一株卵形鲳鲹肠道源粪肠球菌的分离鉴定与生物学特性研究

Isolation,Identification and Biological Characteristics Analysis of Enterococcus faecalis from the Intestinal Tract of Trachinotus ovatus

【目的】近年来鱼源益生菌在水产养殖中的应用越来越广泛,然而卵形鲳鲹(Trachinotus ovatus)源的益生菌鲜有报道。从卵形鲳鲹中分离潜在益生菌并对其生物学特性进行分析,为该菌株在卵形鲳鲹中的应用提供理论基础。【方法】无菌采集健康卵形鲳鲹肠道内容物,进行细菌分离与鉴定;对典型菌落做形态观察、革兰染色、生化鉴定、16S rRNA基因序列分析、药敏试验、耐盐能力试验、产酸能力及生长曲线测定和毒力基因鉴定。【结果】根据分离菌株的形态特征、革兰染色、生化特性和16S rRNA基因测序结果比对,分离菌株与粪肠球菌(Enterococcus faecalis)相似度达100%,确定其为粪肠球菌。该菌株菌落呈淡灰白色,为革兰氏阳性球菌,七叶苷、纤维二塘、麦芽糖、甘露醇、水杨苷等生化检测结果均显示阳性。药敏试验结果表明,分离菌株对哌拉西林、多粘菌素B、青霉素、复方新诺明、苯唑西林等药物呈现出不同程度的耐药性;对环丙沙星、麦迪霉素、头孢曲松呈中等敏感,对万古霉素、头孢哌酮、氧氟沙星等11种药物敏感。分离菌株可在0%~8%NaCl浓度下生长,具有一定耐盐性。产酸能力和生长曲线测定结果表明,随着菌株大量繁殖,菌液pH值快速下降,其产酸能力增强。毒力基因鉴定结果表明,分离菌株包含efaA、agg、as、fsrA 4种毒力基因。【结论】该研究成功从卵形鲳鲹肠道内分离到一株潜在益生菌菌株,鉴定为粪肠球菌。该菌株具有良好的耐盐和产酸能力,并含有与肠道黏附相关的毒力基因,可为该菌株的后续应用奠定基础。

【Objective】In recent years,probiotics have been widely used in aquaculture,but the probiotics derived from Trachinotus ovatus been rarely reported.In the study,a strain was isolated from the intestine of Trachinotus ovatus and its biological characteristics were analyzed,which provided a theoretical basis its application in T.ovatus culture.【Method】The bacteria were isolated and identified through the sterile collection of intestinal contents from the healthy T.ovatus.And the morphological observation,gram staining,biochemical identification,16S rRNA gene sequence analysis,drug sensitivity test,salt tolerance test,acid production ability,growth curve determination,and virulence gene identification of the typical bacterial colonies were performed.【Result】Based on the morphological characteristics,gram staining results biochemical characteristics,and 16S rRNA gene sequence alignment of the isolated bacterium,the results showed that it shared 100%similarities with Enterococcus faecalis,therefore,the isolated bacterium was identified as E.faecalis.The colony of the isolated strain was light grayish white,and Gram-positive.The biochemical indicators including esculin,cellobios,maltose,mannose and salicylic acid were tested positive.The results of the drug sensitivity test showed that the isolated bacterium exhibited varying degrees of resistance to drugs such as piperacillin,polymyxin B,penicillin,trimethoprim-sulfamethoxazole and oxacillin,exhibited intermediate sensitivity to ciprofloxacin,medemycin and ceftriaxone and sensitive to 11 drugs such as vancomycin,cefoperazone,ofloxacin and etc.The results of the salt tolerance test showed that the isolated strain could grow at 0%-8%NaCl concentrations,showing salt tolerance to some extent.The results of acid production capacity and growth curve measurements indicated that the pH of the culture media rapidly decreased and the acid production capacity was enhanced as the proliferation of the strain.The identification results of virulence genes showed that the isolated strains possessed four virulence genes including efaA,agg,as and fsrA.【Conclusion】A potential probiotic strain was successfully isolated from the intestine of T.ovatus and identified as E.faecalis.The isolated strain has good salt tolerance and acid production capability,and it contains functional genes associated with intestinal adhesion,which will provide a theoretical basis for further application of the strain.