鲮鱼源无乳链球菌的鉴定、血清型分析及药敏试验

Identification, serotype analysis and drug sensitivity test of Streptococcus agalactiae from Cirrhinus molitorella

【目的】确诊患病鲮鱼的发病原因,并对其病原菌进行分子血清型及药敏性分析,为有效防治该病提供参考依据,进而促进鲮鱼养殖业健康持续发展。【方法】以常规方法进行患病鲮鱼的病原菌分离纯化,通过形态观察、生理生化鉴定、16S rRNA基因序列分析及多位点序列分型(MLST分型)进行鉴定,并采用药敏纸片扩散法进行药敏试验。【结果】从患病鲮鱼体内分离获得1株优势菌株(GYCm081),为呈链状排列的革兰氏阳性球菌,其16S rRNA基因片段与无乳链球菌16S rRNA序列(F0393392和NR_102871)的同源性最高,相似性均为100%,系统发育进化树也显示GYCm081菌株与无乳链球菌亲缘关系最近,可确定为无乳链球菌。GYCm081菌株分子血清型为Ib型,MLST型为ST261型,对氨苄西林、氯霉素、壮观霉素、红霉素、恩诺沙星、氧氟沙星、利福平、环丙沙星、呱拉西林、新生霉素、头孢唑啉、四环素和呋喃妥因等13种药物高度敏感而对磺胺甲噁唑、磺胺甲异噁唑和青霉素G已经产生耐药性。【结论】引起广东阳江某鲮鱼养殖场发病死亡的病原菌为无乳链球菌,其分子血清型为Ib-ST261,实际生产中可选用氨苄西林、恩诺沙星和氧氟沙星等药物进行防治。

[Objective]The present experiment was conducted to investigate pathogenesis of diseased Cirrhinus molitorella and analyze serotype and drug sensitivity of pathogen isolated from diseased C.molitorella,in order to provide reference for controling disease and promoting sustainable development of C.molitorella aquaculture industry.[Method]The pathogen was isolated from diseased C.molitorella.And the isolated pathogen was identified through morphologic observation,physiological and biochemical properties determination,and 16 S rRNA gene sequence and multilocus sequence typing（MLST） analysis.Then the drug sensitivity of isolated pathogen were determinated through Kirby- Bauer test.[Result]The results showed that,a dominant strain（GYCm081） was isolated from diseased C.molitorella,which was Gram-positive streptococcus,and formed into chain-like configuration.And its 16 S rRNA gene sequence shared the highest nucleotide sequence homology（100%） with Streptococcus agalactiae（F0393392 and NR_102871）.The phylogenetic tree showed that GYCm081 strain was most closely related to S.agalactiae,so which was identified as S.agalactiae,with moleculan serotype of Ib-ST261.The drug sensitivity test showed that the GYCm081 strain was highly sensitive to 13 drugs viz.,ampicillin,chloroamphenicol,spectinomycin,erythromycin,enrofloxacin,ofloxacin,rifampin,iprofloxacin,piperacillin,novobiocin,cefazolin,tetracycline,nitrofurantoin,while which was tolerant to 4 drugs resistant to sulfamethoxazole,sulfamethoxazolum and penicillin G.[Conclusion]S.agalactiae is major pathogen causing diseasedness and death of C.molitorella with molecular serotype of Ib-ST261 on aquatic farm.Ampicillin,enrofloxacin and ofloxacin are candidates to prevent and control such a pathogen in actual production.