罗非鱼无乳链球菌毒力检测及药敏试验

Toxicity and Drug Sensitivity Testing of Streptococcus agalactiae from Tilapia

无乳链球菌病是罗非鱼养殖过程中一个严重的问题,给罗非鱼养殖业带来不可估量的经济损失。本试验以从广东省肇庆市某罗非鱼养殖场中分离出的罗非鱼无乳链球菌(ZQ0910)作为研究对象,对其进行毒力检测和药敏试验,以期为后续研究提供理论基础。使用腹腔注射的方法感染小鼠,测定无乳链球菌的半数致死量(LD_(50));利用PCR检测无乳链球菌的致病基因;使用微量肉汤2倍稀释法测定12种抗菌药对无乳链球菌的最小抑菌浓度(MIC),再采用棋盘法对其中5种抑菌作用较好的抗菌药进行体外联合药敏试验。结果显示:无乳链球菌ZQ0910的LD_(50)为402.35 CFU/mL;PCR检测无乳链球菌致病基因hylB、ponA和cfb的结果均呈阳性,致病基因sip和cpsE的检测结果呈阴性。单药药敏试验结果显示,阿莫西林对无乳链球菌的抑制效果最好,MIC为0.125μg/mL;多西环素次之,MIC为0.5μg/mL;无乳链球菌对青霉素、林可霉素耐药性严重,MIC>128μg/mL。体外联合药敏试验结果提示,环丙沙星分别与多西环素、替米考星和氟苯尼考体外联合结果为相加作用;阿莫西林分别与多西环素、替米考星和氟苯尼考体外联合结果以及氟苯尼考与替米考星的体外联合结果均为拮抗作用;环丙沙星联合阿莫西林的体外联合结果为无关作用。

Streptococcus agalactiae is a significant cause of economic losses in tilapia breeding industry.In this study,S.agalactiae(ZQ0910)was isolated from a tilapia farm in Zhaoqing,Guangdong province,and its toxicity and drug susceptibility were tested.LD_(50) of S.agalactiae was determined in mice after intraperitoneal injection.PCR was used to detect the presence of pathogenic genes in S.agalactiae.The minimum inhibitory concentration(MIC)of 12 antibiotics against S.agalactiae was determined by microbroth double dilution method,and 5 antibiotics with better antimicrobial activity were tested by checkerboard method in vitro.The results revealed that S.agalactiae ZQ0910 had the LD_(50) of 402.35 CFU/mL by Bliss method,and possessed pathogenic genes hylB,ponA and cfb,but not sip and cpsE.Based on single drug susceptibility test,S.agalactiae ZQ0910 was inhibited by amoxicillin with MIC of 0.125 g/mL,followed by doxycycline with MIC of 0.5 g/mL,but was largely resistant to penicillin and lincomycin,with MIC>128 g/mL.The combined susceptibility test in vitro indicated that a combination of ciprofloxacin with doxycycline,telmicorin and flurbenicol was additive;a combination of amoxicillin with doxycycline,telmicorin and flurbenicol,or a combination of flurbenicol and telmicorin was antagonistic;and a combination of ciprofloxacin and amoxicillin was independent.