动物源大肠埃希菌gyrA基因突变与氟喹诺酮耐药相关性分析

Analysis of Mutations in gyrA Gene Associated with Fluoroquinolone Resistance in Escherichia coli Isolates from Animals

为了解动物源大肠埃希菌对氟喹诺酮类药物的耐药情况及其与gyrA基因突变的联系,采用微量肉汤稀释法测定200株猪鸡源大肠埃希菌对恩诺沙星和氧氟沙星的最低抑菌浓度(MIC);对大肠埃希菌的gyrA基因片段进行PCR扩增,产物进行变性高效液相色谱(DHPLC)检测和序列测定。结果表明,200株动物源大肠埃希菌对恩诺沙星和氧氟沙星的耐药率分别为44.5%和41.0%;144株大肠埃希菌gyrA基因产物的DHPLC图谱有异常峰型,即核苷酸突变率为72.0%;测序证实gyrA基因存在5个突变位点,其中2个位点的突变可导致Ser83→Leu和Asp87→Asn/Tyr,突变率分别为54.0%和24.0%,其余3个位点为同义突变点;结合恩诺沙星耐药情况可知,敏感菌株gyrA基因的氨基酸突变率最低(18.5%),其次为中介菌株(56.5%),耐药菌株最高(78.6%),氧氟沙星测试结果与此趋势一致。由此可知动物源大肠埃希菌gyrA基因突变引起的氨基酸替代与氟喹诺酮耐药高度相关。

To investigate the relationship between fluoroquinolone resistance and the mutations in gyrA gene among Escherichia coli isolates from animals,a total of 200 E.coli isolates from swine and chickens were studied.Antimicrobial susceptibility test was carried out by broth microdilution method to determine the minimum inhibitory concentrations(MICs)of E.coli isolates towards enrofloxacin and ofloxacin.PCR was performed to amplify gyrA gene,and screening for mutations in gyrA gene was done by DHPLC and DNA sequencing.The results showed that the enrofloxacin and ofloxacin resistance rates were 44.5%and 41.0%.DHPLC detected different profiles of 144 isolates as the mutation rate was 72.0%.The results of DNA sequencing showed that there were 5 mutation sites in gyrA gene,while 2 mutations led the alteration in Ser83→Leu and Asp87→Asn/Tyr,and the other 3 mutations led no substitution.Overall,54.0%E.coli isolates had a mutation in gyrA gene in codon 83,and 24.0%in codon 87.Also,the isolates susceptible to enrofloxacin had the lowest mutation rate in gyrA gene(18.5%),followed by the intermediate isolates(56.5%)and the resistant isolates(78.6%).It showed the same trend in ofloxacin test.Our findings revealed a high level of fluoroquinolone resistance associated with the mutations that caused substitution of amino acids in gyrA gene.