猪源致病性沙门氏菌四环素耐药基因tetC的PCR扩增及序列分析

PCR Application and Nucleotide Sequencing of the tetC Gene of Pathogenic Salmonella from Swine

四环素类抗生素作为一类广谱抗生素,现已产生严重的耐药性。作为人兽共患病原菌的沙门氏菌,也存在大量这样的耐药菌株,使得本病的发病率和死亡率都不断上升。本研究对规模化猪场分离的经动物试验和生化试验鉴定的致病性沙门氏菌16株、药敏质控菌ATCC25922和沙门氏菌标准株C79-13对进行了四环素、金霉素、土霉素的药敏试验,结果表明:16株致病性沙门氏菌对四环素类抗生素表现出普遍耐受性,耐药率达100%。其中MIC值>128μg/mL的高耐药菌株13株,高耐药率81.25%;MIC值为32μg/mL的低耐药菌株3株,低耐药率18.75%。设计沙门氏菌四环素抗性基因tetC的引物,对16株致病性沙门氏菌的tetC基因做PCR扩增,结果12株菌获得以质粒为模板长约400bp的特异性扩增产物,未能从染色体扩增到产物。分别对临床分离的1株低耐药菌株(DY1)和1株高耐药菌株(SL1-3)的tetC基因扩增产物进行序列分析,结果表明:菌株DY1和SL1-3的tetC的核苷酸同源率为97.7%;菌株DY1与质粒pBR322中tetC的核苷酸同源率为98.7%;菌株SL1-3与质粒pBR322中tetC的核苷酸同源率为98.4%,说明对于耐药程度和地方来源各不相同的菌株,在核苷酸序列上同源率很高。本文用PCR技术对规模化猪场分离的致病性沙门氏菌的四环素耐药基因进行了研究。

The detection on tetracyclines-resistance of16salmonella strains showed the resistance-rate arrived at100%.The tetC gene of16salmonella strains was amplified by polymerase chain reaction.Twelve strains were positive,and this gene located on plasmids.Compared with the result of the drug-suspective test,The PCR method obtained75%concordance in positive rate.In this study,the method of PCR was established,and it was applied to surveillance of the molecular epidemiology of tetracyclines-resistance.