title: 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: Infection 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 high-throughput method for analyzing QRDR mutations by pyrosequencing is a powerful tool for epidemiological studies of fluoroquinolone resistance in bacteria.
authors.affiliation: Department of Microbiology and Infectious Diseases, Toho University School of Medicine, Tokyo, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Aoike
authors.forename: Tomoo
authors.initials: T
authors.lastname: Saga
authors.forename: Ryuji
authors.initials: R
authors.lastname: Sakata
authors.forename: Ayumi
authors.initials: A
authors.lastname: Yoshizumi
authors.forename: Soichiro
authors.initials: S
authors.lastname: Kimura
authors.forename: Morihiro
authors.initials: M
authors.lastname: Iwata
authors.forename: Sadako
authors.initials: S
authors.lastname: Yoshizawa
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Sugasawa
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Tateda
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D016000:Cluster Analysis; D027081:DNA Gyrase / Q000235:genetics*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000453:epidemiology / Q000382:microbiology*; D005838:Genotype; D006801:Humans; D007564:Japan / Q000453:epidemiology; D017720:Molecular Epidemiology; D058885:Multilocus Sequence Typing; D020125:Mutation, Missense; D010802:Phylogeny; D015363:Quinolones / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D015363:Quinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase