Jennifer M. Fettweis

The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies

BackgroundCharacterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits.ResultsWe observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions.ConclusionsBias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.

Differences in vaginal microbiome in African American women versus women of European ancestry.

Women of European ancestry are more likely to harbour a Lactobacillus-dominated microbiome, whereas African American women are more likely to exhibit a diverse microbial profile. African American women are also twice as likely to be diagnosed with bacterial vaginosis and are twice as likely to experience preterm birth. The objective of this study was to further characterize and contrast the vaginal microbial profiles in African American versus European ancestry women. Through the Vaginal Human Microbiome Project at Virginia Commonwealth University, 16S rRNA gene sequence analysis was used to compare the microbiomes of vaginal samples from 1268 African American women and 416 women of European ancestry. The results confirmed significant differences in the vaginal microbiomes of the two groups and identified several taxa relevant to these differences. Major community types were dominated by Gardnerella vaginalis and the uncultivated bacterial vaginosis-associated bacterium-1 (BVAB1) that were common among African Americans. Moreover, the prevalence of multiple bacterial taxa that are associated with microbial invasion of the amniotic cavity and preterm birth, including Mycoplasma, Gardnerella, Prevotella and Sneathia, differed between the two ethnic groups. We investigated the contributions of intrinsic and extrinsic factors, including pregnancy, body mass index, diet, smoking and alcohol use, number of sexual partners, and household income, to vaginal community composition. Ethnicity, pregnancy and alcohol use correlated significantly with the relative abundance of bacterial vaginosis-associated species. Trends between microbial profiles and smoking and number of sexual partners were observed; however, these associations were not statistically significant. These results support and extend previous findings that there are significant differences in the vaginal microbiome related to ethnicity and demonstrate that these differences are pronounced even in healthy women.

Species-level classification of the vaginal microbiome.

BackgroundThe application of next-generation sequencing to the study of the vaginal microbiome is revealing the spectrum of microbial communities that inhabit the human vagina. High-resolution identification of bacterial taxa, minimally to the species level, is necessary to fully understand the association of the vaginal microbiome with bacterial vaginosis, sexually transmitted infections, pregnancy complications, menopause, and other physiological and infectious conditions. However, most current taxonomic assignment strategies based on metagenomic 16S rDNA sequence analysis provide at best a genus-level resolution. While surveys of 16S rRNA gene sequences are common in microbiome studies, few well-curated, body-site-specific reference databases of 16S rRNA gene sequences are available, and no such resource is available for vaginal microbiome studies.ResultsWe constructed the Vaginal 16S rDNA Reference Database, a comprehensive and non-redundant database of 16S rDNA reference sequences for bacterial taxa likely to be associated with vaginal health, and we developed STIRRUPS, a new method that employs the USEARCH algorithm with a curated reference database for rapid species-level classification of 16S rDNA partial sequences. The method was applied to two datasets of V1-V3 16S rDNA reads: one generated from a mock community containing DNA from six bacterial strains associated with vaginal health, and a second generated from over 1,000 mid-vaginal samples collected as part of the Vaginal Human Microbiome Project at Virginia Commonwealth University. In both datasets, STIRRUPS, used in conjunction with the Vaginal 16S rDNA Reference Database, classified more than 95% of processed reads to a species-level taxon using a 97% global identity threshold for assignment.ConclusionsThis database and method provide accurate species-level classifications of metagenomic 16S rDNA sequence reads that will be useful for analysis and comparison of microbiome profiles from vaginal samples. STIRRUPS can be used to classify 16S rDNA sequence reads from other ecological niches if an appropriate reference database of 16S rDNA sequences is available.

A New Era of the Vaginal Microbiome: Advances Using Next-Generation Sequencing

Until recently, bacterial species that inhabit the human vagina have been primarily studied using organism‐centric approaches. Understanding how these bacterial species interact with each other and the host vaginal epithelium is essential for a more complete understanding of vaginal health. Molecular approaches have already led to the identification of uncultivated bacterial taxa associated with bacterial vaginosis. Here, we review recent studies of the vaginal microbiome and discuss how culture‐independent approaches, such as applications of next‐generation sequencing, are advancing the field and shifting our understanding of how vaginal health is defined. This work may lead to improved diagnostic tools and treatments for women who suffer from, or are at risk for, vaginal imbalances, pregnancy complications, and sexually acquired infections. These approaches may also transform our understanding of how host genetic factors, physiological conditions (e.g., menopause), and environmental exposures (e.g., smoking, antibiotic usage) influence the vaginal microbiome.

Identification of a gene in Mycoplasma hominis associated with preterm birth and microbial burden in intraamniotic infection

OBJECTIVE Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta. STUDY DESIGN We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M hominis. RESULTS We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were truncated severely in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was associated significantly with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor. CONCLUSION A gene in M hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth.

The Changing Landscape of the Vaginal Microbiome

Deep sequence analysis of the vaginal microbiome is revealing an unexpected complexity that was not anticipated as recently as several years ago. The lack of clarity in the definition of a healthy vaginal microbiome, much less an unhealthy vaginal microbiome, underscores the need for more investigation of these phenomena. Some clarity may be gained by the careful analysis of the genomes of the specific bacteria in these women. Ongoing studies will clarify this process and offer relief for women with recurring vaginal maladies and hope for pregnant women to avoid the experience of preterm birth.

An Emerging Mycoplasma Associated with Trichomoniasis, Vaginal Infection and Disease

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as “Mnola.” In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name “Candidatus Mycoplasma girerdii” for this potential new pathogen.

Effects of combined oral contraceptives, depot medroxyprogesterone acetate and the levonorgestrel-releasing intrauterine system on the vaginal microbiome

OBJECTIVES Prior studies suggest that the composition of the vaginal microbiome may positively or negatively affect susceptibility to sexually transmitted infections (STIs) and bacterial vaginosis (BV). Some female hormonal contraceptive methods also appear to positively or negatively influence STI transmission and BV. Therefore, changes in the vaginal microbiome that are associated with different contraceptive methods may explain, in part, effects on STI transmission and BV. STUDY DESIGN We performed a retrospective study of 16S rRNA gene survey data of vaginal samples from a subset of participants from the Human Vaginal Microbiome Project at Virginia Commonwealth University. The subset included 682 women who reported using a single form of birth control that was condoms, combined oral contraceptives (COCs), depot medroxyprogesterone acetate (DMPA) or the levonorgestrel-releasing intrauterine system (LNG-IUS). RESULTS Women using COCs [adjusted odds ratio (aOR) 0.29, 95% confidence interval (CI) 0.13-0.64] and DMPA (aOR 0.34, 95% CI 0.13-0.89), but not LNG-IUS (aOR 1.55, 95% CI 0.72-3.35), were less likely to be colonized by BV-associated bacteria relative to women who used condoms. Women using COCs (aOR 1.94, 95% CI 1.25-3.02) were more likely to be colonized by beneficial H2O2-producing Lactobacillus species compared with women using condoms, while women using DMPA (aOR 1.09, 95% CI 0.63-1.86) and LNG-IUS (aOR 0.74, 95% CI 0.48-1.15) were not. CONCLUSIONS Use of COCs is significantly associated with increased vaginal colonization by healthy lactobacilli and reduced BV-associated taxa. IMPLICATIONS COC use may positively influence gynecologic health through an increase in healthy lactobacilli and a decrease in BV-associated bacterial taxa.

Skin-to-Skin Care and the Development of the Preterm Infant Oral Microbiome

OBJECTIVE The oral cavity represents an initial entry way for oral and gut indigenous colonization. Skin-to-skin (STS) care, in which the mother holds the diaper clad naked preterm (PT) infant between her breasts, is associated with improved digestive function, decreased stress, and improved survival. This study evaluated the development of oral microbial colonization repertoires and health characteristics in PT infants with or without STS exposure. METHODS Saliva from 42 PT infants (<32 weeks of gestation at birth) was collected prospectively at 1 month and/or at discharge. High-throughput 16S rRNA sequencing identified microbial diversity and prevalence of bacterial signatures correlated with clinical STS or non-STS care. RESULTS Corrected for gestational age (CGA) at sampling, bacterial taxa demonstrated increased Streptococcus as a signature of oral repertoire maturation. STS was associated with increased Streptococcus (p < 0.024), while non-STS was associated with greater Corynebacterium (p < 0.023) and Pseudomonas (p < 0.019) in infants ≤ 32 weeks CGA. In infants > 32 weeks CGA, Neisseria and Acinetobacter were more prevalent, 50 vs. 16.7% and 40 vs. 0%, respectively. STS care was associated with shorter hospitalization (p < 0.039). CONCLUSION STS care during earlier gestation was associated with a distinct microbial pattern and an accelerated pace of oral microbial repertoire maturity.

Changes in vaginal community state types reflect major shifts in the microbiome

ABSTRACT Background: Recent studies of various human microbiome habitats have revealed thousands of bacterial species and the existence of large variation in communities of microorganisms in the same habitats across individual human subjects. Previous efforts to summarize this diversity, notably in the human gut and vagina, have categorized microbiome profiles by clustering them into community state types (CSTs). The functional relevance of specific CSTs has not been established. Objective: We investigate whether CSTs can be used to assess dynamics in the microbiome. Design: We conduct a re-analysis of five sequencing-based microbiome surveys derived from vaginal samples with repeated measures. Results: We observe that detection of a CST transition is largely insensitive to choices in methods for normalization or clustering. We find that healthy subjects persist in a CST for two to three weeks or more on average, while those with evidence of dysbiosis tend to change more often. Changes in CST can be gradual or occur over less than one day. Upcoming CST changes and switches to high-risk CSTs can be predicted with high accuracy in certain scenarios. Finally, we observe that presence of Gardnerella vaginalis is a strong predictor of an upcoming CST change. Conclusion: Overall, our results show that the CST concept is useful for studying microbiome dynamics.