J Med Microbiol 57 (2008), 332-337; DOI: 10.1099/jmm.0.47576-0
© 2008 Society for General Microbiology
ISSN 1473-5644
High-level carbapenem resistance in a Citrobacter freundii clinical isolate is due to a combination of KPC-2 production and decreased porin expression
Rong Zhang1,
Lijiang Yang2,
Jia Chang Cai1,
Hong Wei Zhou1 and
Gong-Xiang Chen1
1 Second Affiliated Hospital of Zhejiang University, Zhejiang University, 88 JieFang Road, Hangzhou 310009, PR China
2 Huazhong University of Science & Technology, Wuhan, Hubei 430074, PR China
Correspondence
Gong-Xiang Chen
chengong218{at}163.com
Received 17 August 2007
Accepted 8 November 2007
An imipenem-resistant isolate of Citrobacter freundii ZJ163 (MIC 256 µg ml–1) isolated from a Chinese hospital was investigated. The C. freundii ZJ163 isolate exhibited high-level resistance to carbapenems, penicillins, cephalosporins, cefoxitin, aztreonam, quinolones and aminoglycosides. Isoelectric focusing (IEF) demonstrated three β-lactamases with pIs of 5.4 (TEM-1), 6.7 (KPC-2) and 7.9 (CTX-M-14). Two different transconjugants (types A and B) were obtained by conjugation studies. The type A transconjugant exhibited reduced susceptibility or resistance to penicillins, cephalosporins and aztreonam, but was susceptible to carbapenems, quinolones and aminoglycosides. The antimicrobial susceptibility patterns of the type B transconjugant were similar to that of type A, except for its significantly reduced carbapenem susceptibility (imipenem MIC 2 µg ml–1). IEF, specific PCRs and DNA sequence analysis indicated that the type A transconjugant produced CTX-M-14 β-lactamase with a pI of 7.9, that the type B transconjugant produced KPC-2 β-lactamase with a pI of 6.7 and that the β-lactamase with a pI of 5.4 was TEM-1. PCR analysis and sequencing confirmed the presence of the ampC gene in the chromosomal DNA from C. freundii ZJ163, although no activity of AmpC β-lactamase was detected by IEF. Urea/SDS-PAGE analysis of outer-membrane proteins revealed that the levels of the 41 and 38 kDa porins were decreased in C. freundii ZJ163. It was concluded that production of KPC-2 combined with decreased expression of porins contributes to high-level resistance to carbapenems in C. freundii ZJ163.
Abbreviations: ESBL, extended-spectrum β-lactamase; pI, isoelectric point; OMP, outer-membrane protein.
The GenBank/EMBL/DDBJ accession numbers for the blaKPC-2 and ampC gene sequences of Citrobacter freundii ZJ163 are EF062508 and EF426097.
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INTRODUCTION
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Citrobacter freundii is an opportunistic pathogen that can cause diarrhoea, septicaemia, meningitis, and urinary tract and respiratory system infection, especially in high-risk groups. C. freundii has now become one of the most important nosocomial infection pathogens in China. According to monitoring data during 1994–2001 for the Chinese Net for the Nosocomial Pathogens Resistance Surveillance, C. freundii was ranked tenth among Gram-negative organisms.
Increased emergence of members of the Enterobacteriaceae possessing extended-spectrum β-lactamases (ESBLs) has been accompanied by the widespread use of cephalosporins. Carbapenems are considered to be one of the few remaining therapies for infections caused by multidrug-resistant isolates, especially strains producing high-level AmpC or ESBLs. Over the past two decades, whilst carbapenem resistance has become a serious problem for non-lactose-fermenting bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Flavobacterium meningosepticum, it has remained uncommon in the Enterobacteriaceae. However, recently identification of carbapenem-resistant members of the Enterobacteriaceae has increased. The production of carbapenemase is a common mechanism of carbapenem resistance.
Carbapenem-hydrolysing KPC β-lactamases are a group of recently identified carbapenemases that belong to Bush group 2f, molecular class A. KPCs are capable of hydrolysing carbapenems, penicillins, cephalosporins and aztreonam, and are inhibited by clavulanic acid and tazobactam. The initial report of one of these β-lactamases, KPC-1, was from carbapenem-resistant Klebsiella pneumoniae isolated in North Carolina, USA (Yigit et al., 2001). KPC-2 was then found in isolates of K. pneumoniae (Moland et al., 2003), Salmonella enterica (Miriagou et al., 2003), Klebsiella oxytoca (Yigit et al., 2003) and Enterobacter sp. (Hossain et al., 2004). Soon after, KPC-3 was found in K. pneumoniae (Woodford et al., 2004) and Enterobacter cloacae (Bratu et al., 2005) from New York, USA. Recently, KPC-2 was found outside the USA in France (Naas et al., 2005), Israel (Navon-Venezia et al., 2006) and South America (Villegas et al., 2006), and KPC-2 was first identified in P. aeruginosa isolates, outside the family Enterobacteriaceae (Villegas et al., 2007). KPC-2 has emerged in China. A K. pneumoniae isolate from Hangzhou, China, producing KPC-2 was reported (Wei et al., 2007). Almost simultaneously, we identified KPC-2 in three isolates of Serratia marcescens from the same city but a different hospital (Zhang et al., 2007).
However, there has been no report of KPC in C. freundii so far. In this report, we describe a clinical isolate of carbapenem-resistant C. freundii that produces carbapenem-hydrolysing KPC-2 and expresses a reduced level of porins. This is the first report of detection of the plasmid-mediated carbapenem-hydrolysing KPC-2 in C. freundii.
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METHODS
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Bacterial strains.
A clinical isolate of carbapenem-resistant C. freundii ZJ163 was collected from a urine sample of a patient in the rehabilitation ward in the Second Affiliated Hospital of Zhejiang University in June 2006. The patient was initially diagnosed with an aneurysm at the end of May 2006. The patient received surgery in another hospital in Shanghai and was then transferred back to our hospital for rehabilitation at the end of June. Levofloxacin was provided immediately after hospitalization and C. freundii ZJ163 was then isolated from the patient on the second day. The identity of the isolate was confirmed using a VITEK system (bioMérieux). Escherichia coli EC600 (LacZ– NalR RifR) was used as a recipient in conjugal transfer experiments. E. coli ATCC 25922 was used for quality control in antimicrobial susceptibility tests.
Antimicrobial susceptibility testing.
MICs were determined using an agar dilution method according to the recommendations of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) (NCCLS, 2006).
Conjugal transfer experiments.
Conjugation experiments were carried out in mixed broth cultures. Rifampicin-resistant E. coli EC600 was used as the recipient. Overnight cultures of donor strain (200 µl) and recipient strain (100 µl) were mixed with 600 µl fresh Luria–Bertani broth and incubated for 4 h at 35 °C. The mixture was then inoculated on Mueller–Hinton agar plates containing rifampicin (700 µg ml–1) plus cefotaxime (2 µg ml–1), or rifampicin (700 µg ml–1) plus imipenem (0.5 µg ml–1) for 24 h at 35 °C. Colonies that grew on the selective medium were identified by a VITEK system. Plasmid DNA preparations were obtained by an alkaline lysis technique using an AxyPrep Plasmid Miniprep kit (Axygen Scientific). Plasmids were separated by electrophoresis in a 0.8 % agarose gel containing ethidium bromide in 0.5x TBE buffer at a constant voltage for 1 h.
Isoelectric focusing (IEF) of β-lactamases.
Crude β-lactamase extracts were prepared by ultrasonic treatment of bacterial cells. IEF was performed on a PhastGel polyacrylamide gel (Amersham Biosciences) using the PhastSystem (Pharmacia Biotech) following the method of Mathew et al. (1975). β-Lactamase activity was visualized by staining the gel with nitrocefin (Oxoid). The isoelectric points (pIs) were determined after comparison with the known β-lactamases TEM-1 (pI 5.4), SHV-1 (pI 7.6) and SHV-5 (pI 8.2).
PCR amplification and DNA sequence analysis of bla genes.
The primers used to amplify bla genes, class I integrase and class I integron are listed in Table 1
. PCR was carried out using approximately 0.3 µg template DNA, 0.5 µM each primer, 10 mM Tris/HCl (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM dNTPs and 2.5 U Taq DNA polymerase (Promega) in a total volume of 50 µl. Amplification was conducted in a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems). The PCR products were cloned into a pGEMT-Easy vector (Promega) and recombinant plasmids were transformed into E. coli DH5
as described previously (Chen et al., 2006). Inserts were sequenced using an ABI 3730 Sequencer (Applied Biosystems). The DNA sequences were identified using the BLAST program (www.ncbi.nlm.nih.gov/blast/).
Analysis of outer-membrane proteins (OMPs).
Protein concentration was determined with a Coomassie Brilliant Blue protein assay kit (Bio-Rad). The OMPs of C. freundii ATCC 8090 and C. freundii ZJ163 were isolated as described by Hernández-Allés et al. (1999). Electrophoretic analysis of OMPs by urea/SDS-PAGE was performed in 11.6 % acrylamide/0.4 % bisacrylamide/0.1 % SDS gels containing 20 % urea. Samples were boiled for 5 min in sample buffer before electrophoresis. The 0.75 mm thickness mini gel was run at a constant current of 20 mA for 85 min using a Mini-PROTEAN 3 slab electrophoresis cell (Bio-Rad). The gel was visualized by staining with Coomassie Brilliant Blue.
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RESULTS AND DISCUSSION
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Antimicrobial susceptibility
The results of susceptibility testing are shown in Table 2. C. freundii ZJ163 exhibited high-level resistance to imipenem and meropenem, with MICs for both drugs of 256 µg ml–1. The isolate was also resistant to penicillins, cephalosporins, cefoxitin, aztreonam, quinolones and aminoglycosides.
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Table 2. Antimicrobial susceptibility results for C. freundii ZJ163, Escherichia coli EC600 and transconjugants A and B
CTX, cefotaxime; RIF, rifampicin; IPM, imipenem.
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Plasmids and transfer of imipenem resistance
Transfer of β-lactam resistance from the C. freundii ZJ163 isolate to E. coli EC600 by conjugation was successful. Several single colonies that grew on the plate containing rifampicin plus cefotaxime were reinoculated simultaneously onto a plate containing rifampicin plus cefotaxime and a plate containing rifampicin plus imipenem. Bacteria from the same colony that grew on the plate containing rifampicin plus cefotaxime but not on the plate containing rifampicin plus imipenem were named transconjugant A, whilst those that grew on both plates were named transconjugant B. Both transconjugants were identified as E. coli by the VITEK system. Transconjugant A was resistant or had intermediate resistance to penicillins, cephalosporins and aztreonam, but was susceptible to carbapenems, cefoxitin, quinolones and aminoglycosides. The antimicrobial susceptibility patterns of transconjugant B were similar to those of transconjugant A, except for its significantly reduced carbapenem susceptibility, with MICs of 2 µg ml–1 (Table 2
). This suggested that the plasmid that existed in transconjugant B may play an important role in resistance to carbapenems in C. freundii ZJ163.
Plasmid profile results indicated that the C. freundii ZJ163 isolate harboured several plasmids. Transconjugants A and B both had a plasmid with a size of approximately 60 kb (Fig. 1).
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Fig. 1. Plasmid profiles of C. freundii ZJ163 and transconjugants. Plasmids prepared from E. coli V517 (54.2, 7.3, 5.6, 5.2, 3.9, 3.0, 2.7 and 2.1 kb) (lane 1), C. freundii ZJ163 (lane 2), transconjugant A (lane 3) and transconjugant B (lane 4).
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IEF analysis
IEF analysis demonstrated that the C. freundii ZJ163 isolate produced three β-lactamases with pIs of 5.4, 6.7 and 7.9, respectively. As shown in Fig. 2, transconjugant A produced a pI 7.9 β-lactamase and transconjugant B produced a pI 6.7 β-lactamase.
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Fig. 2. IEF patterns of crude β-lactamase extracts from C. freundii ZJ163 and transconjugants. Lanes: 1, C. freundii ZJ163; 2, transconjugant A; 3, transconjugant B; 4, strain producing TEM-1 (pI 5.4), SHV-1 (pI 7.6) and SHV-5 (pI 8.2).
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PCR and DNA sequence analysis
The IEF results suggested the presence of blaTEM (pI 5.4), blaKPC (pI 6.7) and blaCTX-M (pI 7.9). These three bla genes were detected in total plasmid DNA from the C. freundii ZJ163 isolate by PCR amplification. Furthermore, blaCTX-M was detected in plasmid DNA from transconjugant A and blaKPC was detected in transconjugant B. These three genes were identified as blaTEM-1 (pI 5.4), blaKPC-2 (pI 6.7) and blaCTX-M-14 (pI 7.9) by comparing their DNA sequences with known genes in GenBank. These results indicated that KPC-2 and CTX-M-14 were encoded on different conjugative plasmids. A transconjugant that produced TEM-1 was not obtained by conjugation experiments using selective medium containing rifampicin (700 µg ml–1) plus ampicillin (50 µg ml–1). blaTEM-1 was detected in total plasmid DNA but not in chromosomal DNA from C. freundii ZJ163, implying that blaTEM-1 was encoded on a third plasmid.
The presence of a chromosomal ampC gene was confirmed by PCR analysis and sequencing. The sequence showed 98.95 % nucleotide similarity (1134/1146) to the chromosomal ampC gene of C. freundii ATCC 6879. There was only one amino acid difference between them: Val-368 in C. freundii strain ATCC 6879 was replaced with Ala in C. freundii ZJ163. However, no activity of AmpC β-lactamase was detected by IEF. This was probably due to low enzyme activity.
Neither class I integrase nor class I integron was detected in the blaKPC-encoding plasmid. Most blaKPC-encoding plasmids can be conjugated into E. coli, except for the blaKPC-1-encoding plasmid described by Yigit et al. (2001). Some also encode blaTEM-1 (Hossain et al., 2004; Miriagou et al., 2003) and various ESBLs (Yigit et al., 2003). However, only a blaKPC gene was identified from the plasmid in transconjugant B in the current study. It is thus evident that blaKPC spreads among different types of plasmid through mobile elements, and that the spread of transmissible plasmids among different clinical members of the Enterobacteriaceae results in the spread of drug resistance. To identify the existence of the integrons, we conducted careful PCR analyses of the plasmid encoding KPC and the total plasmids. We found that class I integrase and the variable regions of integrons were amplified in the total plasmid DNA from C. freundii but not in the plasmid DNA from transconjugant A or B. Fig. 3 shows PCR amplification of class I integrase and the variable regions of integrons from C. freundii and transconjugants A and B. Therefore, we concluded that class I integrons, which may be related to aminoglycoside resistance, were not present on the KPC-encoding plasmid but were present on the TEM-encoding plasmid or other plasmids.
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Fig. 3. PCR amplification of class I integrase (a) and the variable regions of integrons (b) from C. freundii and transconjugants of type A and B. Lanes: 1, positive amplification control (plasmid containing an integron, confirmed by DNA sequencing); 2, total plasmid DNA from C. freundii ZJ163; 3, plasmid DNA from transconjugant A; 4, plasmid DNA from transconjugant B; 5, negative amplification control; M, DNA molecular mass standard (Takara).
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Analysis of OMPs
Transconjugant B, which only produced KPC-2, exhibited significantly reduced susceptibility to imipenem and meropenem (MICs 2 µg ml–1). However, it was not sufficient to explain the high-level carbapenem resistance in C. freundii ZJ163 (MICs 256 µg ml–1), as transconjugant B remained susceptible to carbapenems at NCCLS breakpoints. Therefore, OMPs were examined. Urea/SDS-PAGE analysis of OMPs showed that C. freundii ZJ163 and ATCC 8090 both expressed four major OMPs, with molecular masses of 47, 41, 38 and 32 kDa. The 41 and 38 kDa proteins were considered to be porins (Aoyama et al., 1988). The four OMPs were analogous to E. coli LamB, OmpC, OmpF and OmpA, respectively. We found that the 41 and 38 kDa porins were decreased in C. freundii ZJ163. To confirm whether these two proteins were completely abolished or had decreased expression, we overloaded the sample from C. freundii ZJ163 in electrophoresis. As shown in Fig. 4, even with approximately fourfold overloading of total protein from C. freundii ZJ163, only very faint bands of 41 and 38 kDa were observed compared with the bands from ATCC 8090 (Fig. 4). Therefore, the levels of the 41 and 38 kDa porins were confirmed to be significantly decreased in C. freundii ZJ163 compared with ATCC 8090. We inferred from these results that production of KPC-2 combined with decreased expression of porins confers high-level carbapenem resistance in C. freundii ZJ163.
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Fig. 4. Urea/SDS-PAGE analysis of OMPs extracted from C. freundii ATCC 8090 and C. freundii ZJ163. Lanes: 1, OMPs (2 µg) from C. freundii ATCC 8090; 2, OMPs (5 µg) from C. freundii ZJ163; 3, protein molecular mass standard (MBI Fermentas).
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Carbapenems are important antibiotics for treatment of nosocomial infections. The emergence and spread of β-lactamases capable of hydrolysing carbapenems has caused problems regarding therapy and control. Carbapenem resistance in C. freundii has rarely been reported. In this study, we identified the carbapenem-hydrolysing enzyme KPC-2 and decreased levels of porins in C. freundii. KPCs have been detected in a variety of members of the Enterobacteriaceae (K. pneumoniae, Salmonella enterica, K. oxytoca, Enterobacter sp. and E. coli) and P. aeruginosa in different regions and countries (North Carolina, Maryland, New York and Boston, USA; Paris, France; Israel; South America; and Hangzhou, China). This indicates that the spread of blaKPC genes is no longer a regional problem. Attention should be paid to preventing potential outbreaks of nosocomial infections by pathogens producing KPC.
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TOP
INTRODUCTION
METHODS
