117例梗阻性黄疸患者胆汁细菌群落限制性片段多态性分析

Terminal restriction fragment length polymorphism analysis of microbial community in the bile of 117 patients with obstructive jaundice

目的 分析梗阻性黄疸患者胆汁中的细菌群落结构.方法 选取2010年10月至2013年10月梗阻性黄疸患者117例,其胆汁细菌常规培养和厌氧菌培养结果均为阴性.每例抽取胆汁10 mL,提取胆汁细菌群落DNA,扩增胆汁细菌16S rDNA,进行终端限制性片段长度多态性（T-RFLP）分析,构建克隆文库并行测序和系统分析.研究数据行卡方检验.结果 117例患者中50例胆汁细菌16S rDNA为阳性,总阳性率为42.7％.结石和肿瘤引起的梗阻中胆汁细菌16S rDNA的阳性率分别为97.3％（36/37）和17.5％（14/80）,差异有统计学意义（x2=65.828,P＜0.01）.肝门部和肝门部以上及肝门部以下的梗阻中胆汁细菌16S rDNA的阳性率分别为43.3％（13/30）和42.5％（37/87）,差异无统计学意义（P＞0.05）.结石引起梗阻的细菌群落主要为肠杆菌科（埃希菌属、沙门菌属、克雷伯菌属、变形菌属）、链球菌科（链球菌属）、消化球菌科（消化菌属、消化链球菌属）、微球菌科（葡萄球菌属）、丙酸杆菌科（丙酸杆菌属）、奈瑟球菌科（不动杆菌属）.肿瘤引起梗阻的细菌群落主要为肠杆菌科（克雷伯菌属、埃希菌属、伤寒沙门菌）和微球菌科（葡萄球菌属）.结论 采用T-RFLP方法分析16S rDNA克隆片段能有效评估梗阻性黄疸患者胆汁中细菌群落的存在情况和多样性.

Objective To analyze the structure of microbial community in the bile of patients with obstructive jaundice.Methods From October 2010 to October 2013,117 patients with obstructive jaundice were selected.The results of bile microbial regular culture and anaerobic bacteria culture were both negative.A total of 10 mL bile of each case was aspired and DNA of bile microbial community was isolated.16S rDNA of bile microbial was amplified and underwent terminal restriction fragment length polymorphism （T-RFLP） analysis.Clone libraries were constructed and conducted sequencing and system analysis.Chi-square test was performed for data analysis.Results Among the 117 patients,16S rDNA of 50 cases was positive,and the total positive rate was 42.7 %.The positive rate of bile bacterial 16S rDNA in stone and tumor cased obstruction was 97.3% （36/37） and 17.5% （14/80）,and the difference was statistically significant （x2=65.828,P〈0.01）.There was no statistically significant difference in the positive rate of bile bacterial 16S rDNA among hilar obstruction,above hilar obstruction and below hilar obstruction,which ware 43.3% （13/30） and 42.5%（37/87）,respectively （P〉0.05）.Bile microbial community of obstruction coused by stone was mainly Enterobacteriaceae （Escherichia,Salmonella,Klebsiella,Proteus）,Streptococcus （Streptococcus）,digestive coccaceae （digestive bacteria genus,peptostreptococcus）,Micrococcus branch （Staphylococcus）,Propionibacterium （Propionibacterium） and Neisseriabacteria （Acinetobacter）.Bile microbial community of obstruction caused by tumor was mainly Enterobacteriaceae （Clay Beth spp,Escherichia,Salnonellatyphi） and Micrococcus branch （Staphylococcus）.Conclusion The condition and variety of bile microbial community of patients with obstructive jaundice could be effectively evaluated by 16S rDNA fragment through T RFLP analysis.