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-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6′)-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4– ≥ 32 μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6′)-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6′)-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration 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-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.

The Impact of Mutations in Topoisomerase Genes and the Plasmid-Mediated Quinolone Resistance (PMQR) Determinants on the Resistance to Fluoroquinolones in Klebsiella pneumoniae

“…In Gram-negative bacteria, mutations usually occur in subunit GyrA protein of DNA gyrase which is the first target of fluoroquinolones. DNA sequence analysis of our data showed 5 types of mutations in gyrA gene with amino acid changes among K. pneumoniae isolates which is consistent with the results of previous studies (13,19). Mutation at codon 83 in gyrA was the most frequent one among fluoroquinolone resistant K. pneumonia and substitution of the serine by phenylalanine was the most common changes.…”

“…Mutations at codon 426 and 447 of gyrB usually make a low-level of resistance to fluoroquinolones (11,12). Mutations in the amino acid 78, 80 and 84 positions of parC in K. pneumoniae resistant to fluoroquinolones have also been reported worldwide although with different rates (10,13).…”

Molecular Typing of Klebsiella pneumoniae Isolated from Medical Centers in Kermanshah Using Pulse Field Gel Electrophoresis

“…The results of the above studies are consistent with our results for high genotype diversity among isolates. Furthermore, the research in Asian countries on K. pneumoniae, including studies on 256 and 93 isolates using PFGE, resulted in 136 and 41 different genotypes, respectively (19,20). The results of these studies are also similar to our results in terms of the various genomic patterns of isolates.…”

Genotypes of Ciprofloxacin-ResistantKlebsiella pneumoniaein Korea and Their Characteristics According to the Genetic Lineages

“…All of them had qnr determinants and four of them have coharbored aac(6¢)-Ib-cr. Al-Marzooq et al 1 also reported a K. pneumoniae isolate, which had no mutation in gyrA or parC, but showed ciprofloxacin MIC of ‡32 g/L. This finding again indicates that PMQR is important in ciprofloxacin resistance among K. pneumoniae, although other resistance mechanisms such as active efflux and/or porin loss might be involved in this species.…”