广西罗非鱼源无乳链球菌耐药性及其四环素耐药基因检测  

Detection of antibiotics resistance and tetracycline resistance genes in Streptococcus agalactiae from tilapia in Guangxi

作　　者：梁静真 黄立春 韦慕兰 马沙 黎姗梅 文衍红 蒙兰丽 黄维 黄松 黄钧 LIANG Jing-zhen;HUANG Li-chun;WEI Mu-lan;MA Sha;LI Shan-mei;WEN Yan-hong;MENG Lan-li;HUANG Wei;HUANG Song;HUANG Jun(College of Animal Science and Technology,Guangxi University/Guangxi Aquatic Animal Disease Diagnosis Lab,Nanning 530004,China;Nanning Station of Aquatic Animal Husbandry and Veterinary Technology Extension,Nanning 530001,China;Du’an Station of Aquaculture Technology Extension,Du’an,Guangxi 530700,China;Guangxi General Station of Aquaculture Technology Extension,Nanning 530022,China;Liuzhou Station of Fishery Technology Extension,Liuzhou,Guangxi 545006,China;Tiandong Station of Aquaculture Technology Extension,Tiandong,Guangxi 531500,China;Dahua Station of Aquaculture Management,Dahua,Guangxi 530800,China)

机构地区：[1]广西大学动物科学技术学院/广西水生动物病害诊断实验室,南宁530004 [2]南宁市水产畜牧兽医技术推广站,南宁530001 [3]都安县水产技术推广站,广西都安530700 [4]广西水产技术推广总站,南宁530022 [5]柳州市渔业技术推广站,广西柳州545006 [6]田东县水产技术推广站,广西田东531500 [7]大化县水产管理站,广西大化530800

出　　处：《南方农业学报》2018年第10期2077-2086,共10页Journal of Southern Agriculture

基　　金：农业部主要水产养殖动物病原菌耐药性普查项目(农渔技药函[2015]27号;农渔技药函[2016]22号);广西水产畜牧兽医局专项项目(桂渔牧财[2016]33号);广西教育厅科研项目(KY2015YB014)

摘　　要：【目的】研究分析广西罗非鱼源无乳链球菌的耐药性及其四环素类耐药基因,为指导罗非鱼无乳链球菌病的临床用药和揭示其耐药机制提供参考依据。【方法】采用二倍稀释法测定8种抗生素对广西罗非鱼源无乳链球菌的最小抑菌浓度(MIC),运用PCR检测菌株的四环素类耐药基因(tetM、tetO、tetL和tetS)携带情况,并以体外诱导法测定罗非鱼源无乳链球菌对多西环素耐药性的获得速率。【结果】37株广西罗非鱼源无乳链球菌对氨苄青霉素、多西环素、红霉素和土霉素均敏感,对硫酸新霉素和磺胺二甲嘧啶不敏感(已产生耐药性);仅从8株菌株中检出tetM基因,检出率21.6%(8/37),而tetO、tetL和tetS基因的携带率均为0,即广西罗非鱼源无乳链球菌仅存在tetM+tetO-tetL-tetS-和tetM-tetO-tetL-tetS-两种耐药基因型。经多西环素连续8代的体外传代诱导,6株携带tetM基因的罗非鱼源无乳链球菌MIC由0.24μg/mL上升至3.91μg/mL,即耐药性获得速率约16倍。PCR检测结果显示,各传代菌株均携带有tetM基因,且各传代菌株间的核甘酸相似性为100.0%。【结论】广西罗非鱼源无乳链球菌存在tetM+tetO-tetL-tetS-和tetM-tetOtetL-tetS两种耐药基因型,对氨苄青霉素、多西环素、红霉素和土霉素仍较敏感,对硫酸新霉素和磺胺二甲嘧啶已产生耐药性。携带tetM基因的罗非鱼源无乳链球菌经体外诱导后对多西环素的耐药性大幅度提高,说明未达有效抑菌浓度时极易诱导无乳链球菌产生耐药性。【Objective】Antibiotics resistance and tetracycline resistance genes were studied in Streptococcus agalactiae isolated from tilapia in Guangxi in order to provide references for clinical medication and researching the antibiotics resistance mechanism of tilapia S.agalactiae disease.【Method】The minimal inhibitory concentration(MIC)of eight kinds of antibiotics against S.agalactiae was measured by double dilution method.The carrying situations of tetracycline resistance genes tetM,tetO,tetL and tetS in the strains were detected by PCR.The acquisition rate of doxycycline resistance of tilapia S.agalactiaewas determined by in vitro induction.【Result】The 37 strains of S.agalactiae isolated from tilapia in Guangxi were all sensitive to ampicillin,doxycycline,erythromycin and oxytetracycline,but insensitive to neomycin sulfate and sulfamethazine(antibiotic resistance formed).Only tetM gene was detected from the eight strains,with the detection rate of 21.6%(8/37).The detection rates of tetO,tetL and tetS genes were all 0.Accordingly,there were two kinds of resistance genotypes in tilapia S.agalactiae,including tetM+tetO-tetL-tetS-and tetM-tetO-tetL-tetS-.The MIC values of the six tetM-positive strains of S.agalactiae rose from 0.24μg/mL to 3.91μg/mLafter eight generations of induction of doxycycline and the resistance acquisition rate was approximately 16 times.PCR detection showed that the filial generations of the six strains all carried the tetM gene and that the similarity of nucleotide among the generation were all 100.0%.【Conclusion】Tilapia S.agalactiae of Guangxi contains two kinds of resistance genotypes including tetM+tetO-tetL-tetSand tetM-tetO-tetL-tetS-,and they are sensitive to ampicillin,doxycycline,erythromycin and oxytetracycline,but resistant to neomycin sulfate and sulfamethazine.The doxycycline resistance of tilapia S.agalactiae which carries tetM gene increases by a large margin after in vitro induction,indicating that doxycycline whose dosage does not reach the bacteriostasis level

关 键 词：罗非鱼 无乳链球菌 抗生素 耐药性 四环素类耐药基因 

分 类 号：S965.125[农业科学—水产养殖]