Molecular Characterization of Extraintestinal Escherichia coli Isolates in Japan: Relationship between Sequence Types and Mutation Patterns of Quinolone Resistance-Determining Regions Analyzed by Pyrosequencing

Abstract

bInfection from fluoroquinolone-resistant Enterobacteriaceae is an increasing health problem worldwide. In the present study, we developed a pyrosequencing-based high-throughput method for analyzing the nucleotide sequence of the quinolone resistance-determining regions (QRDRs) of gyrA and parC. By using this method, we successfully determined the QRDR sequences of 139 out of 140 clinical Escherichia coli isolates, 28% of which were nonsusceptible to ciprofloxacin. Sequence results obtained by the pyrosequencing method were in complete agreement with those obtained by the Sanger method. All fluoroquinolone-resistant isolates (n ‫؍‬ 35; 25%) contained mutations leading to three or four amino acid substitutions in the QRDRs. In contrast, all isolates lacking a mutation in the QRDR (n ‫؍‬ 81; 57%) were susceptible to ciprofloxacin, levofloxacin, and nalidixic acid. The qnr determinants, namely, the qnrA, qnrB, and qnrS genes, were not detected in the isolates, and the aac(6=)-Ib-cr gene was detected in 2 (1.4%) of the isolates. Multilocus sequence typing of 34 randomly selected isolates revealed that sequence type 131 (ST131) (n ‫؍‬ 7; 20%) is the most prevalent lineage and is significantly resistant to quinolones (P < 0.01). The genetic background of quinolone-susceptible isolates seemed more diverse, and interestingly, neighboring STs of ST131 in the phylogenetic tree were all susceptible to ciprofloxacin. In conclusion, our investigation reveals the relationship between fluoroquinolone resistance caused by mutations of QRDRs and the population structure of clinical extraintestinal E. coli isolates. This highthroughput method for analyzing QRDR mutations by pyrosequencing is a powerful tool for epidemiological studies of fluoroquinolone resistance in bacteria.

Whole-Genome Analysis of Antimicrobial-Resistant and Extraintestinal Pathogenic Escherichia coli in River Water

“…The numbers of mutations leading to amino acid substitutions in quinolone resistance-determining regions (QRDRs) were closely associated with ciprofloxacin and nalidixic acid resistance; isolates with no mutations were susceptible to both agents, isolates with one or two mutations were resistant to nalidixic acid but not to ciprofloxacin (nonsusceptible to ciprofloxacin according to the ECOFF value), and isolates with three or four mutations were resistant to both agents. These results are consistent with those of studies that analyzed clinical E. coli isolates (27,28). Plasmid-mediated quinolone resistance (PMQR) determinants, i.e., qnrB7, qnrD, qnrS1, qepA, and aac(6=)- …”

“…Fluoroquinolone resistance is conferred mainly by mutations in the gyrA and parC genes (27). The numbers of mutations leading to amino acid substitutions in quinolone resistance-determining regions (QRDRs) were closely associated with ciprofloxacin and nalidixic acid resistance; isolates with no mutations were susceptible to both agents, isolates with one or two mutations were resistant to nalidixic acid but not to ciprofloxacin (nonsusceptible to ciprofloxacin according to the ECOFF value), and isolates with three or four mutations were resistant to both agents.…”

“…The fimH (type 1 fimbrial adhesin gene) allele types were determined following the classification system established previously (59); SNP analysis of fimH can further discriminate isolates genotyped by MLST. The QRDRs of gyrA and parC and chromosomal ampC promoter/attenuator mutations were analyzed as described previously (27,60). PlasmidFinder was used for identification of plasmid replicons (38).…”

Rapid Detection of Genomic Mutations in gyrA and parC Genes of Escherichia coli by Multiplex Allele Specific Polymerase Chain Reaction

“…MICs of ciprofloxacin of these isolates ranged from 0.25 to 2 μg/ml, which were not considered as resistant. But all the seven isolates had remarkable elevated MICs compared to that of the wild‐type isolates, similar to that reported from previous study . Although, a single mutation at codon 83 in gyrA may directly contribute to FQ resistance, this point mutation can also be observed among the susceptible strains .…”

“…However, these isolates demonstrated an elevated ciprofloxacin MICs compared to wild type, in which MICs were very low, as previously observed by Aoike et al. . We observed that all the FQ‐resistant E. coli isolates had at least three point mutations at codons 83 and 87 in gyrA , and 80 in parC .…”