乳杆菌阴道分离株16S rDNA鉴定及优势菌株产H_2O_2和自凝集能力分析

Identification of vaginal Lactobacillus isolates by 16S rDNA sequencing and characterization of the hydrogen peroxide production and autoaggregation activities of the predominant strains

目的从阴道分泌物标本分离鉴定乳杆菌,分析与健康相关优势菌种产过氧化氢（H2O2）和自凝集能力,揭示菌株特异性的潜在益生特性。方法利用MRS固体培养基从41例阴道分泌物标本中分离单菌落,纯培养后提取细菌基因组DNA,PCR扩增16S rDNA序列,依据序列一致性确定细菌种属。利用TMB-HRP-MRS培养基测定优势乳杆菌菌株产H2O2能力,并测定其自凝集能力。结果获得155个细菌分离株,分属乳杆菌属104株（67.1%）、肠球菌属21株（13.5%）、链球菌属28株（18.1%）、葡萄球菌属1株（0.6%）和双歧杆菌属1株（0.6%）。各菌属在无或有妇科临床症状患者标本中的构成比例差异有统计学意义（χ2=7.4918,P=0.0236）,乳杆菌在前者检出率为83.3%,显著高于其在后者47.1%的检出率（χ2=4.4879,P=0.0341）。乳杆菌中卷曲乳杆菌分离株所占比例最高（67.3%,70/104）。来自不同标本的22株卷曲乳杆菌中77.3%菌株产H2O2,且这些菌株具有强弱不同的自凝集能力（27.5%~97.3%）。结论乳杆菌属特别是卷曲乳杆菌种是健康阴道环境中优势菌属,其产H2O2和自凝集能力存在明显菌株特异性,有2株卷曲乳杆菌此两特性均很强可供进一步研发。

Objective To isolate and identify the Lactobacillus spp. from vaginal fluid and study the hydrogen peroxide（ H2O2） production and autoaggregation activities of those health-related predominant strains. Methods Bacterial single colonies were obtained via MRS agar plates from 41 vaginal swab specimens. Genomic DNA was extracted from the pure culture and the 16 S rDNA sequence amplified by PCR was used for direct sequencing and bacterial identification. For the health-related Lactobacillus strains,H2O2-production was detected by a TMB-HRPMRS agar based assay,and the autoaggregation activity was assessed as well. Results A total of 155 bacterial isolates were identified as belonging to the genus of Lactobacillus（ 104,67. 1%）,Enterococcus（ 21,13. 5%）,Streptococcus（ 28,18. 1%）,Staphylococcus（ 1,0. 6%） and Bifidobacterium（ 1,0. 6%） respectively. There was a significant difference in the proportion of various bacterial genera in the vaginal fluid specimens collected from healthy women or from those with gynecological symptoms or signs（ χ2= 7. 4918,P = 0. 0236）. The detection rate of Lactobacillus spp. in the former group was significantly higher than that in the latter one（ χ2= 4. 4879,P = 0. 0341）. L. crispatus was found to be the most frequently isolated Lactobacillus species（ 67. 3%,70 /104）.Among the 22 L. crispatus strains isolated from different specimens,H2O2-production was detected in 77. 3% of the strains and autoaggregation activity（ 27. 5%-97. 3%） was found to vary significantly among the strains. Conclusion Lactobacillus,especially L. crispatus,are the predominant species in healthy vagina. L. crispatus display strain-specific H2O2-production and autoaggregation activities; two of them with strong activities are worthy of further investigation.