Kongsoi Siriporn, Changkwanyeun Ruchirada, Yokoyama Kazumasa, Nakajima Chie, Changkaew Kanjana, Suthienkul Orasa, Suzuki Yasuhiko
Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.
Central Research Laboratory, Kissei Pharmaceutical Co., Ltd, Nagano, Japan.
Drug Test Anal. 2016 Oct;8(10):1065-1070. doi: 10.1002/dta.1910. Epub 2015 Oct 30.
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.
喹诺酮耐药性沙门氏菌的流行已成为一个公共卫生问题。通常在鼠伤寒沙门氏菌DNA促旋酶（GyrA）亚基A的喹诺酮耐药决定区域内发现氨基酸替换。然而，这些替换对喹诺酮耐药性的贡献的直接证据仍有待证明。为了研究鼠伤寒沙门氏菌GyrA中氨基酸替换对喹诺酮耐药性的意义，我们在大肠杆菌中表达了重组野生型（WT）和五种突变DNA促旋酶，并在体外对它们进行了表征。通过混合DNA促旋酶的重组亚基A和B在体外重建WT和突变DNA促旋酶。通过喹诺酮抑制的超螺旋测定评估氨基酸替换与对喹诺酮环丙沙星、左氧氟沙星、萘啶酸和西他沙星的耐药性之间的相关性。与WT DNA促旋酶相比，所有突变DNA促旋酶对所有喹诺酮的敏感性均降低。在密码子83处丝氨酸替换为苯丙氨酸且在87处天冬氨酸替换为天冬酰胺（GyrA-S83F-D87N）的GyrA中具有双氨基酸替换的DNA促旋酶在所有突变DNA促旋酶中表现出最低的喹诺酮敏感性。包括具有GyrA-S83F-D87N的DNA促旋酶在内的突变DNA促旋酶所需的低抑制浓度表明了西他沙星的有效性。我们建议将西他沙星作为治疗由环丙沙星耐药性鼠伤寒沙门氏菌引起的沙门氏菌病的候选药物。版权所有© 2015 John Wiley & Sons, Ltd.
