马链球菌马亚种-MDMC0316株的分离鉴定与遗传进化分析

Isolation,identification and genetic evolution analysis of a strain of Streptococcus equi subspecies.Equi-MDMC0316

从内蒙古锡林浩特市某马场中疑似患有马腺疫的马匹下颌部淋巴结中采集脓汁样本,通过实验室常规检测和分子生物学方法进行细菌分离鉴定和遗传进化分析。采用绵羊血琼脂平板划线法分离和纯化培养可疑细菌,并进行革兰染色和镜检;绘制分离菌生长曲线,并对该分离菌进行常规生化试验;对16S rRNA基因使用标准PCR方法扩增和测序并应用Mega 7.0生物软件绘制出该分离菌的系统发育进化树并确定其种属地位;对纯化的分离菌进行药物敏感性试验,拟筛选出辅助治疗效果最佳的抗生素类药物。结果显示,分离菌在绵羊血琼脂平板上呈现典型的β溶血环,染色特点为革兰阳性链球菌,从生长曲线上可以看出分离菌16 h后达到平台期;在生化试验中,分离菌对蔗糖、葡萄糖、麦芽糖、甘露糖利用呈现阳性,而对乳糖、甘露醇、蕈糖、七叶苷、山梨醇等呈现阴性,对精氨酸脱羧酶分解试验呈阳性,而对硝酸盐(还原)试验、胆汁溶菌试验、马尿酸盐分解试验、VP试验呈现阴性,提示分离菌与典型马链球菌马亚种生化试验标准结果吻合;根据16S rRNA基因BLAST比对分析发现,分离菌与NCBI中已注册的马链球菌马亚种序列同源性为100%,遗传进化树上可以看出分离菌与新疆分离株XJALT-10-M2遗传距离最近,位于相同的分支。为此,将该分离菌命名为马链球菌马亚种MDMC0316株(GenBank登录号:OK254140.1)。最后,根据药物敏感性试验结果,可确定多西环素与红霉素为该分离菌最敏感的抗生素类药物。结果表明,在内蒙古锡林郭勒地区初次分离鉴定了1株马链球菌马亚种,菌株编号为MDMC0316,并进行了遗传进化分析,临床最佳治疗抗生素类药物选定为多西环素与红霉素。

The concentrated purulent fluid samples were collected from the mandibular lymph nodes of horses suspected to be suffering from equine gland disease in a racecourse in Xilinhot, and the bacteria were isolated, identified and analyzed by routine laboratory and molecular biological methods.Suspicious bacteria were isolated, purified and cultured by sheep blood agar plate marking method, and Gram staining and microscopic examination were carried out.The growth curve of the isolated bacteria was drawn, and routine biochemical test on the isolated bacteria was carried out.The 16 S rRNA gene sequence was amplified by standard PCR and sequenced, then the phylogenetic tree of the isolate was plotted and its species status was determined by Mega 7.0 software.In successive experiments, the drug sensitivity test was carried out on the purified isolated bacteria, and the antibiotics with the best adjuvant treatment effect were screened out.The isolated strains showed typical β hemolytic ring on sheep blood agar plate, and the staining characteristic showed that it was Gram-positive Streptococcus.From the growth curve, it could be seen that the isolated strains reached a plateau after 16 h.The biochemical experiments of the isolated bacteria showed positive utilization of sucrose,glucose,maltose and mannose,but negative utilization of lactose,mannitol,muscarinose,aescin and sorbitol,positive utilization of arginine decarboxylase,nitrate(reduction)test,bile bacteriolysis test,hippurate decomposition test and VP test were negative.It is suggested that the biochemical test results of the isolated bacteria were consistent with those of typical Streptococcus equi subsp.equi.According to BLAST analysis of 16SrRNA gene,it was found that the sequence homology between the isolate and the registered Streptococcus equi subsp.equi in NCBI was 100%.It could be seen from the genetic evolutionary tree that the isolate and Xinjiang isolate XJALT-10-M2had the closest genetic distance and were located in the same branch.Therefore,the isolate was named Streptococcus equi subsp.equi MDMC0316,and GenBank registration number was OK254140.1.Finally,according to the results of drug sensitivity test,doxycycline and erythromycin were identified as the most sensitive antibiotics for the isolate.A strain of Streptococcus equi subsp.equi was isolated,identified and genetically analyzed for the first time in Xilingol area.Strain number was MDMC0316,and doxycycline and erythromycin were selected as the best antibiotics for clinical treatment.