人工诱导猪链球菌氟喹诺酮耐药株的靶位突变分析

Mutation analysis of induced fluoroquinolone-resistant mutants of Streptococcus suis in vitro

以4株临床分离的对环丙沙星和恩诺沙星敏感的猪链球菌2型菌株为研究对象,采用体外递增药物浓度的方法分别诱导了其对环丙沙星和恩诺沙星耐药的菌株,按CLSI推荐方法测定了环丙沙星和恩诺沙星对亲本敏感株和诱导耐药株的MIC,测定了亲本株和诱导耐药株的生长曲线,并采用PCR和基因测序的方法分析了诱导耐药株的DNA回旋酶(GyrA和GyrB)和拓扑异构酶Ⅳ(ParC和ParE)耐药决定区(QRDR)的基因突变和氨基酸序列变化。结果表明:浓度递增法成功诱导了猪链球菌对环丙沙星和恩诺沙星耐药性,其MIC分别由0.5 mg.L-1上升至128 mg.L-1;与敏感株比较,恩诺沙星与环丙沙星诱导的耐药菌在gyrA和gyrB,或parC和parE耐药决定区的氨基酸序列有突变,除了已报道的与氟喹诺酮耐药相关的ParC的Ser79Phe,GyrA的Ser81Arg,GyrB的Asp315Asn、Ser285Leu和Glu354Lys及ParE的Pro278Ser点突变外,在诱导菌中还出现了一些不曾报道的突变位点和氨基酸缺失,如GyrA的Gln118His和ParE的Asn297Tyr突变,GyrB的288～291位和ParC的62位氨基酸缺失。结果提示:逐步增加药物浓度可以诱导猪链球菌对氟喹诺酮类抗菌药耐药性,并导致主要靶位发生突变。

To perform a systematic analysis of point mutations in the quinolone resistance determining regions （QRDRs） of the DNA gyrase and topoisomerase genes of Streptococcus suis type 2 strain after in vitro exposure to stepwise increasing concentrations of enrofloxacin and ciprofloxacin. Four parent strains of S, suis type 2 were chosen for stepwise exposure to increasing levels of enrofloxacin and ciprefloxacin followed by selection of resistant mutants. The QRDRs of gyrA, gyrB, parC and pare correlating to mutants with increased MICs were analysed for point mutations. Multiple mutants with significantly increased MICs （ 128 mg·L^-2 ） were generated from each parent strain. Most of the amino acid substitutions identified were Ser79Phe of ParC, Ser81Arg of GyrA, Asp315Asn, Ser285Len, Glu354Lys of GryB and Pro278Ser of ParE, which were consistent to the mechanisms of resistance reported in clinical isolates of S. pneumoniae. However, it has some mutable points Gln118His of GyrA, Asn297Tyr of ParE as well as the deletions 288 -291 amino acids of GyrB and 62 amino acid of ParC, which haven＇t been documented and whether these nuleotide mutations associated with fluoroquinolone resistance are not clear. The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains. The resuhs indicate that stepwise increasing concentration of enrofloxacin and ciprofloxacin can easily select resistant strains of S. suis.