Screening and characterization of vaginal fluid donations for vaginal microbiota transplantation.

Bacterial vaginosis (BV), the overgrowth of diverse anaerobic bacteria in the vagina, is the most common cause of vaginal symptoms worldwide. BV frequently recurs after antibiotic therapy, and the best probiotic treatments only result in transient changes from BV-associated states to "optimal" communities dominated by a single species of Lactobacillus. Therefore, additional treatment strategies are needed to durably alter vaginal microbiota composition for patients with BV. Vaginal microbiota transplantation (VMT), the transfer of vaginal fluid from a healthy person with an optimal vaginal microbiota to a recipient with BV, has been proposed as one such alternative. However, VMT carries potential risks, necessitating strict safety precautions. Here, we present an FDA-approved donor screening protocol and detailed methodology for donation collection, storage, screening, and analysis of VMT material. We find that Lactobacillus viability is maintained for over six months in donated material stored at - 80 °C without glycerol or other cryoprotectants. We further show that species-specific quantitative PCR for L. crispatus and L. iners can be used as a rapid initial screening strategy to identify potential donors with optimal vaginal microbiomes. Together, this work lays the foundation for designing safe, reproducible trials of VMT as a treatment for BV.

Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Antigen Presenting Cells Link the Female Genital Tract Microbiome to Mucosal Inflammation, With Hormonal Contraception as an Additional Modulator of Inflammatory Signatures.

The microbiome of the female genital tract (FGT) is closely linked to reproductive health outcomes. Diverse, anaerobe-dominated communities with low Lactobacillus abundance are associated with a number of adverse reproductive outcomes, such as preterm birth, cervical dysplasia, and sexually transmitted infections (STIs), including HIV. Vaginal dysbiosis is associated with local mucosal inflammation, which likely serves as a biological mediator of poor reproductive outcomes. Yet the precise mechanisms of this FGT inflammation remain unclear. Studies in humans have been complicated by confounding demographic, behavioral, and clinical variables. Specifically, hormonal contraception is associated both with changes in the vaginal microbiome and with mucosal inflammation. In this study, we examined the transcriptional landscape of cervical cell populations in a cohort of South African women with differing vaginal microbial community types. We also investigate effects of reproductive hormones on the transcriptional profiles of cervical cells, focusing on the contraceptive depot medroxyprogesterone acetate (DMPA), the most common form of contraception in sub-Saharan Africa. We found that antigen presenting cells (APCs) are key mediators of microbiome associated FGT inflammation. We also found that DMPA is associated with significant transcriptional changes across multiple cell lineages, with some shared and some distinct pathways compared to the inflammatory signature seen with dysbiosis. These results highlight the importance of an integrated, systems-level approach to understanding host-microbe interactions, with an appreciation for important variables, such as reproductive hormones, in the complex system of the FGT mucosa.

Modeling the temporal dynamics of cervicovaginal microbiota identifies targets that may promote reproductive health.

BACKGROUND: Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations. RESULTS: We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities. CONCLUSIONS: The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa. Video Abstract.

The cervicovaginal mucus barrier to HIV-1 is diminished in bacterial vaginosis.

Bacterial vaginosis (BV), a condition in which the vaginal microbiota consists of community of obligate and facultative anaerobes rather than dominated by a single species of Lactobacillus, affects ~30% of women in the US. Women with BV are at 60% increased risk for HIV acquisition and are 3-times more likely to transmit HIV to an uninfected partner. As cervicovaginal mucus (CVM) is the first line of defense against mucosal pathogens and the home of the resident vaginal microbiota, we hypothesized the barrier function of CVM to HIV may be diminished in BV. Here, we characterized CVM properties including pH, lactic acid content, and Nugent score to correlate with the microbiota community composition, which was confirmed by 16S rDNA sequencing on a subset of samples. We then quantified the mobility of fluorescently-labeled HIV virions and nanoparticles to characterize the structural and adhesive barrier properties of CVM. Our analyses included women with Nugent scores categorized as intermediate (4-6) and BV (7-10), women that were either symptomatic or asymptomatic, and a small group of women before and after antibiotic treatment for symptomatic BV. Overall, we found that HIV virions had significantly increased mobility in CVM from women with BV compared to CVM from women with Lactobacillus crispatus-dominant microbiota, regardless of whether symptoms were present. We confirmed using nanoparticles and scanning electron microscopy that the impaired barrier function was due to reduced adhesive barrier properties without an obvious degradation of the physical CVM pore structure. We further confirmed a similar increase in HIV mobility in CVM from women with Lactobacillus iners-dominant microbiota, the species most associated with transitions to BV and that persists after antibiotic treatment for BV. Our findings advance the understanding of the protective role of mucus and highlight the interplay between vaginal microbiota and the innate barrier function mucus.