阴道内乳杆菌对阴道加德纳菌生物膜影响的初步研究

The effects of lactobacilli on gardnerella vaginalis biofilm

目的：研究阴道加德纳菌（GV）生物膜体外生长过程及阴道来源乳杆菌对GV生物膜的影响。方法：选取GV标准菌株（ATCC14018）体外培养生物膜,在成膜的不同时期检测生物膜生成量及观察生物膜生成形态。选取阴道来源乳杆菌1#（鼠李糖乳杆菌）、2#（乳酪乳杆菌）,分别在GV生物膜生成后的0h、24h、48h加等量乳杆菌共培养24h,用Live/Dead荧光染色剂染色,观察GV生物膜生成情况。结果：（1）同一培养条件下,GV生物膜生成不同时期的OD值有明显差异（P〈0.05）。电镜扫描图片显示,培养4～20h时见散落的单菌落,24h时单独菌落开始连接,48h时形成连接的菌落外面包裹一层胞外聚合物,48h后形态无变化。（2）GV生物膜的荧光染色显示,阴道内乳杆菌对抗生长后0h、24h时,GV生物膜厚度减少,形成松散的菌落,以乳杆菌为主;对抗生长后48h,菌落密度无变化,死菌面积增大,活菌以乳杆菌为主。结论：（1）阴道GV生物膜形成较缓慢,开始成膜时间约为培养后24h,约在48h后形成成熟的生物膜。（2）阴道内乳杆菌可干扰GV生物膜的形成,不同乳杆菌对不同时期GV生物膜的作用不同。

Objective： To observe the growth of Gardnerella vaginalis biofilm in vitro and the effects of lactobacilli isolated from vagina on the formation of Gardnerella vaginalis biofilm. Methods： We cultivated Gardnerella vaginalis ATCC14018 in vitro to form the biofilm and assessed the amount and the morphology of biofilm at different phrases during formation. Then,we added equal amount of 1#Lactobacillus（ L. rhamnosus） and 2^# Lactobacillus（ L. casei） at0 h,24h,48 h after the biofilm had formed and co-cultivated these bacteria for 24h to observe the formation of Gardnerella vaginalis biofilm with LIVE / DEAD Bac Light Bacterial Viability Kit.The five strains of lactobacilli were isolated,purified and identified by the laboratory of gynecology in Peking University First Hospital from the vaginal secretes of health women. Result： There were statistically significant differences in the OD values at various time points under the same cultivated condition by micro-quantitative analysis（ P〈0. 05）. Scanning electron microscope images showed that the scattered single colony existed at 4 ～ 20h and began to aggregate at 24h.The images also showed that the colonies attached mutually were surrounded by extracellular polymeric substance at 48h,but there were no changes after 48h. By fluorescent staining,we observed that biofilm became thinned and dispersed at 0h,24h after adding Lactobacilli. However,there were no differences in density of the colonies. The number of dead bacteria increased and Lactobacilli were the major live bacteria at 48h. Conclusions： Our results suggest that the process of biofilm formation is relatively slow. Gardnerella vaginalis begin to form biofilm at 24 hafter cultivation and the biofilm come to be mature after 48h approximately. Lactobacilli can also interfere the formation of biofilm,and different species of lactobacilli have various effects on the biofilm when in different periods of formation.