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Nozomi Aoike,
Tomoo Saga,
Ryuji Sakata, Ayumi Yoshizumi,
Soichiro Kimura,
Morihiro Iwata,
Sadako Yoshizawa,
Yasuyuki Sugasawa,
Yoshikazu Ishii,
Keizo Yamaguchi,
Kazuhiro Tateda
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ABSTRACT: Fluoroquinolone-resistant Enterobacteriaceae is one of the increasing health problems 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, twenty-eight percent of which were non-susceptible 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 mutation in the QRDR (n = 81; 57%) were susceptible to ciprofloxacin, levofloxacin, and nalidixic acid. The qnr determinants, namely qnrA, qnrB, and qnrS genes, were not detected and aac(6')-Ib-cr gene was detected in 2 (1.4%) isolates. Multilocus sequence typing of 34 randomly selected isolates revealed sequence type (ST) 131 (n = 7; 20%) as the most prevalent lineage and significantly resistant to quinolones (p < 0.01). The genetic background of quinolone susceptible isolates seemed more diverse and, interestingly, neighbor STs of ST131 in the phylogenetic tree were all susceptible to ciprofloxacin. In conclusion, we could reveal the relationship between fluoroquinolone resistance caused by mutations of QRDRs and population structure in the clinical extra-intestinal 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.
Journal of clinical microbiology 03/2013; · 4.16 Impact Factor
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ABSTRACT: We evaluated the efficacy of ethylenediamine-N,N,N',N'-tetraacetic acid, disodium calcium salt (Ca-EDTA), as an inhibitor for New Delhi metallo-β-lactamase-1 (NDM-1) in vitro antibiotic susceptibility and in a mouse model of sepsis caused by Escherichia coli. Ca-EDTA drastically reduced the MICs of carbapenems for all NDM-producing bacteria [imipenem (IPM) ≤1-2 μg/ml; meropenem (MEPM) ≤1-4 µg/ml]. In the neutropenic murine model of sepsis, the bacterial burden was further reduced by combination therapy using imipenem/cilastatin sodium (IPM/CS) and Ca-EDTA to 2.3 × 10(3) CFU/liver, compared with 2.9 × 10(4) CFU/liver for IPM/CS alone. These data demonstrated the possibility of Ca-EDTA for clinical applications. In our understanding, this is the first report examining the effect of Ca-EDTA on a mouse sepsis model caused by NDM-1-producing bacteria.
Journal of Infection and Chemotherapy 12/2012; · 1.80 Impact Factor
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ABSTRACT: We conducted an epidemiological study concerning carbapenem-non-susceptible clinical isolates of Acinetobacter spp. in Japan by molecular procedures including carbapenemase gene identification and amplified ribosomal DNA restriction analysis. Among 598 clinically isolated Acinetobacter spp. in 2007, 27 (4.5%) were non-susceptible to carbapenems. Most carbapenem-non-susceptible Acinetobacter baumannii (13/14) belonged to clonal complex (CC) 92, harbored bla (OXA-51-like) genes, including novel bla (OXA-206), downstream of ISAba1, and were recovered mainly from the Kanto region. Carbapenem-non-susceptible A. baumannii CC92 isolates were further divided by pulsed-field gel electrophoresis into two groups, one of which was characterized by the presence of bla (OXA-23). One A. baumannii CC276 isolate carried bla (IMP-1) and bla (OXA-58). Almost all non-baumannii Acinetobacter isolates (12/13), including Acinetobacter pittii (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU), produced IMP-type metallo-β-lactamases, and were recovered from various regions in Japan. This is the first report describing the nationwide molecular epidemiology of carbapenem-non-susceptible Acinetobacter spp. with genomic species-level identification in Japan.
Journal of Infection and Chemotherapy 02/2012; 18(4):522-8. · 1.80 Impact Factor
