 0.125 0.125 Imipenem+EDTA * * 0.5 ≤0.0625 ≤0.0625 0.125 Meropenem 4 ≤0.0625 0.25 ≤0.0625 ≤0.0625 ≤0.0625 Meropenem+EDTA 0.25 ≤0.0625 ≤0.0625 ≤0.0625 Ertapenem 16 ≤0.0625 0.5 ≤0.0625 ≤0.0625 ≤0.0625 Ertapenem+EDTA 0.5 ≤0.0625 ≤0.0625 ≤0.0625 Ceftazidime >256 ≤0.125 256 ≤0.125 ≤0.125 ≤0.125 Ceftazidime+EDTA 1 ≤0.125 ≤0.125 ≤0.125 Cefotaxime 256 ≤0.125 32 ≤0.125 ≤0.125 ≤0.125 Cefotaxime+EDTA 64 ≤0.125 0.25 ≤0.125 Cefepime 128 ≤0.125 8 ≤0.125 ≤0.125 ≤0.125 Cefoperazone/sulbactam >256 ≤0.125 128 ≤0.125 ≤0.125 ≤0.125 Ampicillin >256 1 128 8 8 8 Piperacillin 256 0.25 4 1 1 1 Piperacillin/tazobactam 16 0.25 2 1 1 1 Cefoxitin >256 >256 4 4 4 Aztreonam 32 ≤0.125 ≤0.125 ≤0.125 ≤0.125 ≤0.125 Ciprofloxacin >32 ≤0.125 ≤0.125 ≤0.125 2 2 Gentamicin 16 0.5 0.5 0.5 1 16 Amikacin 16 0.5 0.5 0.5 1 16 Transfer of antibiotics resistance and plasmid analysis 2 E. cloacae ZY106 failed to transfer carbapenem resistance to E. coli EC600 by conjugation, but it was successful for E. coli C600. The bacteria grew on plates containing streptomycin and ceftazidime were named as E. coli transconjugant A. The plasmid profiles indicated that ZY106 harbored several plasmids and E. coli transconjugant A acquired a plasmid with a size of approximately 40 kb (Fig. 1 1 ). Transfer of ciprofloxacin resistance from E. cloacae ZY106 to E. coli EC600 was successful. Two different E. coli transconjugants were obtained. The one with a 60-kb plasmid was named E. coli transconjugant B 1 , and the other with an extra 6-kb plasmid was named E. coli transconjugant B 2 . As shown in Table 1 1 , MICs of imipenem, meropenem, and ertapenem for E. coli transconjugant A were 0.5, 0.25, and 0.5 μg/ml, respectively, which increased at least 4-fold compared with those for E. coli C600 (MICs of ≤0.0625 μg/ml), although E. coli transconjugant A remained susceptible to carbapenems according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints. E. coli transconjugant A was resistant to several β-lactams but was susceptible to piperacillin, piperacillin/tazobactam, and aztreonam. E. coli transconjugant A had a dramatic decrease in the MICs of carbapenems and cephalosporins in the presence of EDTA. E. coli transconjugant B 1 was intermediately resistant to ciprofloxacin (MIC of 2 μg/ml) and was susceptible to other tested antibiotics. Conjugation experiments and antimicrobial susceptibility results of E. coli transconjugant B suggest the presence of plasmid-mediated quinolone resistance determinants. The antimicrobial susceptibility patterns of E. coli transconjugant B 2 were similar to those of B 1 , except its resistance to aminoglycosides. IEF analysis 2 IEF analysis demonstrated that both E. cloacae ZY106 and E. coli transconjugant A had a band with β-lactamase activity with a pI of approximately 9.0 (Fig. 2 2 ). PCRs and DNA sequence analysis 2 The above-mentioned antibiotic resistance genes were analyzed by PCR and DNA sequencing. E. cloacae ZY106 was positive for bla IMP-1 , bla CTX-M , qnrS , aac(6 ′ )-Ib genes and chromosomal AmpC lactamase gene, while E. coli transconjugant A was only positive for the bla IMP-1 gene, B 1 was positive for the qnrS gene, and B 2 was positive for qnrS and aac(6 ′ )-Ib genes. DNA sequencing results identify the genes as bla IMP-1 , bla CTX-M-3 , qnrS1 , and aac(6 ′ )-Ib . These results indicate that the four genes were carried on separated plasmids. The sizes of plasmids encoding IMP-1, qnrS1 , and aac(6 ′ )-Ib were approximately 40, 60, and 6 kb, respectively, and CTX-M-3 was presumed to be encoded on a 50-kb plasmid (Fig. 1 1 ). AAC(6′)-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, was not found. The aac(6 ′ )-Ib conferred the resistance to gentamicin and amikacin but did not elevate the ciprofloxacin MIC for E. coli transconjugant B 2 compared with that for B 1 . Although E. cloacae ZY106 produced two β-lactamases, only one band with β-lactamase activity was observed in IEF. It was because that IMP-1 and CTX-M-3 had an overlapping band at the same pI of approximately 9.0 (Baldwin et al., 2008 2008 ; Jeong et al., 2005 2005 ). Since the KPC-2 carbapenemase was often detected in carbapenem-resistant Enterobacteriaceae from our hospital (Zhang et al., 2007 2007 ; 2008 2008 ; Cai et al., 2008 2008 ), we had also analyzed the KPC-2 carbapenemase in ZY106. However, we confirmed that this isolate did not produce KPC-2 carbapenemase. Detection of mutations in gyrA and parC genes 2 As shown in Table 1 1 , transconjugants B 1 and B 2 exhibited rather high ciprofloxacin MICs. Usually transconjugants containing qnrS determinants only show ciprofloxacin MICs of less than 0.5 mg/L. To investigate whether other determinants had been contributing to the high ciprofloxacin resistance, we analyzed the partial sequences of the gyrA and parC genes of ZY106. ZY106 had two mutations generating Ser(S)83→Phe(F) and Asp(D)87→Ala(A) changes in GyrA and a Ser(S)83→Ile(I) mutation in ParC (data not shown). Therefore, the resistance to ciprofloxacin of ZY106 is attributed to the mutations in gyrA and parC in the quinolone resistance-determining region (Deguchi et al., 1997 1997 ). Urea-SDS-PAGE analysis of OMPs 2 Urea-SDS-PAGE analysis showed that E. cloacae ATCC13047 expressed four major OMPs, with molecular weights of 47, 41, 38, and 36 kDa. The four OMPs were analogous to E. coli LamB, OmpC, OmpF, and OmpA, respectively (Szabó et al., 2006 2006 ). The 41- and 38-kDa proteins were considered to be porins, and the later was absent in E. cloacae ZY106 (Fig. 3 3 ). DISCUSSION 1 IMP-type MBLs were most frequently detected among gram-negative nonfermenters. IMP-producing Enterobacteriaceae were mainly reported from Japan, Australia, and Taiwan region of China. Production of IMP in Enterobacteriaceae was rare in mainland China, including IMP-4 in C. youngae and K. pneumoniae (Hawkey et al., 2001 2001 ; Mendes et al., 2008 2008 ). In this study, we detected bla IMP-1 and qnrS1 genes along with alternations in gyrA and parC in a clinical isolate of ertapenem-resistant E. cloacae . To our knowledge, no other Enterobacteriaceae harboring such genes were recovered so far. In addition, further studies revealed the loss of an OMP, which enhanced the carbapenem resistance in this isolate. However, for E. cloacae ZY106, the MIC of ertapenem (16 μg/ml) was much higher than those of imipenem (2 μg/ml) and meropenem (4 μg/ml). E. coli transconjugant A that produced IMP-1 exhibited significantly reduced susceptibility to carbapenems (MICs of 0.25 to 0.5 μg/ml). However, it was not sufficient to explain the relatively high carbapenem resistance in E. cloacae ZY106 with imipenem MIC of 2 μg/ml, meropenem MIC of 4 μg/ml, even ertapenem MIC of 16 μg/ml. Therefore, other mechanisms such as deficiency in the OMPs (Bush et al., 1985 1985 ; Lee et al., 1991 1991 ; 1992 1992 ) and existence of efflux pump might also be contributing to the ertapenam resistance (Szabó et al., 2006 2006 ), although the efflux mechanism is not common in the Enterobacteriaceae. The Urea-SDS-PAGE analysis of OMPs proved that ZY106 lacked the OMP (38 kD), which suggests that at least reduced outer membrane permeability might be involved. However, it requires further investigation to prove whether the efflux mechanism was also involved in the ertapenam resistance in ZY106. 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