Molecular Analysis of Ciprofloxacin Resistance Mechanisms in Malaysian ESBL-ProducingKlebsiella pneumoniaeIsolates and Development of Mismatch Amplification Mutation Assays (MAMA) for Rapid Detection ofgyrAandparCMutations

Abstract

Ninety-three Malaysian extended-spectrumβ-lactamase (ESBL)-producingKlebsiella pneumoniaeisolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection ofgyrAandparCmutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes includingaac(6′)-Ib-cr, qepA, andqnr. Ciprofloxacin resistance (MICs4–≥32 μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either ingyrAalone(n=1)or in bothgyrAandparCregions(n=32).aac(6′)-Ib-crwas the most common PMQR gene detected in this study(n=61), followed byqnrBandqnrS(n=55and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carryingqnrBaccompanied by eitheraac(6′)-Ib-cr(n=34)or a singlegyrA83 mutation(n=6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations ingyrAandparCregions. While the isolates harbouringgyrAand/orparCalteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producingK. pneumoniaeisolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.