Metallo-ß-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii

Jing-Jou Yan1, Wen-Chien Ko2, Chin-Luan Chuang1 and Jiunn-Jong Wu3,*

Departments of 1 Pathology, 2 Internal Medicine and 3 Medical Technology, College of Medicine, National Cheng Kung University, No. 1 University Road, Tainan 70101, Taiwan

Received 4 March 2002; returned 6 June 2002; revised 2 July 2002; accepted 9 July 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp. collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- ß-lactamases (MBLs). Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry blaIMP-8 and blaVIM-2, respectively, by colony hybridization, PCR and sequence analysis. The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E. cloacae among different healthcare settings. Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured blaSHV-12, which was on the same transferable plasmids with blaIMP-8. The blaVIM-2-positive isolate and all non-repetitive blaIMP-8-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing. Ribotyping of the IMP-8-producing E. cloacae isolates indicated that the dissemination of blaIMP-8 was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Carbapenems are the most potent agents used for the treatment of infections caused by Gram-negative bacteria because of their broad spectra of activity and stability to hydrolysis by most ß-lactamases, including extended-spectrum ß-lactamases (ESBLs).1,2 The emergence of acquired metallo-ß-lactamases (MBLs) in Gram-negative bacilli is becoming a therapeutic challenge since the enzymes usually possess a broad hydrolysis profile that includes carbapenems and extended-spectrum ß-lactams.37 In the past decade, a number of acquired MBLs have been identified and are categorized into two major groups: IMP- and VIM-type enzymes.313 MBLs can be either chromosomally encoded or plasmid mediated and are all inserted in integrons.312 Although MBL-producing organisms have been detected in many parts of the world,321 the exact prevalence rates in these countries remain unclear except in Japan.1417 In non-fastidious Gram-negative non-fermenters, such as Pseudomonas aeruginosa and Acinetobacter spp., a few outbreaks of infection caused by MBL producers have been reported.8,13,15,16,1921 Only a few MBLs were found in Enterobacteriaceae, and most of them were reported from sporadic isolates, such as IMP-1 in various species of Enterobacteriaceae,18,22 IMP-3 in Shigella flexneri7 and IMP-4 in Citrobacter youngae.9 Endemic outbreaks caused by MBL-producing Enterobacteriaceae isolates remained extremely rare. There have been repeated reports of IMP-1-producing Serratia marcescens isolates from Japan.16,17

Both IMP- and VIM-type MBLs have been detected in Taiwan.11,12 IMP-1 and VIM-2 were found in Pseudomonas putida and Pseudomonas stutzeri, and a variant of the VIM-2 enzyme, VIM-3, was found in P. aeruginosa.12 A variant of the IMP-2 enzyme, IMP-8, was identified from a clinical isolate of Klebsiella pneumoniae, which produced the SHV-12-type ESBL and TEM-1 as well.11 An outbreak caused by IMP-8-producing K. pneumoniae was noted more recently at the intensive care units of a university hospital in 1999 and 2000.23 The present study was conducted to investigate whether IMP- and VIM-type MBLs have spread to other members of the family Enterobacteriaceae in the university hospital during the 2 year period.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Clinical isolates and bacterial strains

A total of 9082 clinical isolates of the family Enterobacteriaceae other than Klebsiella spp. were consecutively collected at the National Cheng Kung University Hospital, a 900 bed hospital in southern Taiwan, in 1999 and 2000. Previous reports showed that all Gram-negative bacilli producing MBLs were resistant to ceftazidime, whereas their susceptibilities to carbapenems covered a wide range.321,23 Therefore, all our isolates judged to be resistant to ceftazidime (inhibition zone diameter <=14 mm) by the standard disc diffusion method were selected for further experiments in the present study.24 The bacterial species and the numbers of clinical isolates that were collected and selected are listed in Table 1. All these isolates were identified by using the conventional techniques25 and/or the API 20E system (bioMérieux, Marcy l’Étoile, France). The bacterial strains used as controls included blaVIM-1-containing P. aeruginosa VR-143/97,5 blaVIM-2-carrying P. putida NTU-91/99,12 blaVIM-3-containing P. aeruginosa NTU-26/99,12 blaIMP-1-carrying P. putida NTU-92/99,12 blaIMP-2-containing Acinetobacter baumannii AC-54/974 and blaIMP-8-carrying K. pneumo-niae KPO787.11


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Table 1.  Prevalence of clinical isolates producing MBLs
 
Colony blot hybridization

Colony blot hybridization was performed with the DNA probes generated by PCR amplification of the entire blaVIM-1, blaVIM-2, blaIMP-1 and blaIMP-2 genes as described previously.23 The probes were labelled with [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Hong Kong, China) by the random priming technique with a commercial kit (Gibco-BRL Life Technologies, Gaithersburg, MD, USA). Since there were only two nucleotide differences between blaVIM-2 and blaVIM-3,12 and four nucleotide differences between blaIMP-2 and blaIMP-8,11 the VIM-3- and IMP-8-producing control strains can also be hybridized with the blaVIM-2- and blaIMP-2-specific probes, respectively.23

PCR detection of MBL genes and DNA sequencing

Plasmids from the isolates were prepared by a rapid alkaline lysis procedure.26 The entire blaIMP-1-, blaIMP-2-, blaVIM-1-, blaVIM-2-, blaSHV- and blaTEM-related genes were amplified by PCR as described previously.23,27 The amplicons were purified with PCR clean-up kits (Roche Molecular Biochemicals, Mannheim, Germany) and were sequenced on an ABI PRISM 310 sequencer analyser (Applied Biosystems, Foster City, CA, USA). The PCR and sequencing primers used have been described elsewhere.23,27 blaIMP-8 and blaVIM-3 can also be amplified with the primers for blaIMP-2 and blaVIM-2, respectively.23,27

Conjugation experiments

Conjugation experiments were performed as described previously.23 The recipient was streptomycin- and rifampicin-resistant Escherichia coli C600.28 Tryptic soy agar plates supplemented with 500 mg/L streptomycin (Sigma Chemical Company, St Louis, MO, USA) or 64 mg/L rifampicin (Sigma) and 10 mg/L ceftazidime (Glaxo Group Research Ltd, Greenford, UK) were used to select transconjugants.

Isoelectric focusing (IEF) and ß-lactamase assays

Crude preparations of ß-lactamases obtained by sonication29 were subjected to IEF by the method of Matthew et al.30 with an LKB Multiphor apparatus (Amersham Pharmacia Biotech Asia Pacific, Hong Kong, China) as described previously.27 Gels were run over a pH range of 3.5–9.5. ß-Lactamase activity was detected by overlaying the gels with 0.5 mM nitrocefin (Oxoid, Basingstoke, UK) in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (pH 7.5) supplemented with 2 mM ZnCl2.4,12

Hydrolysis of 0.1 mM imipenem (Merck Sharp & Dohme, Rahway, NJ, USA) by crude preparations of ß-lactamases from four representative clinical isolates and their E. coli transconjugants was monitored by a Beckman DU-7 ultraviolet spectrophotometer (Palo Alto, CA, USA) at 297 nm and 37°C as described previously.4,19 Inhibition of enzymic activity was determined by measuring the residual carbapenemase activity after incubation of the crude extract with 10 mM EDTA or 50 µM clavulanic acid for 20 min at 25°C. A control without EDTA and clavulanic acid was always run in parallel. Reactivation by Zn2+ was assayed by measuring the carbapenemase activity after incubation of the EDTA-treated crude preparations for 20 min at 25°C in the presence of 2 mM ZnCl2. Controls for the effect of Zn2+ alone on enzyme activity and substrate stability were also included.

Plasmid analysis and Southern hybridization

Plasmids from the E. coli transconjugants were digested with EcoRI (Roche Molecular Biochemicals). Digested DNA samples were analysed by electrophoresis on 0.8% agarose gels. The sizes of transferred plasmids were estimated by adding up restriction fragments. The digested DNA samples were subjected to Southern hybridization according to the original protocol with the DNA probes prepared as described above.23,31

Ribotyping

The chromosomal DNA of the isolates was extracted and purified as described previously.32 The DNA samples were restricted with EcoRI (Roche Molecular Biochemicals), which can generate well-separated, discriminatory patterns among E. cloacae isolates.33 The digests of chromosomal DNA were electrophoresed at 35 V for 18 h on a 0.8% agarose gel, were transferred to a nylon membrane (Amersham Pharmacia Biotech) and then hybridized with a [{alpha}-32P]dCTP-labelled cDNA copy of E. coli rRNA (Roche Molecular Biochemicals) obtained by reverse transcription with avian myeloblastosis virus reverse transcriptase (Gibco-BRL).32

Susceptibility testing

The MICs of seven ß-lactam agents were determined by the standard agar dilution method, and the susceptibilities to eight non-ß-lactam antibiotics were determined by the standard disc diffusion method.24,34 The antimicrobial agents used for the agar dilution tests included aztreonam and cefepime (Bristol-Myers Squibb, New Brunswick, NJ, USA), cefoxitin (Sigma), ceftazidime (Glaxo Group Research Ltd), cefotaxime (Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ, USA), imipenem (Merck Sharp & Dohme) and meropenem (Sumitomo Pharmaceuticals Ltd, Osaka, Japan). Antimicrobial discs were all obtained from Becton Dickinson Microbiology Systems, Cockeysville, MD, USA, including amikacin, chloramphenicol, ciprofloxacin, gentamicin, netilmicin, ofloxacin, tobramycin and trimethoprim–sulfamethoxazole.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Prevalence of MBL-producing isolates

The proportions of MBL-producing Enterobacteriaceae isolates from the National Cheng Kung University Hospital during the two study years are listed in Table 1. Thirty-six of 1261 (2.9%) E. cloacae isolates collected from 20 patients were found to carry blaIMP-8 by colony hybridization, PCR and sequence analysis. Together with our previous study, this finding suggests the occurrence of interspecies spread of blaIMP-8 in Taiwan. All blaIMP-8-positive isolates also carried blaTEM-1 and 32 of them also carried the SHV-12-type ESBL gene. No significant annual increase in the rate of blaIMP-8-positive isolates was observed. One of 340 (0.3%) C. freundii isolates collected was found to harbour blaVIM-2, and the bacterial strain was the first Enterobacteriaceae isolate producing a VIM-type MBL in Taiwan. All the other glucose-fermenting bacilli were negative for the MBL genes tested.

Of the 36 blaIMP-8-positive isolates, 14 (38.9%), 14 (38.9%), three (8.3%), three (8.3%) and two (5.6%) isolates were recovered from samples of urine, sputum, wound, catheter tip and blood, respectively. The blaVIM-2-positive isolate was from a sputum sample. The selected clinical data of the MBL-producing isolates are shown in Table 2. In our previous survey, nearly all IMP-8-producing K. pneumoniae isolates were recovered from patients hospitalized in the intensive care units.23 In the present study, only nine (45%) of the 20 patients with the blaIMP-8-positive isolates were staying at the time of bacterial isolation or had stayed before bacterial isolation in the intensive care units. Seven patients were hospitalized in five different wards at the time of bacterial isolation and one patient was discharged from the neurology ward of the teaching hospital 2 weeks before bacterial isolation. These findings indicate that blaIMP-8 was not confined to the intensive care units as thought before,23 but rather has spread widely in the hospital. Four patients were transferred from four different hospitals <48 h prior to bacterial isolation, implying the spread of IMP-8-producing isolates among different healthcare settings in Taiwan. One isolate from each patient was subjected to further experiments.


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Table 2.  Characteristics of MBL-producing clinical isolates and selected clinical data
 
Conjugation experiments

Resistance to ceftazidime was successfully transferred from 14 of the 20 E. cloacae isolates and the C. freundii isolate to E. coli C600. The sizes of the blaIMP-8-containing plasmids transferred were all >150 kb, and the size of the blaVIM-2-containing plasmid was ~40 kb. The presence of the MBL genes on the transferred plasmids was confirmed by Southern hybridization (data not shown). PCR and sequence analysis indicated that blaSHV-12 and blaTEM-1 were also transferred with blaIMP-8 to E. coli.

Analytical IEF and ß-lactamase assays

The results of IEF are summarized in Table 2. On IEF gels, all blaIMP-8-positive E. cloacae isolates possessed three bands with pIs of 5.4, 8.0 and 8.2. The pI 5.4 band was consistent with the TEM-1 ß-lactamase,34 the pI 8.2 band might represent either IMP-8 alone or a mixture of IMP-8 and SHV-12,11 and the band with pI 8.0 might represent the chromosomal AmpC ß-lactamase of E. cloacae.35,36 The blaVIM-2-positive C. freundii isolate had two bands with pIs of 5.1 and 9.0, which were consistent with VIM-2 and the chromosomal AmpC ß-lactamase,6,35 respectively. Expression of ß-lactamases by the transconjugants was also demonstrated by IEF.

Carbapenemase activity of crude extracts from three IMP-8-producing E. cloacae isolates (K015, Q398 and R947), the VIM-2-producing C. freundii isolate and all their E. coli transconjugants was demonstrated by spectrophotometry. These isolates and transconjugants all hydrolysed imipenem (0.59–0.98 µmol of imipenem/min/mg of protein). Carbapenemase activity was almost completely inhibited by 10 mM EDTA (0.03–0.06 µmol of imipenem/min/mg of protein) but not by clavulanic acid, and the inhibition was reversed by 2 mM ZnCl2 (0.27–0.47 µmol of imipenem/min/mg of protein). The hydrolysis rates were not significantly different among different isolates, between E. cloacae and C. freundii, and between clinical isolates and transconjugants. The ß-lactamase assay confirmed the expression of MBLs by the isolates and their transconjugants.

Ribotyping

The results of ribotyping are listed in Table 2 and the representative patterns are shown in Figure 1. Patterns with at least two discordant bands were considered different.32,37 Four major patterns were obtained by digestion with EcoRI among the 20 E. cloacae isolates. Ribotype E1 was further divided into two subtypes, E1a and E1b, which had only one band difference. The genetically closely related clones with ribotypes E1a and E1b were predominant among the isolates, and were found to circulate in different wards of the university hospital, and probably have also spread in hospitals B and D. The isolates possibly from hospitals A and C had an identical ribotype E3. The ribotyping study indicates that the prevalence of the MBL-producing E. cloacae isolates was largely due to the spread of resistant clones, although horizontal transfer of the MBL gene also occurred.



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Figure 1. Representative ribotypes of IMP-8-producing E. cloacae isolates generated by EcoRI. Ribotypes E1a, E1b, E2, E3 and E4 are from isolates I201, S231, K015, R947 and Y580, respectively.

 
Susceptibility tests

The results of the susceptibility tests are shown in Table 3. All MBL-producing isolates were resistant to ceftazidime, cefotaxime, cefoxitin and cefepime. The 17 E. cloacae isolates harbouring blaIMP-8, blaSHV-12 and blaTEM-1 were also resistant to aztreonam (MICs >= 64 mg/L), whereas the three isolates carrying blaIMP-8 and blaTEM-1 and the VIM-2-producing C. freundii isolate were susceptible (MICs 0.03–0.06 mg/L). Since aztreonam is stable to hydrolysis by MBLs,36 resistance to aztreonam could be due to the co-existence of blaIMP-8 and blaSHV-12 on the same plasmid. All the MBL-producing isolates demonstrated resistance to chloramphenicol, trimethoprim–sulfamethoxazole, and at least one kind of aminoglycoside, and the resistance phenotypes were almost always transferred to their transconjugants, suggesting that the transferred plasmids also contained genetic determinants responsible for resistance to the non-ß-lactam antimicrobial agents. The same finding has been observed in the IMP-8-producing K. pneumoniae isolates from the university hospital.23 Thus, the spread of the blaIMP-8-containing multidrug resistance plasmids has become a great threat to the healthcare setting. The isolates had higher MICs of extended-spectrum ß-lactams than their transconjugants. In E. cloacae, reduced outer membrane permeability is associated with reduced susceptibilities to ß-lactams and some non-ß-lactam antibiotics,38,39 and in conjunction with derepression of intrinsic AmpC cephalosporinases,39 can confer imipenem resistance. Accordingly, it is likely that expression of intrinsic AmpC enzymes reduced permeability to the antibiotics tested, or both might also play a role in reduced susceptibility to the antimicrobial agents tested in our MBL-producing isolates, resulting in higher MICs for the clinical isolates than those for their transconjugants.


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Table 3.  Antimicrobial susceptibility of MBL-producing clinical isolates and their tranconjugants
 
Correlation between carriage of blaIMP-8 and of carbapenem resistance in our isolates was imperfect (Table 3). None of our MBL-producing isolates was resistant to carbapenems based on the susceptibility tests; however, hydrolysis of imipenem by crude extracts from the selected strains indicated that the MBL genes were expressed. Similar results were obtained in many studies on MBLs,321 including our previous study on MBL-producing K. pneumoniae.23 It has been suggested that substantive resistance to carbapenems in MBL-producing Gram-negative bacilli might require reduced uptake of carbapenems as well as the presence of MBLs.40,41 Thus, the susceptibility test is not a reliable tool for detection of MBL producers in Enterobacteriaceae.

A disc diffusion test designed by Arakawa et al.22 has been shown to be very useful for screening MBL producers. MBL-producing strains display expansion of the growth-inhibitory zone between a disc containing ceftazidime and a disc containing 2-mercaptopropionic acid. All MBL-producing isolates of Pseudomonas spp. can be detected by the screening test in our previous study.12 We have used the method to screen MBL producers in Enterobacteriaceae in a pilot study; however, an unsatisfactory result was obtained (J. J. Yan & J. J. Wu, unpublished results). A discernible change in the inhibition zone between the ceftazidime and 2-mercaptopropionic acid discs cannot be obtained for the isolates producing both IMP-8 and SHV-12. The result might be due to the fact that SHV-12 exhibits resistance to ceftazidime,27 but unlike MBLs, is not inhibited by 2-mercaptopropionic acid, leading to a minimal change in the inhibition zone of the ceftazidime disc.

In conclusion, the spread of MBLs in Enterobacteriaceae is becoming a clinical concern in Taiwan. Consistent and constant surveillance of the MBL producers will be the prime measure to prevent their further dissemination. However, the detection of MBLs is a great problem to healthcare settings. Routine susceptibility testing cannot be used to predict the presence of MBLs, and the problem might be complicated by co-existence of other ß-lactamases in a bacterial strain. Molecular biology techniques, such as hybridization assays with gene-specific probes or PCR with gene-specific primers,16,17 are therefore needed for the purposes of epidemiology and infection control.


    Acknowledgements
 
The authors are grateful to Dr G. M. Rossolini for providing the IMP-2-containing A. baumannii strain AC-54/97 and the VIM-1-containing P. aeruginosa strain VR-143/97. This work was partially supported by grant DOH91-DC1033 from the Center for Disease Control, the Department of Health, the Executive Yuan, and grant NSC 91-2314-B-006-002 from the National Science Council, Taiwan.


    Footnotes
 
* Corresponding author. Tel: +886-6-2353535-5605; Fax: +886-6-2363956; E-mail: jjwu{at}mail.ncku.edu.tw Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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