JAC Advance Access originally published online on November 16, 2007
Journal of Antimicrobial Chemotherapy 2008 61(1):219-220; doi:10.1093/jac/dkm444
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Research letters |
Detection of blaVIM-2 carbapenemase in Pseudomonas aeruginosa in Ireland
Department of Clinical Microbiology, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland
* Corresponding author. Tel: +353-1-896-3620; E-mail: walshf1{at}tcd.ie
Keywords: P. aeruginosa , metallo-β-lactamases , MBLs
Pseudomonas aeruginosa is one of the most frequently isolated Gram-negative pathogens associated with hospital-acquired infections. The carbapenems, imipenem and meropenem, have a broad spectrum of activity and are considered the agents of choice for the treatment of P. aeruginosa infections. However, metallo-β-lactamases (MBLs), which readily hydrolyse most β-lactams, including carbapenems, are increasingly being identified among P. aeruginosa clinical isolates. The VIM-type MBLs have been widespread in Asia, Southern Europe and North America. 1 Since 2000, the Antibiotic Resistance Monitoring and Reference Laboratory has received approximately 80 isolates, mostly Pseudomonas species, from the UK, which tested positive for bla VIM (Dr Neil Woodford, Health Protection Agency, London, personal communication). This study describes the first identification of bla VIM-2 in Ireland.
P. aeruginosa isolates 32297 and 15488 were isolated from a swab and a sputum sample, respectively, from the same patient at St James's Hospital, Dublin, Ireland. These isolates were 74.3% similar with more than three band differences, as determined by XbaI restriction digest and separation using PFGE, and thus considered unrelated. Both isolates were resistant to imipenem and meropenem using the CLSI guidelines. 2 Isolate 32297 had imipenem and meropenem MICs of 64 and 16 mg/L, respectively. Isolate 15488 had imipenem and meropenem MICs of >128 mg/L for both carbapenems. Both isolates had a reduction in imipenem and meropenem MICs of greater than 3 doubling dilutions in the presence of EDTA (320 mg/L), suggesting the presence of an MBL. This was confirmed by the MBL Etest (AB Biodisk, Solna, Sweden). bla VIM- and bla IMP-positive controls were included in each test. Isoelectric focusing was performed using the methods of Matthew et al. 3 bla VIM- and bla IMP-positive controls were included in the gels. The bla VIM control had pI values of 8.65, 6.85 and 6.75. Isolate 15488 had pI values of 8.65, 7.2 and 6.65 and isolate 33075 had values of 8.15 and 7.85. Both isolates were screened by PCR for the presence of bla VIM, bla IMP, bla SPM and bla GIM genes. Isolate 15488 was bla VIM-positive by PCR. The bla VIM gene was amplified by PCR and sequenced using primers forward 5'-GGC ATC CAA GCA GCA AG-3' and reverse 5'-AAG CAG ACT TGA CCT GA-3' for the amplification of the entire gene, which yield an 800 bp PCR product. The PCR product of 15488 had 100% identity with the bla VIM-2 gene, GenBank accession number AY943084 [GenBank] .1. Sequencing results also revealed that the bla VIM-2 gene of 15488 was inserted as a single gene cassette into a class 1 integron. The two isolates were tested phenotypically for the presence of efflux pumps by measuring the meropenem MIC in the presence of efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (50 mg/L) and phenyl-arginine-β-naphthylamide (20 mg/L). 4,5 There were no changes in the carbapenem MICs in the presence of the efflux pump inhibitors. The outer membrane proteins of the two clinical isolates, the bla VIM and bla IMP controls and the susceptible strain ATCC 27853 were extracted from exponentially growing cells. Both clinical isolates, in contrast to the susceptible ATCC 27853 strain, had loss of a 46 kDa protein band, which corresponds to the OprD porin. Isolate 33075 was PCR-negative for the tested MBLs, although a reduction in carbapenem MIC in the presence of EDTA has been detected. Such false-positive results could be attributed to the EDTA interference with the cell wall permeability as EDTA affects the regulation of the OprD porin and may facilitate the entry of carbapenems. 6
To the best of our knowledge, this is the first report of an MBL-producing P. aeruginosa isolate from Ireland. The MBL was not associated with an outbreak. The integron did not contain other resistance gene determinants. Although this isolate was not associated with an outbreak, the identification of this carbapenemase on a mobile element poses a threat to infection control. Carbapenem-resistant bla VIM-2-producing P. aeruginosa clones have emerged in several countries such as Portugal, Poland, Spain, Italy, Greece and Japan and can be attributed to the spread of bla VIM-2-producing P. aeruginosa. Therefore, continued monitoring and surveillance of P. aeruginosa for MBLs is required to minimize the threat of outbreaks. The identification of bla VIM-2 in Ireland also indicates that MBLs from their original reports in southern Europe have been further spread within Europe.
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This project was funded by the University of Dublin, Trinity College.
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None to declare.
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We wish to thank the staff of St James's Microbiology Department and Dr Neil Woodford for the donation of the bla VIM- and bla IMP-positive controls. Part of this study was presented at the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 2006.
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1 Walsh TR, Toleman MA, Poirel L, et al. Metallo-β-lactamases: the quiet before the storm? Clin Microbiol Rev (2005) 18:306–25.
2 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16 (2006) Wayne, PA, USA: CLSI.
3 Matthew M, Harris AM, Marshall MJ. The use of analytical isoelectric focusing for the detection and identification of β-lactamases. J Gen Microbiol (1975) 88:169–78.[Medline]
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Pournaras S, Maniati M, Spanakis N, et al. Spread of efflux pump-overexpressing, non-metallo-β-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity. J Antimicrob Chemother (2005) 56:761–4.
5 Renau TE, Leger R, Flamme EM, et al. Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin. J Med Chem (1999) 42:4928–31.[CrossRef][Web of Science][Medline]
6 Livermore DM, Brown DFJ. Detection of β-Lactamase-Mediated Resistance. http://www.bsac.org.uk/susceptibility_testing/powerpoint_presentations.cfm (15 October 2007, date last accessed).
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