Illumina测序16S rRNA标签法分析细菌性阴道病患者阴道菌群多样性

Illumina sequencing 16S rRNA tagging reveals diverse vaginal microbiomes associated with bacterial vaginosis

目的从菌群总体角度分析和比较健康育龄期女性和细菌性阴道病(BV)患者阴道菌群差异。方法采集临床Amsel标准及Nugent评分筛选的37例BV患者及37例健康育龄期女性阴道穹窿分泌物标本,提取总基因组DNA,对16S rRNA V6区基因进行扩增,所有扩增的PCR产物经IIlumina测序后,通过BIPES、UCHIME、TSC、GAST最终得到样品的物种信息。结果健康育龄期女性阴道分泌物乳酸杆菌占95%以上,单独以惰性乳酸杆菌或卷曲乳酸杆菌为主,或者两者比例相当,同时存在少量加德纳氏菌属,颗粒链球菌属,链球菌属,普氏菌菌属,埃希氏杆菌属和其它菌属;30例BV患者阴道菌群Alpha多样性明显增加(P<0.001),Beta多样性也发生了明显变化,表现为乳酸杆菌明显减少,其相对比例从45%到1%(24例),以惰性乳酸杆菌为主,部分患者(6例)甚至未检出,同时加德纳氏菌属,普氏菌菌属,颗粒链球菌属,厌氧球菌,微单胞菌属,Peptoniphilus.harei-消化链球菌属,戴阿李斯特杆菌属等相对丰富度增加,不同于既往研究的是7例BV患者以少见的格氏乳杆菌,嗜酸乳杆菌为优势菌群,约占75%～50%。结论健康育龄女性阴道菌群以乳酸杆菌占绝对优势,单独以惰性乳酸杆菌或卷曲乳酸杆菌为主,或者两者相当,并与多种微生物并存;大部分BV患者阴道菌群表现为乳酸杆菌显著减少甚至缺失,小部分BV患者以少见的格氏乳酸杆菌、嗜酸乳酸杆菌为优势菌群。

Objective To analyze and compare vaginal microbiomes in healthy women at child-bearing ages and patients with bacterial vaginosis （BV）. Methods A total of 74 vaginal swabs of the vaginal fornix were collected from 37 BV patients and 37 healthy women. BV status was assessed according to Amsel＇ s clinical criteria for all the subjects and confirmed using Gram-stain criteria （Nugent scores）. Genomic DNA of the samples was extracted for amplifying the 16S rRNA V6 hypervariable region by PCR and pyrosequencing by Illumina. BIPES, UCHIME, TSC and GAST were employed to analyze the information of the species from the samples. Results Lactobacillus was the predominant species in healthy women （more than 95% ）, including mainly L. iners and L. crispatu, with a small quantity of Gardnerella, Granulicatella, Streptococcus, Prevotella, Escherichia and other genus. The a diversity was significantly increased in 30 BV patients （P〈0.001）, and [3 diversity also changed obviously shown by decreased LactobaciUus （varying from 45% to 1%, consisting mainly of L. iners） or even absence Lactobacillus in 6 cases, with increased relative abundance of Gardnerella, Prevotella, Granulicatella, Anaerococcus, Parvimonas, Peptoniphilus.harei, Peptostreptococcus, and Dialister. Different from previous data, 7 BV cases showed a predominance of the rare species L.gasseri and L.acidophilus （75% to 50%）. Conclusion Lactobacillus is the predominant vaginal species in healthy women （mainly L. iners and L. crispatu） co-existing with many other bacteria and a variety of microorganisms. Lactobacillus is significantly decreased and even absent in most of BV patients, and some cases show the predominance of the rare species L. gasseri and L.acidophilus.