title: Amino acid substitutions in GyrA affect quinolone susceptibility in Salmonella typhimurium.
abstract: The prevalence of quinolone-resistant Salmonella has become a public health concern. Amino acid substitutions have generally been found within the quinolone resistance-determining region in subunit A of DNA gyrase (GyrA) of Salmonella Typhimurium. However, direct evidence of the contribution of these substitutions to quinolone resistance remains to be shown. To investigate the significance of amino acid substitutions in S. Typhimurium GyrA to quinolone resistance, we expressed recombinant wild-type (WT) and five mutant DNA gyrases in Escherichia coli and characterized them in vitro. WT and mutant DNA gyrases were reconstituted in vitro by mixing recombinant subunits A and B of DNA gyrase. The correlation between the amino acid substitutions and resistance to quinolones ciprofloxacin, levofloxacin, nalidixic acid, and sitafloxacin was assessed by quinolone-inhibited supercoiling assays. All mutant DNA gyrases showed reduced susceptibility to all quinolones when compared with WT DNA gyrases. DNA gyrase with a double amino acid substitution in GyrA, serine to phenylalanine at codon 83 and aspartic acid to asparagine at 87 (GyrA-S83F-D87N), exhibited the lowest quinolone susceptibility amongst all mutant DNA gyrases. The effectiveness of sitafloxacin was shown by the low inhibitory concentration required for mutant DNA gyrases, including the DNA gyrase with GyrA-S83F-D87N. We suggest sitafloxacin as a candidate drug for the treatment of salmonellosis caused by ciprofloxacin-resistant S. Typhimurium. Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.
authors.forename: Siriporn
authors.initials: S
authors.lastname: Kongsoi
authors.affiliation: Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.
authors.forename: Ruchirada
authors.initials: R
authors.lastname: Changkwanyeun
authors.affiliation: Central Research Laboratory, Kissei Pharmaceutical Co., Ltd, Nagano, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Yokoyama
authors.affiliation: Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.|Hokkaido University The Global Station for Zoonosis Control, Sapporo, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Nakajima
authors.affiliation: Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.
authors.forename: Kanjana
authors.initials: K
authors.lastname: Changkaew
authors.affiliation: Faculty of Public Health, Thammasat University, Rangsit, Thailand.|Faculty of Public Health, Mahidol University, Bangkok, Thailand.
authors.forename: Orasa
authors.initials: O
authors.lastname: Suthienkul
authors.affiliation: Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan. suzuki@czc.hokudai.ac.jp.|Hokkaido University The Global Station for Zoonosis Control, Sapporo, Japan. suzuki@czc.hokudai.ac.jp.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D019943:Amino Acid Substitution; D002939:Ciprofloxacin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D027081:DNA Gyrase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D024841:Fluoroquinolones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D015363:Quinolones / Q000737:chemistry / Q000494:pharmacology*; D012486:Salmonella typhimurium / Q000737:chemistry / Q000187:drug effects*
chemical_list: D024841:Fluoroquinolones; D015363:Quinolones; D002939:Ciprofloxacin; C076246:sitafloxacin; D027081:DNA Gyrase
keywords: Salmonella Typhimurium; amino acid substitutions; quinolone resistance