中国汉族育龄女性阴道内乳杆菌的鉴定与系统发育分析

Identification and phylogenetic analysis of vaginal Lactobacillus in reproductive age Chinese Han women

目的比较基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)与16S rRNA基因测序鉴定女性阴道乳杆菌方法,同时探究不同阴道微生态背景的汉族育龄女性阴道内乳杆菌的种类及其分布情况。方法对260例汉族育龄女性阴道拭子进行阴道微生态分析及乳杆菌的分离培养,根据阴道微生态鉴定结果分为正常组(173例)、中间型细菌性阴道病组(IBV,55例)、细菌性阴道病组(BV,9例)和外阴阴道炎假丝酵母菌病组(VVC,23例),分离乳杆菌并进行纯化培养,纯化的乳杆菌经MALDI-TOF MS和16S rRNA基因测序鉴定并进行聚类分析。结果2种方法鉴定的一致率为95.3%。260例标本中149例(57.3%)检出了乳杆菌,共发现17种乳杆菌,检出的乳杆菌分布于5个乳杆菌系统发育群,其中分离出较多的乳杆菌为卷曲乳杆菌(47例)、加氏乳杆菌(38例)、阴道乳杆菌(21例)。正常组116例(67.1%)标本分离出乳杆菌,IBV组19例(34.5%),BV组2例(22.2%),VVC组12例(52.2%)。正常育龄女性与患BV女性乳杆菌种类与数量差异有统计学意义(χ2=22.9360,P<0.0001),与患VVC女性差异无统计学意义(χ2=1.9830,P=0.1600)。与其他组相比,正常组育龄女性中分离出卷曲乳杆菌的比例显著高于BV组和VVC组(χ2=4.5480,P=0.0330;χ2=5.6050,P=0.0200),VVC组育龄女性分离出加氏乳杆菌的比例显著高于正常组(χ2=5.0900,P=0.0350)。结论对比16S rRNA基因测序与MALDI-TOF MS方法的乳杆菌鉴定结果,除了阴道乳杆菌和人阴道乳杆菌出现不一致的鉴定结果,其余15种乳杆菌鉴定结果一致性均为100%,但基于测序与基于质谱的方法产生的乳杆菌聚类分析结果不完全一致。卷曲乳杆菌可能是反映育龄女性正常阴道微生态的重要标志物,加氏乳杆菌可能与女性VVC的发生有关。

Objective To compare the accuracy of MALDI-TOF MS(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry)vs 16 S rRNA gene sequencing for identification of vaginal Lactobacillus,and investigate the presence of vaginal Lactobacillus species in reproductive age Chinese Han women and their distribution in women with different vaginal microecological status.Methods The vaginal swabs were collected from 260 reproductive-age Chinese Han women.The analysis of vaginal microecological status was performed and Lactobacilli were isolated.According to the analysis of vaginal microecological status,all the subjects were divided into health group(173 cases),intermediate bacterial vaginosis group(IBV,55 cases),bacterial vaginosis group(BV,9 cases),and vulvovaginal candidiasis group(VVC,23 cases).All the isolates were preliminarily detected as Lactobacillus genus by phenotypic analysis.The species-level identification and cluster analysis of Lactobacillus were performed using 16 S rRNA gene sequencing and MALDI-TOF MS.Results The consistency between the two methods was 95.3%.Lactobacillus was detected in 149(57.3%)of the 260 specimens.A total of 17 different Lactobacillus species were identified,which distributed in 5 Lactobacillus phylogenetic groups.Among the isolated Lactobacilli,the top three Lactobacillus species were L.crispatus(47 cases),L.gasseri(38 cases),and L.vaginalis(21 cases).Lactobacilli were detected in 116 cases(67.1%)in the health group,19 cases(34.5%)in IBV group,2 cases(22.2%)in BV group,and 12 cases(52.2%)in VVC group.There was a significant difference in the number of cases of Lactobacilli between Health group and BV group(χ^2=22.9360,P<0.0001)but not VVC group(χ^2=1.9830,P=0.1600).The proportion of L.crispatus isolated from the health group was significantly higher than from BV and VVC groups(χ^2=4.5480,P=0.0330;χ^2=5.6050,P=0.0200),and the proportion of L.gasseri isolated from the VVC group was significantly higher than that from the health group(χ^2=5.0900,P=0.0350).Conclusion MALDI-TOF MS has high consistency with 16 S rRNA gene sequencing,except that it failed to identify a small portion of L.vaginalis and L.coleohominis.The dendrogram of cluster analysis of Lactobacillus based on ribosomal sequence data and phenotypic protein profiles indicate that the two methods are not completely consistent in taxonomy.L.crispatus may be an important marker reflecting healthy vaginal microecology of reproductive-age women,while L.gasseri may be associated with the occurrence of VVC.