Turner Arthur K, Nair Satheesh, Wain John
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1SA, UK.
J Antimicrob Chemother. 2006 Oct;58(4):733-40. doi: 10.1093/jac/dkl333. Epub 2006 Aug 8.
To determine the contribution to fluoroquinolone resistance of point mutations in the gyrA and parC genes of Salmonella Typhi.
Point mutations that result in Ser-83-->Phe, Ser-83-->Tyr and Asp-87-->Asn amino acid substitutions in GyrA and Glu-84-->Lys in ParC were introduced into a quinolone-susceptible, attenuated strain of Salmonella Typhi using suicide vector technology. This is the first time that this approach has been used in Salmonella and abrogates the need for selection with quinolone antibacterials in the investigation of resistance mutations.
A panel of mutants was created using this methodology and tested for quinolone resistance. The ParC substitution alone made no difference to quinolone susceptibility. Any single GyrA substitution resulted in resistance to nalidixic acid (MIC >or= 512 mg/L) and increased by up to 23-fold the MIC of the fluoroquinolones ofloxacin (MIC <or= 2 mg/L) ciprofloxacin (MIC <or= 1 mg/L) and gatifloxacin (MIC <or= 0.38 mg/L). Among the double substitution mutants, those with a substitution in ParC were less prone to killing with ciprofloxacin. The triple substitution mutants (Ser-83-->Phe or Tyr and Asp-87-->Asn in GyrA with Glu-84-->Lys in ParC) showed high levels of resistance to all the fluoroquinolones tested (MICs: gatifloxacin, 3-4 mg/L; ofloxacin, 32 mg/L; ciprofloxacin, 32-64 mg/L).
In Salmonella Typhi the fluoroquinolones tested act on GyrA and, at higher concentrations, on ParC. The point mutations conferred reduced susceptibility to ofloxacin and ciprofloxacin, and also reduced susceptibility to gatifloxacin. Three mutations conferred resistance to ofloxacin (32 mg/L), ciprofloxacin (32 mg/L) and to the more active fluoroquinolone gatifloxacin (MIC >or= 3 mg/L). These results predict that the use of ofloxacin or ciprofloxacin will select for resistance to gatifloxacin in nature.
确定伤寒沙门菌gyrA和parC基因点突变对氟喹诺酮耐药性的影响。
利用自杀载体技术，将导致GyrA中Ser-83→Phe、Ser-83→Tyr和Asp-87→Asn氨基酸替换以及ParC中Glu-84→Lys的点突变引入对喹诺酮敏感的伤寒沙门菌减毒株。这是该方法首次应用于沙门菌，且在耐药性突变研究中无需使用喹诺酮类抗菌药物进行筛选。
使用该方法构建了一组突变体，并检测其对喹诺酮的耐药性。单独的ParC替换对喹诺酮敏感性无影响。任何单个GyrA替换均导致对萘啶酸耐药（MIC≥512mg/L），并使氟喹诺酮类药物氧氟沙星（MIC≤2mg/L）、环丙沙星（MIC≤1mg/L）和加替沙星（MIC≤0.38mg/L）的MIC增加高达23倍。在双替换突变体中，ParC有替换的突变体对环丙沙星的杀伤作用较不敏感。三替换突变体（GyrA中Ser-83→Phe或Tyr以及Asp-87→Asn，同时ParC中Glu-84→Lys）对所有测试的氟喹诺酮均表现出高水平耐药（MIC：加替沙星3 - 4mg/L；氧氟沙星32mg/L；环丙沙星32 - 64mg/L）。
在伤寒沙门菌中，所测试的氟喹诺酮作用于GyrA，在较高浓度下作用于ParC。这些点突变使对氧氟沙星和环丙沙星的敏感性降低，对加替沙星的敏感性也降低。三个突变导致对氧氟沙星（32mg/L）、环丙沙星（32mg/L）以及活性更高的氟喹诺酮加替沙星（MIC≥3mg/L）耐药。这些结果预示，在自然环境中使用氧氟沙星或环丙沙星会筛选出对加替沙星耐药的菌株。
