Selection, Phenotyping and Identification of Acid and Hydrogen Peroxide Producing Bacteria from Vaginal Samples of Canadian and East African Women

The common but poorly understood condition known as bacterial vaginosis (BV) increases vulnerability to HIV infection and is associated with the absence of H2O2-producing Lactobacillus. Vaginal lactic acid bacteria (LAB) produce anti-HIV factors such as organic acids and hydrogen peroxide (H2O2), and may bind and inactivate HIV particles during scavenging of mannose. These factors define potential criteria for initial selection of candidate probiotics to block heterosexual transmission of HIV. Therefore, the primary goal of this study was to characterize acid production on mannose and H2O2 production in vaginal isolates from Canadian adolescents (192 isolates, 16 individuals) and commercial sex workers in Nairobi, Kenya (576 isolates, 96 individuals). Selection of isolates from H2O2-detecting media suggested an idiosyncratic individual-level profile and extensive phenotypic diversity, including the identification of a subset of “double-strong” acid- and H2O2-producers with phenotypes similar to well-characterized probiotic strains. Molecular fingerprinting of all isolates by capillary electrophoresis of 16S-23S rRNA interspacer amplicons was coupled with chaperonin-60 universal target (cpn60 UT) sequencing in a subset, tentatively identifying 96% of isolates although only 19% were sequenced. Most isolates belonged to Lactobacillus, Streptococcus, Bifidobacterium or Gardnerella, with a total of 37 species in 15 genera, as well as 5 potentially novel organisms, identified in this study. This sensitivity was likely enhanced by phenotype-based selection on two chromogenic media formulations. Identification of double-strong isolates may provide a rational basis for selection and further characterization of vaginal probiotics, with potential application as part of HIV prevention initiatives in western Canada and East Africa.这种常见却鲜有深入认知的细菌性阴道病（bacterial vaginosis, BV）会提升HIV感染易感性，且与产过氧化氢（H₂O₂）的乳酸杆菌缺失密切相关。阴道乳酸菌（lactic acid bacteria, LAB）可产生有机酸、过氧化氢（H₂O₂）等抗HIV因子，在清除甘露糖的过程中还能结合并灭活HIV病毒颗粒。上述特性为筛选可阻断HIV异性传播的候选益生菌提供了潜在评判标准。因此，本研究的核心目标是，对加拿大青少年（16名受试者，192株分离菌）以及肯尼亚内罗毕商业性工作者（96名受试者，576株分离菌）的阴道分离株，开展甘露糖产酸能力与H₂O₂产生能力的表征分析。基于H₂O₂检测培养基的分离株筛选结果显示，个体层面存在独特的菌群特征，且表型多样性丰富，其中还鉴定出一类兼具强产酸与强产H₂O₂能力的“双强”分离株，其表型与已被充分表征的益生菌菌株相似。本研究通过16S-23S rRNA基因间隔区扩增子毛细管电泳对所有分离株进行分子指纹图谱分析，并对其中部分分离株开展伴侣蛋白60通用靶点（chaperonin-60 universal target, cpn60 UT）测序；尽管仅完成了19%分离株的测序，但仍初步鉴定出96%的分离株。绝大多数分离株隶属于乳酸杆菌属、链球菌属、双歧杆菌属或加德纳菌属，本研究共鉴定出15个菌属下的37个菌种，以及5株潜在的新型微生物。该高鉴定率可能得益于基于两种显色培养基配方的表型筛选。本次“双强”分离株的鉴定，可为阴道益生菌的筛选与后续表征提供合理依据，有望应用于加拿大西部与东非地区的HIV预防工作中。