The Vaginal Microbiome: Disease, Genetics and the Environment

The vagina is an interactive interface between the host and the environment. Its surface is covered by a protective epithelium colonized by bacteria and other microorganisms. The ectocervix is nonsterile, whereas the endocervix and the upper genital tract are assumed to be sterile in healthy women. Therefore, the cervix serves a pivotal role as a gatekeeper to protect the upper genital tract from microbial invasion and subsequent reproductive pathology. Microorganisms that cross this barrier can cause preterm labor, pelvic inflammatory disease, and other gynecologic and reproductive disorders. Homeostasis of the microbiome in the vagina and ectocervix plays a paramount role in reproductive health. Depending on its composition, the microbiome may protect the vagina from infectious or non-infectious diseases, or it may enhance its susceptibility to them. Because of the nature of this organ, and the fact that it is continuously colonized by bacteria from birth to death, it is virtually certain that this rich environment evolved in concert with its microbial flora. Specific interactions dictated by the genetics of both the host and microbes are likely responsible for maintaining both the environment and the microbiome. However, the genetic basis of these interactions in both the host and the bacterial colonizers is currently unknown. _Lactobacillus_ species are associated with vaginal health, but the role of these species in the maintenance of health is not yet well defined. Similarly, other species, including those representing minor components of the overall flora, undoubtedly influence the ability of potential pathogens to thrive and cause disease. Gross alterations in the vaginal microbiome are frequently observed in women with bacterial vaginosis, but the exact etiology of this disorder is still unknown. There are also implications for vaginal flora in non-infectious conditions such as pregnancy, pre-term labor and birth, and possibly fertility and other aspects of women’s health. Conversely, the role of environmental factors in the maintenance of a healthy vaginal microbiome is largely unknown. To explore these issues, we have proposed to address the following questions:

*1.	Do the genes of the host contribute to the composition of the vaginal microbiome?* We hypothesize that genes of both host and bacteria have important impacts on the vaginal microbiome. We are addressing this question by examining the vaginal microbiomes of mono- and dizygotic twin pairs selected from the over 170,000 twin pairs in the Mid-Atlantic Twin Registry (MATR). Subsequent studies, beyond the scope of the current project, may investigate which host genes impact the microbial flora and how they do so.
*2.	What changes in the microbiome are associated with common non-infectious pathological states of the host?* We hypothesize that altered physiological (e.g., pregnancy) and pathologic (e.g., immune suppression) conditions, or environmental exposures (e.g., antibiotics) predictably alter the vaginal microbiome. Conversely, certain vaginal microbiome characteristics are thought to contribute to a woman’s risk for outcomes such as preterm delivery. We are addressing this question by recruiting study participants from the ~40,000 annual clinical visits to women’s clinics of the VCU Health System.
*3.	What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions?* We hypothesize that susceptibility to infectious disease (e.g. HPV, _Chlamydia_ infection, vaginitis, vaginosis, etc.) is impacted by the vaginal microbiome. In turn, these infectious conditions clearly can affect the ability of other bacteria to colonize and cause pathology. Again, we are exploring these issues by recruiting participants from visitors to women’s clinics in the VCU Health System.

Three kinds of sequence data are generated in this project: i) rDNA sequences from vaginal microbes; ii) whole metagenome shotgun sequences from vaginal samples; and iii) whole genome shotgun sequences of bacterial clones selected from vaginal samples. The study includes samples from three vaginal sites: mid-vaginal, cervical, and introital. The data sets also include buccal and perianal samples from all twin participants. Samples from these additional sites are used to test the hypothesis of a per continuum spread of bacteria in relation to vaginal health. An extended set of clinical metadata associated with these sequences are deposited with dbGAP. We have currently collected over 4,400 samples from ~100 twins and over 450 clinical participants. We have analyzed and deposited data for 480 rDNA samples, eight whole metagenome shotgun samples, and over 50 complete bacterial genomes. These data are available to accredited investigators according to NIH and Human Microbiome Project (HMP) guidelines. The bacterial clones are deposited in the Biodefense and Emerging Infections Research Resources Repository ("http://www.beiresources.org/":http://www.beiresources.org/). 

In addition to the extensive sequence data obtained in this study, we are collecting metadata associated with each of the study participants. Thus, participants are asked to complete an extensive health history questionnaire at the time samples are collected. Selected clinical data associated with the visit are also obtained, and relevant information is collected from the medical records when available. This data is maintained securely in a HIPAA-compliant data system as required by VCU’s Institutional Review Board (IRB). The preponderance of these data (i.e., that judged appropriate by NIH staff and VCU’s IRB are deposited at dbGAP ("http://www.ncbi.nlm.nih.gov/gap":http://www.ncbi.nlm.nih.gov/gap). Selected fields of this data have been identified by NIH staff as ‘too sensitive’ and are not available in dbGAP. Individuals requiring access to these data fields are asked to contact the PI of this project or NIH Program Staff. 
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A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.https://doi.org/10.1186/1750-1172-8-10

Phylometagenomics: a new framework for uncovering microbial community diversity

Microbial communities are recognized as major drivers of global biogeochemical processes. However, the genetic diversity and composition, as well as processes leading to the origin and diversification of these communities in space and time, are poorly understood. Character- ization of microbial communities using high-throughput sequencing of 16S tags shows that Operational Taxonomic Unit (OTU) abundances can be approximated by a gamma distribu- tion, which suggests structuring around small numbers of highly abundant OTUs and a large proportion of rare OTUs. The current methods used to characterize how communities are structured rely on multivariate statistics, which operate on pair-wise distance matrices. My analyses demonstrate that use of these methods, by reducing a highly-dimensional data set (tens of samples, thousands of OTUs), results in a significant loss of information. I demon- strate that, in some cases, up to 80% of the least abundant OTUs may be removed while still recovering the same community relationships; this indicates these metrics are biased toward the highly abundant OTUs. I also demonstrate that the observed patterns of OTU abundance detected from microbial communities can be robustly modeled using techniques similar to those used to model the presence and absence of genes in genome evolution. Using simulation studies, I show that general Markov models in a Bayesian inference framework out- perform traditional, multivariate ecological methods in recovering true community structure. Applying this new methodology to Atlantic Ocean communities uncovered a distance-decay effect which was not revealed by the traditional methods; applying to communities discov- ered on Hog Island point toward mechanisms of thicket establishment. Although the ocean data set operated on a much larger, continental scale, characterization of the sequence data generated from the nutrient-poor soil on Hog Island, a barrier island off the Virginia coast, allows for a better characterization of the processes affecting these communities on a much smaller scale. Finally, using 16S data from the Human Vaginal Microbiome Project, gener- ated here at VCU under the umbrella of the overall NIH HMP initiative, I give examples of the quality control, analysis and visualization pipeline that I developed to support the efforts of this project. In conclusion, my analyses of the metagenomic sequence data from bacterial communities sampled from different environments demonstrate that the proper identification of the biological processes influencing these communities requires the development and im- plementation of new statistical and computational methodologies that take advantage of the extensive amount of information generated in next-generation, high-throughput sequencing projects

Data from: Evolutionary genomics of gypsy moth populations sampled along a latitudinal gradient

The European gypsy moth (Lymantria dispar L.) was first introduced to Massachusetts in 1869 and within 150 years has spread throughout eastern North America. This large-scale invasion across a heterogeneous landscape allows examination of the genetic signatures of adaptation potentially associated with rapid geographic spread. We tested the hypothesis that spatially divergent natural selection has driven observed changes in three developmental traits that were measured in a common garden for 165 adult moths sampled from six populations across a latitudinal gradient covering the entirety of the range. We generated genotype data for 91,468 single nucleotide polymorphisms (SNPs) based on double digest restriction-site associated DNA sequencing (ddRADseq) and used these data to discover genome-wide associations for each trait, as well as to test for signatures of selection on the discovered architectures. Genetic structure across the introduced range of gypsy moth was small in magnitude (FST = 0.069), with signatures of bottlenecks and spatial expansion apparent in the rare portion of the allele frequency spectrum. Results from applications of Bayesian sparse linear mixed models were consistent with the presumed polygenic architectures of each trait. Further analyses were indicative of spatially divergent natural selection acting on larval development time and pupal mass, with the linkage disequilibrium like component of this test acting as the main driver of observed patterns. The populations most important for these signals were two range-edge populations established less than 30 generations ago. We discuss the importance of rapid polygenic adaptation to the ability of non-native species to invade novel environments

Data from: The role of hybridization during ecological divergence of southwestern white pine (Pinus strobiformis) and limber pine (P. flexilis)

Interactions between extrinsic factors, such as disruptive selection, and intrinsic factors, such as genetic incompatibilities among loci, often contribute towards the maintenance of species boundaries. The relative roles of these factors in the establishment of reproductive isolation can be examined using species pairs characterized by gene flow throughout their divergence history. We investigated the process of speciation and the maintenance of species boundaries between Pinus strobiformis and P. flexilis. Utilizing ecological niche modeling, demographic modeling, and genomic cline analyses, we illustrated a divergence history with continuous gene flow. Our results supported an abundance of advanced generation hybrids and a lack of loci exhibiting steep transition in allele frequency across the hybrid zone. Additionally, we found evidence for climate-associated variation in the hybrid index and niche divergence between parental species and the hybrid zone. These results are consistent with extrinsic factors, such as climate, being an important isolating mechanism. A buildup of intrinsic incompatibilities and of co-adapted gene complexes is also apparent, although these appear to be in the earliest stages of development. This supports previous work in coniferous species demonstrating the importance of extrinsic factors in facilitating speciation. Overall, our findings lend support to the hypothesis that varying strengths and directions of selection pressures across the long lifespans of conifers, in combination with their life history strategies, delay the evolution of strong intrinsic incompatibilities

Population genomics supports speciation with gene flow, not genomic islands of differentiation, in sky-island populations of southwestern white pine

This file contains background, inferences and hypothesis testing results, and conclusions based on demographic modeling analyses conducted on ddRAD-seq SNP data from southwestern white pine (<i>Pinus strobiformis</i>) core and periphery (hybrid zone) lineages, plus limber pine (<i>P. flexilis</i>), presented in the format of a scientific poster. The first author (JCB) recently presented this poster to colleagues at the Evolution 2017 Meeting held from June 23-27 in Portland, OR. Results shown here form the demographic modeling portion of a broader integrative project studying ecological speciation and the influence of hybridization in maintaining species boundaries in the southwestern white pine–limber pine study system using i) species distribution modeling and niche divergence tests, ii) demographic modeling (statistical phylogeography), and iii) Bayesian genomic cline analysis.<br