JAC Advance Access originally published online on January 6, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 397-400
© 2003 The British Society for Antimicrobial Chemotherapy
Characterization of a new integron containing VIM-2, a metallo- ß-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae
1 Departments of Clinical Pathology, 7 Internal Medicine and 8 Therapeutic Radiology, Kosin University College of Medicine, Amnam-Dong 34, Seo-Ku, Busan; 2 Research Institute of Bacterial Resistance and 3 Department of Clinical Pathology, Yonsei University College of Medicine, Seoul; 4 Department of Genetic Engineering, Youngdong University, Youngdong; 5 SJ Hightech, Busan; 6 Department of Clinical Pathology, Dong-A University College of Medicine, Busan, South Korea
Received 7 March 2002; returned 19 June 2002; revised 14 October 2002; accepted 18 October 2002
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Abstract |
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We report the first description of a new integron containing blaVIM-2, a metallo-ß-lactamase gene from Enterobacter cloacae KU680, which was isolated from peritoneal fluid of a liver cirrhosis patient in South Korea. Antibiotic susceptibility testing, and modified Hodge and EDTA-disc synergy tests, were carried out to screen for metallo-ß-lactamase-producing strains. PCR and sequence analysis were used to identify and analyse the blaVIM-2-containing integron. The isolate was resistant to most ß-lactams, including imipenem, and demonstrated a positive modified Hodge and EDTA-disc synergy test, which are findings suggesting a metallo-ß-lactamase. Preliminary PCR-based experiments detected the metallo-ß-lactamase gene blaVIM-2. Sequencing of the 4392 bp cloned PCR amplicon, containing the gene cassette blaVIM-2, revealed the structure of the class 1 integron. The integron also contained additional insert gene cassettes, aadA, and unknown open reading frames ‘orfII’ and ‘orfIII’. To the best of our knowledge, this is the first time that this metallo-ß-lactamase gene has been detected in E. cloacae.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Enterobacter cloacae is a well-recognized nosocomial pathogen that causes significant infections. This microorganism is intrinsically resistant to ampicillin and narrow-spectrum cephalosporins, due to a chromosomal cephalosporinase. Additional resistance to broad-spectrum cephalosporins and aztreonam is usually related to the mutational over production of the species-specific cephalosporinase, or production of plasmid-mediated extended-spectrum ß-lactamases. But imipenem-resistant clinical isolates of E. cloacae are unusual and have been described in strains with porin alterations combined with hyperproduction of chromosomal cephalosporinase, and in strains producing class A carbapenem-hydrolysing non-metallo-ß-lactamases, such as NmcA ß-lactamase and IMI-1 ß-lactamase.1
However, class B metallo-ß-lactamases have been reported recently in several strains of Gram-negative bacilli.2 These enzymes possess the broadest substrate hydrolysis range among ß-lactamases of Gram-negative bacilli, including penicillins, cephalosporins, cephamycins and carbapenems, but not monobactams. Since the first finding of IMP-1 metallo-ß-lactamase in a clinical isolate of Pseudomonas aeruginosa in Japan in 1988, IMP-2, VIM-1 and VIM-2 metallo-ß-lactamases have been reported in various Gram-negative bacilli. The blaVIM and blaIMP genes occur in mobile gene cassettes inserted in the variable regions of integrons.3 Gene cassettes are mobile elements, so that horizontal spread of resistance can be anticipated, in addition to clonal spread.
In 2000, an imipenem-resistant strain of E. cloacae was isolated in a tertiary care hospital in Busan, Korea, from the peritoneal fluid of a patient with liver cirrhosis, which carried a blaVIM-2-containing integron. To the best of our knowledge, E. cloacae strains carrying metallo-ß-lactamase genes have not been reported before. Therefore, we undertook this study to analyse the structure of the blaVIM-2-containing integron.
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Materials and methods |
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Bacterial strains and susceptibility testing
The E. cloacae KU680 isolate was identified by the conventional method, and with the API 20E system (bioMérieux Vitek, Marcy-l’Étoile, France). Antibiotic-containing discs (BBL, Cockeysville, MD, USA) were used for routine antibiograms by disc diffusion assay.4 MICs of antimicrobial agents were determined by the agar dilution method.5 Escherichia coli ATCC 25922 was used as MIC reference strain. Modified Hodge and EDTA-disc synergy tests were performed for the screening of metallo-ß-lactamase-producing strains.6
Conjugation and isoelectric focusing of ß-lactamase
The filter mating method was used to determine the transferability of the carbapenem resistance determinant, as previously described.7 The isoelectric points of ß-lactamases were determined by loading cell sonicates to precast pH 3 to 10 gels. The gel was overlaid with a filter paper soaked in 20 mM EDTA for 5 min, before the imipenem (0.5 mg/L)-containing Mueller–Hinton agar was added. In this manner, inhibition of imipenem-hydrolysing activities could be observed.
Molecular techniques
Searches for blaIMP-1 and blaVIM-2 genes in E. cloacae KU680 were performed by PCR amplification with the following sets of primers: for the blaIMP-1 gene, IMP-1F (5'-CATGGTTTGGTGGTTCTTGT-3') and IMP-1R (5'-ATAATTTGGCGGACTTTGGC-3'); for the blaVIM-2 gene, VIM-2F (5'-ATGTTCAAACTTTTGAGTAAG-3') and VIM-2R (5'-CTACTCAACGACTGAGCG-3'); for the class 1 integron, 5'CS-F (5'-CTTCTAGAAAACCGAGGATGC-3') and 3'CS-R (5'-CTCTCAAGATTTTAATGCGGATG-3'). PCR amplification was carried out as previously described,7 as was the preparation of recombinant plasmids containing PCR product, and transformation of them into E. coli DH5
.7 Plasmids from successful clones were used to determine the sequence of the integron by the dideoxynucleotide-chain termination method, with an automatic DNA sequencer (ABI 3700, Perkin-Elmer, Foster City, CA, USA). The determination of the sequence was repeated with more than two clones from independent amplicons. Both strands were sequenced.
Nucleotide and amino acid sequence analysis
Nucleotide sequence analysis was performed with DNASIS for Windows (Hitachi Software Engineering America Ltd, San Bruno, CA, USA). Database similarity searches for nucleotide and deduced amino acid sequences were carried out at the NCBI website (http://www.ncbi.nlm.nih.gov).
Nucleotide sequence accession number
The nucleotide sequences of the blaVIM-2 gene of E. cloacae KU680 have been assigned to the GenBank nucleotide sequence database under accession number AF305559.
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Results |
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Properties of E. cloacae KU680
Disc diffusion testing revealed that E. cloacae KU680 isol- ate was resistant to most ß-lactams, including ampicillin, ampicillin–sulbactam, piperacillin, piperacillin–tazobactam, cefalothin, cefoxitin, cefotaxime, ceftazidime and aztreonam. The isolate was also resistant to tobramycin, intermediate to gentamicin, but susceptible to amikacin and ciprofloxacin. MICs of imipenem and meropenem for the isolate were 4 mg/L, and that of aztreonam was 64 mg/L. MICs of ampicillin, ampicillin–sulbactam, piperacillin, piperacillin–tazobactam, cefalothin, cefoxitin, cefotaxime and ceftazidime were >128 mg/L.
Isoelectric focusing of extract of the isolate showed two ß-lactamase bands of pI 
5.3 and 9.0. The isolate showed positive modified Hodge and EDTA-disc synergy tests, and the only pI 5.3 band was no longer present when the gels were overlaid with EDTA, which are findings suggesting a metallo-ß-lactamase. The band of pI 9.0 was likely to be chromosomal AmpC cephalosporinase. Carbapenem resistance was not transferred by conjugation. A plasmid harbouring a carbapenem resistance determinant was not detected (data not shown). These results suggest that a metallo-ß-lactamase gene may be located on the chromosome.
Sequence analysis of the blaVIM-2-containing integron
Preliminary PCR-based experiments detected blaVIM-2 but failed to detect blaIMP-1. Sequence analysis of the 4392 bp cloned PCR amplicon revealed the structure of the class 1 integron, such as the 5'-CS element containing an IntI1 integrase gene with its own promotor region, an attI1 recombination site, and the 3'-CS element containing qacE
1. The integron contained insert gene cassettes blaVIM-2, aadA1, and unknown open reading frames (ORFs) ‘orfII’ and ‘orfIII’. The blaVIM-2 gene was located immediately downstream of the IntI1 integrase gene. The initiation codon of the blaVIM-2 gene was proceeded by a putative promoter region of Pc. The guanine at the second base in the –35 consensus of the promoter Pc was replaced by thymidine, and the guanine at the fourth base in the –10 consensus was replaced by adenine. The blaVIM-2 gene cassettes had a 59 bp element, and the qacE1 gene also had a core site at its 5' end. The sequences of orfII and orfIII did not show a typical cassette structure, i.e. possession of the consensus core sequence of GTTRRRY on the 5' end (Figure 1).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Since 1989, metallo-ß-lactamases have spread among glucose non-fermenting Gram-negative bacilli, including Pseudomonas aeruginosa, Pseudomonas putida, Alcaligenes (Achlomobacter) xylosoxidans, Acinetobacter baumannii and Acinetobacter genomospecies 3.7,8 However, these enzymes were rarely found in members of the family Enterobacteriaceae. The presence of the mobile blaVIM-2-containing integron in E. cloacae suggests that the resistance can spread to other members of the family Enterobacteriaceae.
MICs of imipenem (4 mg/L) and meropenem (4 mg/L) for E. cloacae KU680 were relatively low compared with those for VIM-2-producing P. aeruginosa COL-1 (128 mg/L) and Serratia marcescens YMC 00/4/1591 (64 mg/L).8,9 The difference in permeability coefficients of carbapenems between bacterial species may play a role in the low level of resistance to carbapenems.10
The Pc promoter for the blaVIM-2 gene is a strong promoter, due to the point mutations in the –35 and –10 consensus regions.9 The P2 promoter was not found upstream of the blaVIM-2 gene. P2 expression may be responsible for up to 90% of blaVIM-2 transcription, as described for other integron-located genes.9 Inactive P2 promoter may also play a role in the low expression of resistance to carbapenems.
VIM-2 metallo-ß-lactamase has no hydrolytic activity against aztreonam, but the MIC of aztreonam for E. cloacae KU680 was 64 mg/L, which is higher than the resistant breakpoint.9 This result was possibly due to production of a chromosomal cephalosporinase (pI 9.0). The isolate was found to be resistant to tobramycin and intermediate to gentamicin, but the new class 1 integron in this study contained only a streptomycin/spectinomycin adenyltransferase gene (aadA1) besides blaVIM-2 cassettes, suggesting that the gene(s) responsible for the aminoglycoside resistance resided in another location.
The structure of the new integron (intI1 [blaVIM-2 aadA1 orfII orfIII] qacE1) was similar to that of an integron in P. putida YMC 97/8/322 (intI1 [blaVIM-2 aacA7 aadA1] qacE1; GenBank accession number AF327064), that of an integron in Acinetobacter genomospecies 3 YMC 99/11/160 (intI1 [blaVIM-2 aacA4 aadA1 orfII orfIII] qacE1; GenBank accession number AF369871), and that of an integron in P. aeruginosa YMC 95/1/704 (intI1 [aacA4 blaVIM-2 orfI aadA1 orfII orfIII] qacE1; GenBank accession number AY029772), all of which were detected in South Korea. The nucleotide sequences of the two unknown ORFs (orfII and orfIII) were very similar to those of P. aeruginosa (99.8% and 99.7% identity), Acinetobacter genomospecies 3 (99.9% and 100% identity) and S. marcescens (both 99.7 % identity) strains previously isolated in Korea, which were arbitrarily named orfII and orfIII.8 Since several bacterial species carrying blaVIM-2-containing integrons (which show similarity in their possession of orfII and orfIII as well as blaVIM-2) have been isolated in Korea, this may suggest the horizontal spread of blaVIM-2. As a result of partial DNA sequencing we also found one Morganella morganii isolate containing an integron similar to that of E. cloacae KU680.
In conclusion, E. cloacae KU680, which carries a blaVIM-2 gene, represents an emerging threat. The blaVIM-2-containing integrons are mobile, so horizontal spread of imipenem resistance to more frequent pathogens, such as E. coli, can be anticipated. The spread of blaVIM-2 could compromise the future usefulness of carbapenems for the treatment of infections caused by Gram-negative bacilli.
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Acknowledgements |
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This work was supported by a research grant of the Korea Science and Engineering Foundation (1999-2-211-002-5).
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Footnotes |
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* Corresponding author. Tel: +82-51-990-6373; Fax: +82-51-990-3034; E-mail: kscpjsh{at}ns.kosinmed.or.kr
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References |
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