title: Development and validation of a multiplex TaqMan real-time PCR for rapid detection of genes encoding four types of class D carbapenemase in Acinetobacter baumannii.
abstract: A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D β-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87 %) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2 %) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.
authors.affiliation: Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: Xiao-Zhe
authors.initials: XZ
authors.lastname: Huang
authors.affiliation: Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: Dana M
authors.initials: DM
authors.lastname: Cash
authors.affiliation: Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: Mohamad A
authors.initials: MA
authors.lastname: Chahine
authors.affiliation: Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: Mikeljon P
authors.initials: MP
authors.lastname: Nikolich
authors.affiliation: Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Craft
mesh_terms: D000151:Acinetobacter Infections / Q000382:microbiology*; D040981:Acinetobacter baumannii / Q000187:drug effects* / Q000201:enzymology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001618:beta-Lactamases; C063912:carbapenemase