氟喹诺酮类抗菌药对临床分离猪链球菌的防耐药变异浓度测定及一步耐药突变株的筛选  

Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of Streptococcus suis and Screen of Single-step Resistant Mutants

作　　者：张雨菡[1] 王丽平[1] 陆承平[1] 

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《畜牧兽医学报》2011年第1期71-76,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30972220);公益性行业(农业)科研专项(200803016)

摘　　要：分别用微量肉汤稀释法(CLSI规定的标准方法)和琼脂二倍稀释法测定了4种氟喹诺酮抗菌药(环丙沙星、恩诺沙星、氧氟沙星、甲磺酸培氟沙星)对临床分离的32株氟喹诺酮敏感的猪链球菌的体外最小抑菌浓度(MIC)和防耐药变异浓度(MPC),比较二者的关系;分别与利血平和氰氯苯腙(CCCP)联合用药,检测了各抗菌药突变选择窗(MSW)内富集的一步耐药突变株是否存在主动外排泵机制;采用PCR和基因测序的方法检测在不同药物突变选择窗内筛选出的猪链球菌一步耐药突变株的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,探明猪链球菌耐氟喹诺酮类药物的作用机制,分析不同氟喹诺酮药物在抑制猪链球菌时的特点,为临床用药提供依据。结果显示:4种药的MIC90值从小到大依次为环丙沙星=恩诺沙星<氧氟沙星<甲磺酸培氟沙星,MPC90值从小到大依次为恩诺沙星<氧氟沙星<环丙沙星<甲磺酸培氟沙星,选择指数(MPC/MIC)除了环丙沙星为16外,其余药物均为2;只在环丙沙星的耐药突变窗内筛选到了耐药株,但其DNA回旋酶(gyrA和gyrB)和拓扑异构酶IV(parC和parE)耐药决定区(QRDR)没有碱基或氨基酸的突变;与利血平联合用药时检测到了外排机制。结论:环丙沙星在治疗猪链球菌感染时很容易筛选出一步耐药突变株,从而导致猪链球菌对其产生耐药性,耐药机制可能是由主动外排泵介导产生。The mutant prevention concentration（MPC） represents a threshold above which the selective proliferation of resistant mutants is expected to occur only rarely.Measurement of the minimal inhibitory concentration（MIC） and the MPC for 32 veterinary clinical strains of Streptococcus suis revealed that ofloxacin and enrofloxacin are more potent than ciprofloxacin and pefloxacin mesylate to restrict the selective enrichment of resistant mutants.Based on their potential for restricting the selection of resistant mutants,the four fluoroquinolones,in descending order,were found to be enrofloxacin ofloxacin ciprofloxacin pefloxacin mesylate.For several compounds,all of the clinical isolates lacked a known resistance mutation in the quinolone resistance-determining region（QRDR） of gyrA,gyrB,parC and parE.Additionally,the isolates were tested for CCCP or reserpine-sensitive efflux.The MICs of ciprofloxacin,enrofloxacin,ofloxacin and pefloxacin mesylate for each single-step mutants were determined by agar dilution on Mueller-Hinton agar plates plus 5% sheep blood in the presence and absence of CCCP（40 mg·L-1） or reserpine（50 mg·L-1）.As a result,isolates showed no reduction in MIC in the presence of CCCP and considered negative for CCCP-sensitive efflux.Nevertheless,it revealed a four or more-fold decrease of ciprofloxacin accumulation in the presence of reserpine in the ciprofloxacin first-step mutants compared to that in the parent resistant strain,and it′s considered that there is a reserpine-sensitive efflux to decrease the level of ciprofloxacin accumulation in single-step mutants.

关 键 词：氟喹诺酮类抗菌药 猪链球菌 MIC MPC 耐药突变株 主动外排泵 

分 类 号：R378.12[医药卫生—病原生物学] S852.611[医药卫生—基础医学]