Identification Of Vaginal Lactobacillus And Analysis Of Vaginal Microbiota In Reproductive-age Women

Objective1.The accuracy of Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF MS)and 16S r RNA gene sequencing for identification of vaginal Lactobacillus was compared.The presence of vaginal Lactobacillus species of reproductive-age women and their distribution in women with different vaginal microecological status was investigated.2.High-Throughput Sequencing was performed to detect the abundance of Lactobacillus and profile of vaginal microbiota.in women’s vagina.The clustering of the vaginal microbiota in reproductive-age women with different vaginal microecological status was determined.Methods1.The vaginal swabs were collected from 260 reproductive-age Han Chinese women.The analysis of the vaginal microecological status was performed and the Lactobacillus were isolated.According to the analysis of the vaginal microecological status,all 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 isolates were preliminarily detected as the Lactobacillus genus by phenotypic analysis.The species-level identification and cluster analysis of Lactobacillus were performed by 16S r RNA gene sequencing and MALDI-TOF MS2.The above 148 cases of vaginal swabs were divided into the healthy group(97cases),IBV group(29cases),BV group(8 cases),VVC group(14 cases)according to the results of vaginal microecological analysis,while these cases were also divided into Lactobacillus group(L group)(82 cases)and Lactobacillus-free group(NL group)(66cases)according to the results of Lactobacillus culturing.High-throughput sequencing and bioinformatics analysis were performed on all these samples.Results1.The consistency between the two methods was 96.9%(249/257).Lactobacillus was detected in 149(57.3%)of the 260 specimens.A total of 17 different Lactobacillus species were identified,which are distributed in 5 Lactobacillus phylogenetic groups.Among the isolated Lactobacilli,the most isolated Lactobacillus species were L.crispatus(41 cases),L.gasseri(38 cases),and L.vaginalis(21 cases).The number of cases of Lactobacilli detected in the health group,IBV group,BV group,VVC group were 116(67.0%),19(34.5%),2(22.2%)and 12(52.2%)respectively.There was a significant difference in the number of cases of Lactobacilli between healthy reproductive-age Han Chinese women and those with bacterial vaginosis(χ~2=22.9360,P<0.0001),and no significant difference with those with vulvovaginal candidiasis(χ~2=1.9830,P=0.1600).The proportion of L.crispatus isolated from the health group was significantly higher than that of the other 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 of the health group(χ~2=5.0900,P=0.0350).2.A total of 13 Lactobacillus species were found in 148 specimens,among which the most abundant Lactobacillus species were L.crispatus,L.iners,L.jensenii/L.fornicalis,L.gasseri,L.delbrueckii and L.paracasei in order.On the aspect of single specimen,Lactobacillus was isolated from 90(92.8%)cases in the healthy group,18(62.1%)cases in the IBV group,4(50.0%)cases in the BV group,and 12(85.7%)cases in the VVC group.There were statistically significant differences in the number of Lactobacillus detected between the healthy group,IBV group and BV group(χ~2=22.3400,P<0.0001).With the worsening of the severity of BV,the detection rate of L.iners increased(χ~2=6.7400,P=0.0340),while the detection rate of L.crispatus in the VVC group was significantly lower than that in the healthy group(χ~2=4.4020,P=0.0360).On the aspect of different groups,the relative abundance of Lactobacillus in the vaginal microbiota in the health group,IBV group,BV group and VVC group was 82.5%,42.7%,3.6%and 58.0%respectively.The most abundant Lactobacillus in the healthy group and the IBV group is L.crispatus,while the most abundant Lactobacillus in the BV group and VVC group is L.iners.The vaginal microbiota is divided into five Vagitypes,namely VT1(46/148,31.08%)dominated by L.crispatus,VT2(33/148,22.30%)dominated by L.iners,VT3(11/148,7.43%)dominated by other Lactobacillus species and lactic acid bacteria,VT4(38/148,25.68%)dominated by Gardnerella vaginalis and VT5(20/148,13.5%)dominated by a variety of other bacteria.ConclusionThe results of the culture-dependent method and the culture-independent method to identify and clustering Lactobacillus species are not completely consistent,and the two types of methods can complement each other in detecting the Lactobacillus species qualitatively and quantitatively.The normal vaginal microbiota of reproductive-age women is mainly dominated by L.crispatus and L.iners.L.crispatus may be a marker reflecting the healthy vaginal microecology of reproductive-age women,while L.iners may be a marker intimating the increased risk of BV and VVC.