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

A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D b-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 IMPresistant 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_OXAgenes. 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 multidrugresistant Gram-negative bacteria.