健康女性阴道乳杆菌抑菌功能的初步研究

The preliminary study of antimicrobial activity of vaginal Lactobacillus isolated from healthy women

目的:研究健康女性阴道乳杆菌对阴道常见致病菌的抑制作用及其机制。方法:选取北京大学第一医院体检中心18～45岁的育龄期健康女性17例,从其阴道分泌物中分离、纯化乳杆菌并进行鉴定。采用spot on lawn和双层牛津杯法筛选出对粪肠球菌、微小消化链球菌、大肠埃希菌和白假丝酵母菌有抑制作用的阴道乳杆菌菌株。测定乳杆菌的生长曲线及代谢物内的乳酸含量。将乳杆菌与白假丝酵母菌共培养,观察乳杆菌菌体和菌液粗提物在抑制白假丝酵母菌的过程中各自所起的作用。结果:17例健康女性阴道分泌物中分离出23株乳杆菌,其中6株具有抑菌作用,分别是加氏乳杆菌、詹氏乳杆菌、阴道乳杆菌、卷曲乳杆菌、发酵乳杆菌和德氏乳杆菌,其中加氏乳杆菌增殖能力较强,詹氏乳杆菌最弱。6株乳杆菌代谢物内乳酸的含量不同,发酵乳杆菌最高为(2.72±0.035)mg/ml,卷曲乳杆菌和德氏乳杆菌较低,分别为(2.14±0.035)mg/ml和(2.13±0.025)mg/ml。加氏乳杆菌、詹氏乳杆菌、阴道乳杆菌的菌液、菌体及菌液粗提物均可以抑制白假丝酵母菌的生长(P≤0.05);发酵乳杆菌和德氏乳杆菌的菌液和菌体可以抑制白假丝酵母菌的生长(P≤0.05);卷曲乳杆菌对白假丝酵母菌则无抑制作用。结论:健康女性阴道乳杆菌的多个菌株均具有抑制大肠埃希菌、粪肠球菌、微小消化链球菌或白假丝酵母菌的功能。詹氏乳杆菌粗提物中细菌素类物质和过氧化氢的联合作用可以抑制白假丝酵母菌,加氏乳杆菌和阴道乳杆菌主要依靠过氧化氢起作用。乳杆菌产生的乳酸没有发挥抑菌作用。

Objective ： To investigate the mechanism and the inhibitory effect of vaginal lactobacillus species of healthy female on common vaginal pathogens. Methods： 17 cases of healthy reproductive female aged 18 -45 years old were collected in medical examination center of Peking University First Hospital, and Lactobacilli were isolated and purified from their vaginal secretions. Growth inhibition of Enterococcus faecalis, Micromonas micros, Escherichia coli and Candida albicans were screened using the spot on the lawn and double agar diffusion method. The growth curve of lactobacillus strains and the production of lactic acid were determined. Lactobacilli and Candida albicans were co-cultured to observe the antimicrobial activity of lacto- bacillus bacteria and cell-free culture supernatants （CFCs）respectivly by using spread plate method. Results：23 strains of Lactobacillus were isolated from 17 cases of healthy female vaginal secretions and 6 strains including L. gasseri, L. jensenii, L. vaginalis, L. crispatus, L. fermentum and L. debrueckii showed inhibitory effect. The proliferation ability of L. gasseri was strong, while L. jensenii had the lowest proliferation ability. Six Lactobacillus strains produced varying amounts of lactic acid. The maximum level of lactic acid was 2.72±0.035mg/ml in L. fermentum and lower levels were 2.14±0. 035mg/ml and 2.13±0. 025mg/ml,in L. crispatus and L. debrueckii. Laetobacillus cultures,bacteria and CFCs of L. gasseri, L. jensenii and L. vaginalis exhibited antimicrobial activity against Candida albicans （P≤0.05） , L. fermentum and L. de-brueckii cultures and bacteria also showed inhibitory activity（P^0.05 ）. L. crispatus played no role in inhibiting Candida albicans. Conclusions：Many vaginal Lactobacillus stains of healthy female can inhibit Enterococcus faecalis, Micromonas micros, Escherichia coli and Candida albicans. The antimicrobial ability of L. jensenii CFCs is attributed to hydrogen peroxide and bacteriocin-like substance, while the antimicrobial ability of L. gasseri and L. vaginalis depended mainly on hydrogen peroxide. Lactic acid alone developed inhibitory activity against candida albicans at concentrations is higher than that present in CFCs, and it points out that lactic acid displays no antimicrobial activity.