Isolation of Vaginal Lactobacilli and Characterization of Anti-Candida Activity

Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. Metabolic profiles of lactobacilli supernatants were studied by 1H-NMR analysis. Metabolome was found to be correlated with both taxonomy and activity score. Exclusion, competition and displacement experiments were carried out to investigate the interference exerted by lactobacilli toward the yeast adhesion to HeLa cells. Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants. Inhibition of histone deacetylases was hypothesised to support the antifungal activity of vaginal lactobacilli. Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.健康阴道菌群以乳杆菌属（Lactobacillus spp.）为核心优势类群，它们是对抗包括假丝酵母菌属（Candida spp.）在内的病原体的关键防御屏障。本研究旨在筛选具备体外抗假丝酵母菌属活性的阴道乳杆菌，并解析其抗真菌作用机制。研究菌株从健康绝经前女性的阴道拭子中分离获得，通过16S rRNA基因测序将分离株鉴定至物种水平，分别为卷曲乳杆菌（L. crispatus）B1-BC8、加氏乳杆菌（L. gasseri）BC9-BC14以及阴道乳杆菌（L. vaginalis）BC15-BC17。所有菌株均可产生过氧化氢与乳酸。采用肉汤微量稀释法，评估菌株对白假丝酵母菌（C. albicans）、光滑假丝酵母菌（C. glabrata）、克柔假丝酵母菌（C. krusei）、热带假丝酵母菌（C. tropicalis）、近平滑假丝酵母菌（C. parapsilosis）以及卢西塔尼亚假丝酵母菌（C. lusitaniae）的抑菌与杀菌活性。卷曲乳杆菌BC1、BC4、BC5与阴道乳杆菌BC15展现出最广泛的抗菌谱，可对所有分离株的白假丝酵母菌与卢西塔尼亚假丝酵母菌发挥杀菌活性。通过氢核磁共振（1H-NMR）分析，对乳杆菌上清液的代谢谱进行了研究，结果显示代谢组与菌株的分类学地位及活性评分均存在相关性。通过排斥、竞争与置换实验，探究乳杆菌对假丝酵母菌黏附HeLa细胞的干扰作用，结果表明多数乳杆菌菌株可通过上述三种机制显著降低白假丝酵母菌的黏附能力。其中，卷曲乳杆菌BC2、加氏乳杆菌BC10与加氏乳杆菌BC11在降低病原体黏附方面活性最强，其作用同时由菌体细胞与上清液共同介导。研究推测，组蛋白去乙酰化酶（histone deacetylases）抑制作用可辅助增强阴道乳杆菌的抗真菌活性。本研究结果为开发基于益生菌的新型治疗制剂，用于假丝酵母菌感染的预防与辅助治疗提供了重要前提。