1 Division of Medical Microbiology, Institute of Infectious Diseases & Molecular Medicine, Medical School, University of Cape Town, Anzio Road, Observatory 7925, Cape Town; 2 National Health Laboratory Service, Groote Schuur Hospital, Cape Town, South Africa
Keywords: blaOXA-23 , carbapenem resistance , ß-lactamases
Sir,
In 2002, we saw the emergence of carbapenem resistance in clinical isolates of Acinetobacter baumannii isolated from patients at two local hospitals, Groote Schuur Hospital (GSH) and Tygerberg Hospital (TH), Western Cape, South Africa. The high levels of imipenem (16256 mg/L) and meropenem (>32 mg/L) resistance observed in these A. baumannii isolates suggested the presence of a metallo-ß-lactamase (MBL) or an oxacillinase, since these carbapenemases are considered the major mechanism of carbapenem resistance in these organisms.1
To screen for the presence of MBLs, Etest strips (AB Biodisk, Solna, Sweden) were used according to the manufacturer's instructions. Etest MBL strips contain increasing concentrations of imipenem (IP) on one end and imipenem overlaid with EDTA (IPI) on the other. The EDTA chelates the zinc ions required by MBLs to catalyse hydrolysis of imipenem and meropenem, thereby inhibiting MBL activity. A reduction in the imipenem MIC in the presence of EDTA of greater than or equal to eight-fold (IP/IPI 8) is interpreted as indicating MBL activity. A control Pseudomonas aeruginosa strain expressing a metallo-ß-lactamase VIM-2 (gift from P. Nordmann, France) had an IP/IPI ratio of >64. Of the 49 A. baumannii isolates included in this study (GSH, n = 13; TH, n = 36), all had an IP/IPI ratio of 864, suggesting the presence of an MBL in each of these strains.
To elucidate which enzyme was responsible for the observed MBL activity, PCR assays were carried out using primers for amplification of MBL-encoding genes blaIMP and blaVIM. Although PCR products of the expected size were obtained from P. aeruginosa strains carrying blaIMP-1 (gift from Y. Hirakata, Japan) and blaVIM-2, no PCR products were obtained from any of the 49 carbapenem-resistant A. baumannii isolates screened. This suggested that the strains tested do not contain IMP- or VIM-type MBLs.
Notwithstanding the Etest MBL result, PCR assays were carried out to screen for the presence of genes encoding oxacillinases. Amplicons of the expected size were obtained from a control A. baumannii strain containing OXA-23 (gift from L. Poirel, France) and from all 49 test strains. PCR products from a representative strain from each hospital were purified and sequenced. Analysis of the sequencing data obtained showed that the blaOXA genes were identical to blaOXA-23. It is likely, therefore, that the production of OXA-23 is the major mechanism of carbapenem resistance in the GSH and TH isolates, since this enzyme confers high levels of imipenem and meropenem resistance (MIC > 32 mg/L) on an A. baumannii host.2
One explanation for the anomalous results obtained with the Etest MBL strips may come from the data presented by Danel et al.3 These authors showed that OXA-10 and OXA-14 exist as highly active dimers and less active monomers. Whether OXA-23 requires dimerization to be fully active has not been tested. However, that this enzyme exists in two forms would explain the observations that the OXA-2327 group of enzymes have weak activity against carbapenems but confer high-level resistance to imipenem (1632 mg/L) and meropenem (32>128 mg/L) on their A. baumannii hosts.2,4 Danel et al.3 showed that metal ions such as Zn2+, Cu2+ and Ca2+ stabilize dimerization of OXA-10 and OXA-14 and prevent interconversion of these enzymes to less active monomeric forms. It is the metal ion stabilized dimeric form of the enzymes that are more active against the carbapenems and other ß-lactams. It follows, therefore, that in the presence of the metal chelator, EDTA, OXA enzymes are converted to a less active monomeric state, with a concomitant reduction in carbapenemase activity. Thus, the eight- to 64-fold reduction in IP/IPI observed when using Etest MBL strips may indicate conversion of the OXA-23 to the less active monomeric form, rather than MBL activity, and further work is required to test this hypothesis.
These data suggest that results obtained with Etest MBL strips should be interpreted with caution, particularly when the test organism is A. baumannii, and perhaps other pathogens including P. aeruginosa, which are known to contain oxacillinases. In these cases it may be necessary to carry out PCR assays designed for the detection of MBL and OXA genes.
Acknowledgements
This work was supported by grants from the University of Cape Town and the Medical Research Council, South Africa.
References
1. Livermore DM. The impact of carbapenemases on antimicrobial development and therapy. Curr Opin Investig Drugs 2002; 3: 21824.[Medline]
2.
Dalla-Costa LM, Coelho JM, Souza HAPHM et al. Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. J Clin Microbiol 2003; 41: 34036.
3. Danel F, Paetzel M, Strynadka NC. Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D ß-lactamases from Pseudomonas aeruginosa. Biochemistry 2001; 40: 941220.[CrossRef][ISI][Medline]
4.
Afzal-Shah M, Woodford N, Livermore DM. Characterization of OXA-25, OXA-26, and OXA-27, molecular class D ß-lactamases associated with carbapenem resistance in clinical isolates of Acinetobacter baumannii. Antimicrob Agents Chemother 2001; 45: 5838.
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