山羊源无枝菌酸棒状杆菌(Corynebacterium amycolatum)的分离鉴定与耐药性分析

Isolation,identification and antibiotic resistance analysis of Corynebacterum amycolatum from goat

为了对1起羊腹泻病例进行诊断,从腹泻病羊粪便中分离到10株革兰阳性棒状杆菌YN21083~YN210812,对分离株进行形态学和16S rDNA基因鉴定并研究其药物敏感性和耐药基因特征。NCBI数据库BLAST比对鉴定结果显示,10株分离株与无枝菌酸棒状杆菌(Corynebacterium amycolatum)德国分离株FDAARGOS_991的同源性为99.3%~100.0%。16S rDNA基因进化分析显示10株分离株与C.amycolatum参考株形成同一个进化分支,与C.amycolatum参考株之间的同源性为97.2%~100.0%,与C.amycolatum模式菌株ATCC49368之间的同源性为98.3%~99.2%,将10株分离株鉴定为C.amycolatum。药敏试验结果显示,10株分离菌株对青霉素、氨苄西林、阿莫西林克拉维酸、头孢噻吩、头孢呋辛、氟苯尼考、阿米卡星和四环素敏感,对卡那霉素和磺胺甲恶唑耐药。耐药基因PCR检测结果显示,共检测到8种耐药基因aadA1、aadA2、tetA、tetM、qnrS、Sul1、Sul2和Sul3,其检出率分别为30.0%,30.0%,100.0%,60.0%,60.0%,100.0%,10.0%和20.0%。本研究首次报道羊C.amycolatum感染,对羊C.amycolatum感染的预防和控制具有重要指导意义。

To diagnose a case of diarrhea disease in goat,ten strains of Gram positive,coryneform bacteria,designated YN21083 to YN210812,were isolated from faeces of goats with diarrhea,and their morphological identification,16 S rDNA gene identification,antibiotic resistance and antibiotic resistance genes were studied.BLAST analysis of 16 S rDNA against NCBI database indicated that the 10 isolates showed sequence similarities of 99.3%-100.0% to C.amycolatum German isolate FDAARGOS91.Phylogenetic analysis of the 16 S rDNA gene showed that the 10 isolates formed a cluster with 10 C.amycolatum reference isolates,and shared sequence similarity of 97.2%-100.0% with them,and shared sequence similarity of 98.3%-99.2% with C.amycolatum type strain ATCC49368,respectively,and therefore the 10 isolates were identified as C.amycolatum.The results of antibiotic sensitivity test showed that the 10 isolates were sensitive to penicillin,ampicillin,amoxicillin-clavulanicacid,cephalothin,cefuroxime,florfenicol,amikacin and tetracyclines,and resistant to kanamycin and sulfamethoxazole.Antibiotic resistant genes(ARGs) detection results showed that eight ARGs,including aad A1,aadA2,tetA,tetM,qnrS,Sull,Sul2 and Sul3 with detection rate of 30.0%,30.0%,100.0%,60.0%,60.0%,100.0%,10.0% and 20.0 %,respectively,were detected.To our knowledge,this is the first isolation of C.amycolatum from goat and confirmed the presence of C.amycolatum infection in goat.These results have important guiding significance for the prevention and control of C.amycolatum infection in goat.