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

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 (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. 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 inhibitingMBL activity. A reduction in the imipenemMIC 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-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 from P. aeruginosa strains carrying blaIMP-1 (gift fromY. 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 IMPor 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. One explanation for the anomalous results obtained with the EtestMBL stripsmay come from the data presented byDanel et al. These authors showed that OXA-10 and OXA-14 exist as highly active dimers and less activemonomers.Whether OXA-23 requires dimerization to be fully active has not been tested. However, that this enzyme exists in two formswould explain the observations that the OXA-23–27 group of enzymes have weak activity against carbapenems but confer high-level resistance to imipenem (16– 32 mg/L) and meropenem (32–>128 mg/L) on their A. baumannii hosts. Danel et al. showed thatmetal ions such as Zn, Cu and Ca 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 b-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 eightto 64-fold reduction in IP/IPI observed when using Etest MBL strips may indicate conversion of the OXA-23 to the less activemonomeric form, rather thanMBL 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.