细菌性阴道病患者阴道菌群特征的非培养分析

Diversity of Human Vaginal Bacterial Communities in Healthy Women and Bacterial Vaginosis

细菌性阴道病(bacterial vaginosis,BV)是一种阴道菌群失调所致的常见病,许多阴道菌群为厌氧菌,培养条件要求苛刻,很难纯培养和分离鉴定。本研究旨在通过非培养方法,明确BV患者阴道菌群特征。根据Amsel标准和Nugent评分筛选BV患者和健康育龄期妇女各30例,提取细菌基因组DNA,构建细菌16SrRNA基因文库。同时,应用特异性PCR方法扩增标本中的阴道加德纳菌、阴道阿托波菌、乳酸杆菌等8种阴道常见细菌,比较两组间各菌种的检出率。结果显示:BV患者阴道分泌物涂片以革兰染色不定或阴性的小球杆菌为主,并形成特征性的线索细胞。特异性PCR显示,BV患者阴道加德纳菌、阴道阿托波菌、普雷沃氏菌属、纤毛菌属、巨球菌属和羞怯动弯杆菌的检出率显著高于健康人群,而卷曲乳酸杆菌检出率显著低于健康人群,差异有统计学意义。文库构建显示BV患者菌群构成呈多样化,个体差异明显,共检出34种细菌,克隆子百分比排列在前5位是:Gardnerella vaginalis(23.0%),Prevotella species(18.57%),Megasphaera spe-cies(14.3%),Leptotrichia species(10.57%),Atopobium vaginae(6.4%)。与之相比,健康人群阴道菌群构成单一,以卷曲乳酸杆菌和惰性乳酸杆菌为主。结论:BV患者阴道菌群表现为乳酸杆菌显著减少甚至缺失,而以阴道加德纳菌、阴道阿托波菌、普雷沃氏菌属、纤毛菌属和巨球菌属占优势,同时多种其他厌氧致病菌共存的病态表现。而健康育龄期妇女阴道分泌物菌群表现为以乳酸杆菌占绝对优势、尤以卷曲乳酸杆菌为主、同时多种细菌共同存在的一种稳态。

Bacterial vaginosis（BV） is a polymicrobial infestation of the human vagina,thus an accurate understanding of the composition and ecology of the ecosystem is essential for understanding the aetiology of BV.Traditional cultivation technologies are limitative for most of the microbial species that reside in the vagina,while the method of construction 16S rRNA library is credible for illuminate the diversity of human vaginal bacterial communities.Broad-range polymerase-chain-reaction（PCR） amplification of 16S rRNA and construction of 16S rRNA library were used to analyze the vaginal flora diversity of 30 healthy women and 30 BV positive ones,and 8 species were detected with idiographic primers,in order to know the positive rates.The results showed that diversity and kinds of organisms that comprise the vaginal microbial community varied among women.As detected by broad-range PCR of 16SrRNA,lactobacillus was the predominant species in healthy women,especially L.crispatus.BV positive ones had greater bacterial diversity,arranged from 6 to 15 species in each sample,where Gardnerella vaginalis（23.0%）,Prevotella species（18.57%）,Megasphaera species（14.3%）,Leptotrichia species（10.57%）,Atopobium vaginae（6.4%）were strongly associated with BV.8 species can be detected;however,the positive rates differ from each other.Vaginal flora predominant with Lactobacillus crispatus is a normal status,while BV positive ones have a predominant species of Gardnerella vaginalis,Prevotella,Megasphaera species,Leptotrichia species and Atopobium vaginae,et al.