More Easily Cultivated Than Identified: Classical Isolation with Molecular Identification of Vaginal Bacteria Targeted Locus (Loci)

Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria that have been identified with molecular methods and associated with BV. It is unclear if this is due to the inability to adequately propagate these bacteria, or to correctly identify them in culture. Vaginal fluid from 15 women was plated on six different media using classical cultivation approaches. Individual isolates were identified by 16S rRNA gene sequencing, and compared with validly published species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene PCR and pyrosequencing. Pyrosequencing reads and bacterial isolates from the same set of samples were compared to evaluate cultivation efficacy. We isolated and identified 101 distinct bacterial strains spanning six phyla. Bacteria isolated include: 1) novel strains with <98% 16S rRNA sequence identity to published species (Dialister sp. Type 2, Eggerthella sp. Type 1), 2) phylogenetically diverse bacteria (Prevotella spp.), 3) bacteria previously isolated from other body sites but not the vagina (Atopobium deltae, Fenollaria massiliensis), and 4) bacteria formerly isolated from the vagina (Dialister micraerophilus, Veillonella montpellierensis). Pyrosequencing showed that novel strains Peptoniphilaceae bacterium DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. Genomes of 31 strains are being sequenced by the Human Microbiome Project. We isolated a diverse set of novel and clinically significant anaerobes from the vagina using conventional approaches with systematic molecular identification. Several previously “uncultivated” bacteria from the human vagina are amenable to cultivation using conventional methods.

患有细菌性阴道病（bacterial vaginosis, BV）的女性体内定植着复杂的厌氧细菌群落。目前已有多种细菌通过分子生物学方法被鉴定出来，并与BV发病相关，但尚未获得其可培养分离株。目前尚不明确这一现象的成因：究竟是无法充分培养此类细菌，还是无法在培养体系中正确鉴定它们。

研究人员采用经典培养方法，将15名女性的阴道分泌物接种至6种不同培养基中。通过16S rRNA基因测序对单个分离株进行鉴定，并与已正式发表的物种进行比对。同时，利用广谱16S rRNA基因PCR和焦磷酸测序技术，测定阴道样本的细菌群落组成谱。通过对比同一样本组的焦磷酸测序读段与细菌分离株，评估该培养方法的有效性。

本研究共分离并鉴定出101株不同的细菌菌株，隶属于6个细菌门。所分离得到的细菌可分为四类：1）与已发表物种的16S rRNA序列同源性低于98%的新型菌株（如Dialister sp. Type 2、Eggerthella sp. Type 1）；2）系统发育多样性丰富的细菌（如普雷沃氏菌属Prevotella spp.）；3）此前从其他身体部位分离得到、但从未从阴道样本中分离的细菌（如Atopobium deltae、Fenollaria massiliensis）；4）此前已从阴道样本中分离得到的细菌（如Dialister micraerophilus、Veillonella montpellierensis）。

焦磷酸测序结果显示，新型菌株Peptoniphilaceae bacterium DNF01163与Prevotellaceae DNF00733在BV患者体内呈高流行状态。31株菌株的基因组正由人类微生物组计划（Human Microbiome Project, HMP）开展测序工作。

本研究通过结合系统性分子鉴定的传统培养方法，从阴道样本中分离得到了一系列具有多样性的新型菌株及具有临床意义的厌氧菌。多种此前被标记为‘未培养’的人类阴道细菌，可通过传统培养方法实现成功培养。