Failure to predict and understand the causes of preterm birth, the leading cause of neonatal morbidity and mortality, have limited effective interventions and therapeutics. From a cohort of 2000 pregnant women, we performed a nested case control study on 107 well-phenotyped cases of spontaneous preterm birth (sPTB) and 432 women delivering at term. Using innovative Bayesian modeling of cervicovaginal microbiota, seven bacterial taxa were significantly associated with increased risk of sPTB, with a stronger effect in African American women. However, higher vaginal levels of β-defensin-2 lowered the risk of sPTB associated with cervicovaginal microbiota in an ethnicity-dependent manner. Surprisingly, even in Lactobacillus spp. dominated cervicovaginal microbiota, low β-defensin-2 was associated with increased risk of sPTB. These findings hold promise for diagnostics to accurately identify women at risk for sPTB early in pregnancy. Therapeutic strategies could include immune modulators and microbiome-based therapeutics to reduce this significant health burden.

Background Taxonomic profiles of vaginal microbial communities can be sorted into a discrete number of categories termed community state types (CSTs). This approach is advantageous because collapsing a hyper-dimensional taxonomic profile into a single categorical variable enables efforts such as data exploration, epidemiological studies, and statistical modeling. Vaginal communities are typically assigned to CSTs based on the results of hierarchical clustering of the pairwise distances between samples. However, this approach is problematic because it complicates between-study comparisons and because the results are entirely dependent on the particular set of samples that were analyzed. We sought to standardize and advance the assignment of samples to CSTs. Results We developed VALENCIA (VAginaL community state typE Nearest CentroId clAssifier), a nearest centroid-based tool which classifies samples based on their similarity to a set of reference centroids. The references were defined using a comprehensive set of 13,160 taxonomic profiles from 1975 women in the USA. This large dataset allowed us to comprehensively identify, define, and characterize vaginal CSTs common to reproductive age women and expand upon the CSTs that had been defined in previous studies. We validated the broad applicability of VALENCIA for the classification of vaginal microbial communities by using it to classify three test datasets which included reproductive age eastern and southern African women, adolescent girls, and a racially/ethnically and geographically diverse sample of postmenopausal women. VALENCIA performed well on all three datasets despite the substantial variations in sequencing strategies and bioinformatics pipelines, indicating its broad application to vaginal microbiota. We further describe the relationships between community characteristics (vaginal pH, Nugent score) and participant demographics (race, age) and the CSTs defined by VALENCIA. Conclusion VALENCIA provides a much-needed solution for the robust and reproducible assignment of vaginal community state types. This will allow unbiased analysis of both small and large vaginal microbiota datasets, comparisons between datasets and meta-analyses that combine multiple datasets.

Analysis of metagenomic and metatranscriptomic data is complicated and typically requires extensive computational resources. Leveraging a curated reference database of genes encoded by members of the target microbiome can make these analyses more tractable. In this study, we assemble a comprehensive human vaginal non-redundant gene catalog (VIRGO) that includes 0.95 million non-redundant genes. The gene catalog is functionally and taxonomically annotated. We also construct a vaginal orthologous groups (VOG) from VIRGO. The gene-centric design of VIRGO and VOG provides an easily accessible tool to comprehensively characterize the structure and function of vaginal metagenome and metatranscriptome datasets. To highlight the utility of VIRGO, we analyze 1,507 additional vaginal metagenomes, and identify a high degree of intraspecies diversity within and across vaginal microbiota. VIRGO offers a convenient reference database and toolkit that will facilitate a more in-depth understanding of the role of vaginal microorganisms in women's health and reproductive outcomes.

Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.

This study describes the community structure of the microbial eukaryotic community from hypersaline Lake Tyrrell, Australia, using near full length 18S rRNA sequences. Water samples were taken in both summer and winter over a 4-year period. The extent of eukaryotic diversity detected was low, with only 35 unique phylotypes using a 97% sequence similarity threshold. The water samples were dominated (91%) by a novel cluster of the Alveolate, Apicomplexa Colpodella spp., most closely related to C. edax. The Chlorophyte, Dunaliella spp. accounted for less than 35% of water column samples. However, the eukaryotic community entrained in a salt crust sample was vastly different and was dominated (83%) by the Dunaliella spp. The patterns described here represent the first observation of microbial eukaryotic dynamics in this system and provide a multiyear comparison of community composition by season. The lack of expected seasonal distribution in eukaryotic communities paired with abundant nanoflagellates suggests that grazing may significantly structure microbial eukaryotic communities in this system.