对阴道加德纳菌具有抑制作用的乳酸杆菌的初步筛选  

Screening of lactobacilli for ability to inhibit Gardnerella vaginalis

作　　者：柳迎春[1] 朱德全[1] 郑慧琦[1] 孟祥晨[1] 

机构地区：[1]东北农业大学乳品科学教育部重点实验室,黑龙江哈尔滨150030

出　　处：《中国病原生物学杂志》2014年第5期412-416,共5页Journal of Pathogen Biology

摘　　要：目的从健康妇女阴道分泌物中分离并筛选出对阴道加德纳菌具有抑菌活性的乳酸杆菌(简称乳杆菌),为开发治疗细菌性阴道炎的微生态制剂奠定基础。方法采集20例健康育龄妇女阴道分泌物,采用选择性培养基De Man-Rogosa-Sharpe(MRS)分离乳杆菌,采用乳杆菌上清液与指示菌株阴道加德纳菌(Gardnerella vaginalis,Gv)ATCC14018共培养的方法筛选具有抑菌活性的乳杆菌。通过生理生化、糖发酵试验和16SrDNA基因测序比对的方法对筛选出的具有抑菌活性的菌株进行鉴定。然后用5mol/L的NaOH将具有较强抑菌活性的乳杆菌无细胞上清液pH值调至pH 7.0后,再次进行抑菌试验。结果利用乳杆菌上清液对致病菌进行抑菌试验,分离得到的18株乳酸菌均具有抑菌活性,其中3号、N7号、15号乳杆菌对病原指示菌的抗菌活性最高,对照组MRS中Gv菌落数的log值为7.26,加入上述3株菌的无细胞上清液后Gv菌落数的log值分别为5.09、5.42和5.44;加入商业对照菌株DM8909无细胞上清液后Gv菌落数的log值为5.95。3株菌的抑菌活性与德式乳杆菌DM8909比较差异有统计学意义(P<0.05)。经生理生化、糖发酵试验结合16SrDNA基因比对确定3株乳杆菌均为植物乳杆菌。pH值调至7.0后的无细胞上清液几乎无抑菌活性,由此推断上清液中的有机酸起主要抑菌作用。结论筛选获得的3株植物乳杆菌具有一定的抑菌作用,可作为治疗细菌性阴道炎的候选菌株。Objectives To select lactobacilli that inhibit Gardnerella vaginalis from vaginal secretions of healthy women and to lay the foundation for development of probiotics for use in treating bacterial vaginosis. Methods Vaginal swab samples were collected from 20 healthy women of childbearing age and lactobacilli were isolated from those samples using De Man-Rogosa-Sharpe （MRS） medium. Stmins of lactobacilli were grown in MRS broth for 24 h at 37 C. Cell-free cul- ture supernatant （CFCS） was obtained by centrifugation at 8,000 r/min for 10 min at 4~C. After the CFCS was centrifuged, it was passed through a sterile 0.22μm Millex GS filter unit. Laetobacillus stmins were screened for antimierobial activity by coculturing G. vaginalis ATCC 14018 with CFCS. The suspension was spread on an agar plate to determine the number of viable bacteria according to the bacterial colony count. Selected stmins were identified using physiological and biochemical tests, 16S rDNA nucleotide sequencing, and a sugar fermentation test. Five mol/L NaOH was added to increase the pH of the CFCS to a pH of 7.0 for another antibacterial test. Results The lactobacillus supernatant was tested for its inhibition of pathogens. Lactohacilli were examined for its antibacterial action against vaginal isolates of G. vaginalis. Antibacterial action was measured using the bacterial colony count 24 h after G. vaginalis was cocultured with CFCS. Results indicated that the CFCS reduced the viability of G. vaginalis with differing levels of effectiveness. Stmins with the highest activity against G. vaginalis were 3, N7, and 15. The control had log 7.26 CFU/ml of G. val4inalis. Adding CFCS to BHI broth containing stmin 3 resulted in log 5.09 CFU/ml of G. vaginalis, adding CFCS to BHI broth containing stmin N7 resulted in log 5.42 CFU/ml of G. vaginalis, and adding CFCS to BHI broth containing stmin 15 resulted in log 5.44 CFU/ml of G. vaginalis. Adding CFCS to BHI broth containing the commercial stmin DM8909 resulted in log 5.95 CFU/ml of G. vaginalis

关 键 词：乳杆菌 阴道加德纳菌 抑菌作用 

分 类 号：R378[医药卫生—病原生物学]