Use of Etest MBL strips for the detection of carbapenemases in Acinetobacter baumannii

Abstract

Sir, In2002,wesawtheemergenceofcarbapenemresistanceinclinical 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 (16–256 mg/L) and meropenem (>32 mg/L) resistance observed in these A. baumannii isolates suggested the presence of a metallo-b-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 inhibitingMBLactivity.AreductionintheimipenemMICinthepresence 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-b-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 8–64, 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 fromP. aeruginosastrainscarryingblaIMP-1(giftfromY.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 VIMtype 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