Diversity Of Urogenital Microbiota Associated With Female Lower Genital Tract Infcctions

Part1Molecular analysis of the diversity of vaginal microbiota associated with female lower genital tract infectionsBackground The female genital tract (FGT) harbors different species of bacteria in very large numbers that play an important role on health. Bacterial vaginosis (BV) is an ecological disorder of the vaginal microbiota that affects millions of women annually, and is associated with numerous adverse health outcomes including pre-term birth and the acquisition of sexually transmitted infections. However, little is known about the overall structure and composition of vaginal microbial communities; most of the earlier studies focused on predominant vaginal bacteria in the process of BV. In addition, there is no convincing evidence that the microbiota in FGT is associated with cervicitis and vulvovaginal candidiasis (VVC).Methods In the present study, the diversity and richness of vaginal microbiota in50BV positive and50healthy women from China were investigated using culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and barcoded454pyrosequencing methods, and validated by quantitative PCR (qPCR). While vaginal swabs and ectocervical swabs were collected from100participants including30women with BV (BV group),22women with cervicitis (Cer group),18 women with BV in combination with cervicitis (BC group) and30healthy control women (CN group) from China. The diversity and richness of cervicovaginal microbiota were investigated with culture-independent PCR-DGGE and qPCR targeting11microorganisms that have been associated with FGT infections, In addition, the composition and diversity of vaginal microbiota from48VVC women and32women with BV in combination with VVC (BVC) were explored by PCR-DGGE.Results Our data demonstrated that there was a profound shift in the absolute and relative abundances of bacterial species present in the vagina when comparing populations associated with healthy and diseased conditions. In spite of significant interpersonal variations, the diversity of vaginal microbiota in the two groups could be clearly divided into two clusters. A total of246,359high quality pyrosequencing reads was obtained for evaluating bacterial diversity and24,298unique sequences represented all phylotypes. The most predominant phyla of bacteria identified in the vagina belonged to Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria. The higher number of phylotypes in BV positive women over healthy is consistent with the results of previous studies and a large number of low-abundance taxa which were missed in previous studies were revealed. Although no single bacterium could be identified as a specific marker for healthy over diseased conditions, three phyla-Bacteroidetes, Actinobacteria and Fusobacteria, and eight genera including Gardnerella, Atopobium, Megasphaera, Eggerthella, Aerococcus, Leptotrichia/Sneathia, Prevotella and Papillibacter were strongly associated with BV (p<0.05). These genera are potentially excellent markers and could be used as targets for clinical BV diagnosis by molecular approaches. However, except for H2O2-and lactic acids-producing Lactobacillus, there was not sufficient evidence that the cervicovaginal microbiota including vaginal microbiota and cervical microbiota is directly involved in the development of cervicitis. And there was no significantly correlation between vaginal predominant microbiota and VVC.Conclusions The data presented here have clearly profiled the overall structure of vaginal communities and clearly demonstrated that BV is associated with a dramatic increase in the taxonomic richness and diversity of vaginal microbiota. The study also provides the most comprehensive picture of the vaginal community structure and the bacterial ecosystem, and significantly contributes to the current understanding of the etiology of BV. However, there was no significantly correlation between vaginal predominant microbiota and VVC, and between cervicovaginal predominant microbiota and cervicitis.Part2Host responses to vaginal microbiota in vaginal lavage fluid of women with BVBackground The host determinants of susceptibility to BV are poorly understood. Infectogenomics and infectoproteomics in the vagina might play important roles in the development of BV.Methods In order to explore the host responses to vaginal microbiota in women with BV in a deeper level, we investigated the gene expression profiles of vaginal epithelial cells with Affymetrix GENECHIP and the associated cytokines with Bio-Plex Pro Human Cytokine27-plex Assay from vaginal lavage fluid obtained from10women with BV and10women without BV.Results After gene expression analysis with microarray,2397genes changed more that2fold, while1234genes upregulated and1163genes downregulated, which were participated in inflammatory associated signaling pathway such as TNF-a, NF-κB, IFN-y, IL-5and apoptosis. With cytokines analysis, IL-1β, IL-5, IL-12, IFN-y, RANTES and TNF-a were changed significantly in BV development (p<0.05). Conclusions Host responses especially the increase of these cytokines such as IL-1β, IL-5, IL-12, IFN-γ, RANTES and TNF-α in vaginal lavage fluid might be involved in defending against invasion by BV-related microorganisms.Part3Vaginal microbiota restoration for bacterial vaginosis treated with metronidazole and Lactobacillus intravaginallyBackground Alterations of bacterial composition and diversity in the vagina of healthy and infected women have been fully elucidated with molecular techniques. However, its overall structure and composition after actively treatment was still not investigated so far.Methods and Results We applied pyrosequencing of over4,30016S rRNA gene V3region amplicons per subject to characterize the vaginal microbiota from55subjects after treated with metronidazole (30cases,7days) and probiotic Lactobacillus (25cases,10days) for one short-term course and60control subjects (30BV positive control and30BV negative control). After completion of therapy,83.3%in metronidazole-treated group and88.0%in Lactobacillus-treated group was free of bacterial vaginosis (BV). Metronidazole treatment reduced the microbial diversity and eradicated the BV-associated microbiota, while probiotic Lactobacillus treatment could not alter its bacterial diversity and only inhibit those bacteria overgrowth. Despite significant interpersonal variations, the microbiota of actively treated groups or participants constituted a unique profile that was separable from those untreated. Along with the decrease of those potential pathogenic bacteria such as Gardnerella, Atopobium, Prevotella, Megasphaera, Coriobacterineae, Lachnospira, Mycoplasma and Sneathia, a Lactobacillus-dominated vaginal microbiota was restored and new vaginal eubiosis was re-established (p<0.05). Acting as vaginal sentinels and biomarkers, the relative abundance of Lactobacillus and those pathogenic bacteria determined the consistency of BV clinical and microbiological cured rate, and the recurrence rate after treatment.Conclusions Our data indicated that intravaginal administration of metronidazole and exogenous probiotic Lactobacillus could restore a normal vaginal microbiota, while Lactobacillus could maintain a long-term vaginal eubiosis. These results help to clarify the role of those potential pathogenic populations in BV pathogenesis and provide new insights into BV treatment in the future.