title: Evaluation of a new Etest for detecting metallo-beta-lactamases in routine clinical testing.
abstract: Several Etest (AB BIODISK, Solna, Sweden) gradient formats were developed for detection of metallo-beta-lactamases based on the reduction of imipenem (IP) or ceftazidime (TZ) MICs in the presence of EDTA or 2-mercaptopropionic acid (MPA). The Etest metallo-beta-lactamase (Etest MBL) strips consisted of a double-sided seven-dilution range of IP or TZ (4 to 256 microg/ml) and IP or TZ (1 to 64 microg/ml) overlaid with a constant concentration of EDTA or MPA. The prototype strips were evaluated on several agar media (brain heart infusion agar, Isosensitest agar, nutrient agar, and Mueller-Hinton agar for aerobes and brucella blood agar for anaerobes) with 138 challenge strains: Acinetobacter spp. (n = 9), Aeromonas spp. (n = 8), Chryseobacterium spp. (n = 28), Escherichia coli (n = 1), Klebsiella pneumoniae (n = 4), Pseudomonas aeruginosa (n = 14), Proteus mirabilis (n = 3), Serratia spp. (n = 10), Stenotrophomonas maltophilia (n = 43), Sphingobacterium spp. (n = 3), and Bacteroides fragilis group (n = 15). PCR analysis using specific primers for IMP-1, L1, CcrA, and bla(B/C) confirmed the presence of the metallo-beta-lactamase genes. Enzyme assays were also performed with IP as an indicator substrate followed by EDTA inhibition profiles. EDTA was found to be a better inhibitor of metallo-beta-lactamases, especially for anaerobes. IP was a better than TZ. Mueller-Hinton agar was the preferred medium, particularly when compared to Isosensitest agar, which frequently produced falsely low MICs for IP. Etest IP plus IP-EDTA with Mueller-Hinton agar had a sensitivity of 94% (79 of 84) and specificity of 95% (124 of 130). The Etest MBL strip appears to be an acceptable diagnostic reagent to detect metallo-beta-lactamase phenotypes in the clinical microbiology laboratory.
authors.affiliation: University of Bristol, United Kingdom. t.r.walsh@bristol.ac.uk
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Walsh
authors.forename: Anne
authors.initials: A
authors.lastname: Bolmström
authors.forename: Anette
authors.initials: A
authors.lastname: Qwärnström
authors.forename: Ana
authors.initials: A
authors.lastname: Gales
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology*; D002442:Ceftazidime / Q000378:metabolism* / Q000494:pharmacology; D003470:Culture Media; D006801:Humans; D015378:Imipenem / Q000378:metabolism* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D016133:Polymerase Chain Reaction; D011934:Reagent Strips; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D011934:Reagent Strips; D015378:Imipenem; D002442:Ceftazidime; D001618:beta-Lactamases