艰难梭菌中粪肠球菌污染的去除方法  

Clinical Isolation of Enterococcus faecalis Contamination from Clostridium difficile

作　　者：饶凤琴 程玉梅[2] 张婷[1] 周青帅 崔古贞 齐晓岚[1] 洪伟[1] RAO Fengqin;CHEN Yumei;ZHANG Ting;ZHOU Qingshuai;CUI Guzhen;QI Xiaolan;HONG Wei(Key Laboratory of Endemic and Ethnic Diseases (Ministry of Education),and Key Laboratory of Medical Molecular Biology (Guizhou Province),Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of ICU, the Affiliate Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学地方病与少数民族疾病教育部重点实验室&贵州省分子生物学重点实验室,贵州贵阳550004 [2]贵州医科大学附院综合ICU,贵州贵阳550004 [3]贵州医科大学基础医学院,贵州贵阳550025

出　　处：《贵州医科大学学报》2019年第10期1122-1127,1133,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(31560318,31601012,31760318,U1812403);贵州省科技计划项目[黔科合平台人才团队(2017)5652]

摘　　要：目的:建立从粪肠球菌-艰难梭菌混合培养物中分离艰难梭菌的方法。方法:取不同厂家脑心浸出液(BHI)培养基、不同抗生素和是否添加羊血(或血清)3种组分进行组合,加入粪肠球菌-艰难梭菌混合物进行培养;显微镜观察不同组分培养基中细菌形态,将不同组分培养基中获得的混合培养物划线固体平板培养基并培养、观察菌落形态及菌落气味,采用16SrDNA测序技术评价不同组合培养基对艰难梭菌的纯化效果。结果:艰难梭菌抗生素抗性实验结果显示,艰难梭菌对氯霉素、克拉霉素和氨苄青霉素敏感,对四环素、D-环丝氨酸和头孢西丁具有抗性;在相同艰难梭菌接种量条件下,不同生产厂家BHI-酵母粉培养基(BHIS)均有艰难梭菌典型的沉淀生成,并伴有浓烈臭鸡蛋气味;显微镜观察发现纯化前艰难梭菌与粪肠球菌共培养物有大量球菌和链球菌、少量杆菌;BHIS-BDC培养基所有视野中无球菌,仅见杆状细菌;BHIS培养基上艰难梭菌仅在平板边缘形成零星菌落,BHIS-BDC培养基表面形成典型艰难梭菌菌落;16SrDNA测序结果表明1~2mm菌落为粪肠球菌,3~5mm菌落为艰难梭菌。结论:不同生产厂家的BHIS培养基对艰难梭菌中粪肠球菌的去除没有明显差异,BHIS培养基中添加羊血、D-环丝氨酸与头孢西丁复合抗生素可去除艰难梭菌中粪肠球菌污染。Objective:To explore a method for isolating Clostridium difficile from a mixture of Enterococcus faecalis and C.difficile culture.Methods:Brain heart infusion(BHI)medium from various manufacturers,different antibiotics and whether or not supplying with sheep blood or serum are used for three component combinations;adding the mixture of Enterococcus faecalis and C.difficile for culturing.Microscopic observation was adopted for the bacterial morphs in different components medium;mixed cultures from different component mediums were underlined for solid flat medium for culturing,observing colony morphology and odor of colony;16S rDNA sequencing method was used to evaluate the purification effect of components medium on Clostridium difficile.Results:Antibiotics resistance experiment results indicated that C.difficile was sensitive to chloramphenicol,clarithromycin and ampicillinum,resistant to tetracycline,D-cycloserine and cefoxitin;under the same inoculum dose of C.difficile,BHIS from different manufacturers showed typical C.difficile sedimentation with strong odor of rotten egg.Microscope observation showed that before purification,mixture of Enterococcus faecalis and C.difficile culture had great amount of cocci,streptococci and small amount of bacilli;within vision of BHIS-BDC medium showed no cocci but only bacilliform bacteria;C.difficile of BHIS medium only formed sporadic colony on margins of flat,BHIS-BDC medium surface formed typical C.difficile colony;16S rDNA sequencing results indicated that 1 to 2 mm colony as Enterococcus faecalis,3 to 5 mm colony as C.difficile colony.Conclusion:BHIS medium from different manufacturers showed no obvious differences in isolation Enterococcus faecalis from C.difficile;adding sheep blood and antibiotics to BHI medium is the key factor in removing the contamination of E.faecalis from C.difficile.

关 键 词：肠球菌 粪 梭菌感染 培养基 重症监护病房 结肠炎 细菌 厌氧 

分 类 号：R378.8[医药卫生—病原生物学]