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Antimicrobial Agents and Chemotherapy, August 2007, p. 3038-3039, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00374-07

LETTER TO THE EDITOR

First Occurrence of an Escherichia coli Clinical Isolate Producing the VIM-1/VIM-2 Hybrid Metallo-ß-Lactamase VIM-12

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Gram-negative bacteria producing VIM-type metallo-ß-lactamases (MBLs) are increasingly isolated worldwide (8). The VIM group includes at least 13 variants, clustered into three evolutionary lineages, driven by VIM-1, VIM-2, and VIM-7 (www.lahey.org/studies/other.asp#table1). bla_VIM-12 is a recently identified bla_VIM-1/VIM-2 hybrid gene, originally found in 2005 in a Klebsiella pneumoniae clinical isolate from Greece and ranked as intermediate between bla_VIM-1 and bla_VIM-2 (6). Its gene cassette resides in a class 1 integron, designated In-h12 (6); it has not since been detected in other gram-negative species. Kinetic parameters of the purified VIM-12 enzyme have not been described. We document herein the identification of the bla_VIM-12 gene in an Escherichia coli clinical isolate.

E. coli strain 28 was recovered in February 2006 from a decubitus ulcer infection of an 85-year-old male hospitalized at Hippokration General Hospital, Thessaloniki, Greece, for lower respiratory tract infection. MICs of several ß-lactams, aminoglycosides, ciprofloxacin, tetracycline, and cotrimoxazole were determined by using Etest (AB Biodisk, Solna, Sweden). The isolate was phenotypically screened for MBL production by using Etest MBL (AB Biodisk) and the imipenem-EDTA double disc synergy test (DDST) (5). Pseudomonas aeruginosa ATCC 27853 was used as a control in all susceptibility assays.

PCR detection of various genes encoding MBLs, extended-spectrum beta-lactamases, and AmpC enzymes, including bla_VIM, bla_IMP, bla_SPM, bla_TEM, bla_SHV, bla_GES, bla_CTX-M and bla_CMY/LAT, and PCR integron mapping were performed as described previously (6), followed by DNA sequencing.

Filter mating experiments were performed using E. coli 26R793 (Rif^r) as the recipient. Selection of transconjugants was made on MacConkey agar plates containing rifampin (100 µg/ml) and ceftazidime (2 to 4 µg/ml). Plasmid isolation was performed using the ChargeSwitch Plasmid ER minikit (Invitrogen Corporation, Carlsbad, CA) and a standard alkaline lysis protocol, using E. coli 39R861 as the standard plasmid control. Restriction fragment length polymorphism (RFLP) of the plasmid extract of E. coli strain 28 and the transconjugant derived from Klebsiella pneumoniae 2873 carrying VIM-12 in plasmid p2873 (6) was performed using BamHI. The location of the bla_VIM gene was determined as described previously (3) by Southern blotting of unsheared genomic DNA, followed by gene-specific hybridization using a digoxigenin-labeled bla_VIM-1 probe (7).

E. coli strain 28 was susceptible to imipenem and meropenem (MICs, 1 and 0.25 µg/ml, respectively), aztreonam, ciprofloxacin, and cotrimoxazole but resistant to all other antimicrobials tested (Table 1). The presence of MBL was indicated by positive DDST and Etest MBL. PCR for ß-lactamase genes showed that E. coli strain 28 was positive for bla_VIM and bla_CMY/LAT genes but negative for the remaining genes. PCR assays using primers 5'CS and 3'CS in various combinations with primers bla_VIM and aacA and nucleotide sequencing revealed an integron structure identical with that of integron In-h12, originally found in the bla_VIM-12-producing K. pneumoniae (GenBank accession number DQ143913) (6).

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TABLE 1. Susceptibilities of strain EC28 and its respective transconjugant carrying pEC28

Repeated mating experiments yielded, at a median frequency of 1.8 x 10^–2 per donor cell, transconjugant colonies that had elevated MICs of penicillins, cephalosporins, and aminoglycosides but not of carbapenems and tetracycline (Table 1). Plasmid analysis of both clinical and transconjugant colonies showed a single plasmid of approximately 70 kb. PCR specific for bla_VIM and bla_CMY/LAT in several transconjugant colonies was positive only for the latter gene. RFLP analysis with BamHI showed that the conjugative plasmid of E. coli strain 28 had a different restriction pattern from that of p2873 (6), tested in parallel. The location of In-h12 in the chromosome was indicated by hybridization of the unsheared genomic DNA with the bla_VIM-1 probe.

Production of VIM-12, a VIM-1/VIM-2 hybrid MBL, is documented here for the first time for an E. coli isolate after its original detection in K. pneumoniae. It is noteworthy that several bla_VIM-12-producing K. pneumoniae strains have been identified in the same hospital since the original detection in 2005 (unpublished data), indicating a rather wide dissemination of this gene among our K. pneumoniae isolates. E. coli strain 28 carried an integron identical to In-h12, although not lying in the transferable plasmid p2873, which harbored bla_VIM-12 in K. pneumoniae (6). It could be speculated here that the integron In-h12 containing the bla_VIM-1/VIM-2 hybrid gene bla_VIM-12 has arisen within our hospital settings, where bla_VIM-1- and bla_VIM-2-carrying gram-negative pathogens are common (1, 2, 4, 7). Alternatively, the bla_VIM-12 gene might be independent and have a wide natural distribution in bacterial populations, as was observed previously for other bla_VIM genes (8).

 
Published ahead of print on 21 May 2007.

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1. Galani, I., M. Souli, E. Koratzanis, Z. Chryssouli, and H. Giamarellou. 2006. Molecular characterization of an Escherichia coli clinical isolate that produces both metallo-ß-lactamase VIM-2 and extended-spectrum beta-lactamase GES-7: identification of the In8 integron carrying the bla_VIM-2 gene. J. Antimicrob. Chemother. 58:432-433.[Abstract/Free Full Text]
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2. Giakkoupi, P., A. Xanthaki, M. Kanellopoulou, A. Vlahaki, V. Miriagou, S. Kontou, E. Papafraggas, H. Malamou-Lada, L. S. Tzouvelekis, N. J. Legakis, and A. C. Vatopoulos. 2003. VIM-1 metallo-ß-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals. J. Clin. Microbiol. 41:3893-3896.[Abstract/Free Full Text]
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3. Lauretti, L., M. L. Riccio, A. Mazzariol, G. Cornaglia, G. Amicosante, R. Fontana, and G. M. Rossolini. 1999. Cloning and characterization of bla_VIM, a new integron-borne metallo-ß-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 43:1584-1590.[Abstract/Free Full Text]
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4. Miriagou, V., E. Tzelepi, D. Gianneli, and L. S. Tzouvelekis. 2003. Escherichia coli with a self transferable, multiresistant plasmid coding for metallo-ß-lactamase VIM-1. Antimicrob. Agents Chemother. 47:395-397.[Abstract/Free Full Text]
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5. Oh, E. J., S. Lee, Y. J. Park, J. J. Park, K. Park, S. I. Kim, M. W. Kang, and B. K. Kim. 2003. Prevalence of metallo-ß-lactamase among Pseudomonas aeruginosa and Acinetobacter baumannii in a Korean university hospital and comparison of screening methods for detecting metallo-beta-lactamase. J. Microbiol. Methods 54:411-418.[CrossRef][Medline]
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6. Pournaras, S., A. Ikonomidis, L. S. Tzouvelekis, D. Tokatlidou, N. Spanakis, A. N. Maniatis, N. J. Legakis, and A. Tsakris. 2005. VIM-12, a novel plasmid-mediated metallo-beta-lactamase from Klebsiella pneumoniae that resembles a VIM-1/VIM-2 hybrid. Antimicrob. Agents Chemother. 49:5153-5156.[Abstract/Free Full Text]
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7. Tsakris, A., S. Pournaras, N. Woodford, M. F. Palepou, G. S. Babini, J. Douboyas, and D. M. Livermore. 2000. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J. Clin. Microbiol. 38:1290-1292.[Abstract/Free Full Text]
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8. Walsh, T. R., M. A. Toleman, L. Poirel, and P. Nordmann. 2005. Metallo-ß-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 18:306-325.[Abstract/Free Full Text]

						Alexandros Ikonomidis Maria Labrou Department of Medical Microbiology University Hospital of Larissa 41110 Larissa, Greece Zoe Afkou Department of Microbiology Ippokration Hospital of Thessaloniki Thessaloniki, Greece Antonios N. Maniatis Department of Medical Microbiology University Hospital of Larissa 41110 Larissa, Greece Danai Sofianou Department of Microbiology Ippokration Hospital of Thessaloniki Thessaloniki, Greece Athanassios Tsakris Department of Microbiology University of Athens 11527 Athens, Greece Spyros Pournaras* Department of Medical Microbiology University Hospital of Larissa 41110 Larissa, Greece
						* Phone: (30) 2410 682929 Fax: (30) 2410 681570 E-mail: pournaras{at}med.uth.gr

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Antimicrobial Agents and Chemotherapy, August 2007, p. 3038-3039, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00374-07

This article has been cited by other articles:

• Tokatlidou, D., Tsivitanidou, M., Pournaras, S., Ikonomidis, A., Tsakris, A., Sofianou, D. (2008). Outbreak Caused by a Multidrug-Resistant Klebsiella pneumoniae Clone Carrying blaVIM-12 in a University Hospital. J. Clin. Microbiol. 46: 1005-1008 [Abstract] [Full Text]  

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