1株犬源多重耐药粪肠球菌的分离鉴定及其致病性分析

Isolation and Identification of a Multidrug-resistant Strain of Enterococcus faecalis from Dog and Analysis of Its Pathogenicity

[目的]明确1例犬泌尿生殖道感染的发病原因。[方法]无菌采集病犬尿液,通过细菌分离纯化、革兰染色镜检、生化试验、16S rRNA基因扩增进行鉴定,同时经药敏试验、毒力基因和耐药基因检测及细菌致病性试验测定纯化菌株的生物学特征。[结果]分离得到1株兼性厌氧型革兰阳性球菌;菌落形态如针尖大小、表面光滑、半透明、圆形凸起、边缘整齐;生化鉴定结果显示,致病菌能发酵乳糖、果糖、蔗糖、麦芽糖、葡萄糖、山梨醇、甘露醇、精氨酸,不能发酵木糖、氧化酶、枸橼酸盐、棉籽糖和鸟氨酸脱羧酶;16S rRNA基因序列与已发表的粪肠球菌序列相似性高于99%,鉴定为粪肠球菌,并将其命名为EFGZ23GY01;药敏试验结果显示,分离株仅对万古霉素及氯霉素敏感,对新霉素中度敏感,对克林霉素等25种药物耐药;8种粪肠球菌毒力基因检测结果显示,分离株携带efaA、ace、asa 1及gelE毒力基因;14种耐药基因检测结果显示,分离株携带tetL、sulⅠ、tetC、bla TEM、bla OXA-23、aadA 1和sulⅢ耐药基因;致病性试验结果显示,5只小鼠注射菌液12 h后出现精神萎靡,被毛杂乱等症状,未出现死亡,48 h后死亡1只,表明分离菌对小鼠具有致病性。[结论]本研究成功从犬尿液种分离得到1株粪肠球菌,其携带多种毒力基因和耐药基因,且对小鼠有致病性,研究为病犬的后续治疗提供了参考。

[Objective]This study was to investigate the pathogenesis of a case of canine genitourinary tract infection.[Method]This study aseptically collected urine from sick dog and identified the bacteria by isolation and purification,Gram staining microscopy,biochemical tests,and 16S rRNA gene amplification,and determined the biological characteristics of the purified strains by drug sensitivity test,virulence gene test,drug resistance gene test,and pathogenicity test of the bacteria.[Result]A parthenogenetic anaerobic Gram-positive coccus was isolated.The colony morphology was like a pinpoint,smooth,translucent,rounded raised,and neatly edged.The biochemical characterization showed that the causative agent was able to ferment lactose,fructose,sucrose,maltose,glucose,sorbitol,mannitol,and arginine,but not xylose,oxidase,citrate,cottonseed sugar,ornithine decarboxylase.The 16S rRNA gene sequence showed greater than 99%similarity to the published sequence of Enterococcus faecalis(E.faecalis),the isolate was identified as E.faecalis,and named EFGZ23GY01.The results of drug sensitivity test showed that this isolate was only sensitive to vancomycin and chloramphenicol,moderately sensitive to neomycin,and resistant to 25 drugs including clindamycin.The results of 8 E.faecalis virulence genes showed that this strain of E.faecalis carried efaA,ace,asa 1 and gelE virulence genes.And the results of 14 resistance genes showed that this strain of E.faecalis carried resistance genes,such as tetL,sulⅠ,tetC,bla TEM,bla OXA-23,aadA 1 and sulⅢ.Pathogenicity test showed that 5 mice showed symptoms such as depression and disorganized coat after 12 h of injection,but did not die,while 1 mouse died after 48 h of injection,indicating that the isolate was pathogenic to mice.[Conclusion]In the present study,a strain of E.faecalis was successfully isolated from canine urine species,which carried multiple virulence and resistance genes and was pathogenic to mice.