[Show abstract][Hide abstract] ABSTRACT: In this study, we evaluated the association between high-risk human papillomavirus (hrHPV) and the vaginal microbiome. Participants were recruited in Nigeria between April and August 2012. Vaginal bacterial composition was characterized by deep sequencing of barcoded 16S rRNA gene fragments (V4) on Illumina MiSeq and HPV was identified using the Roche Linear Array® HPV genotyping test. We used exact logistic regression models to evaluate the association between community state types (CSTs) of vaginal microbiota and hrHPV infection, weighted UniFrac distances to compare the vaginal microbiota of individuals with prevalent hrHPV to those without prevalent hrHPV infection, and the Linear Discriminant Analysis effect size (LEfSe) algorithm to characterize bacteria associated with prevalent hrHPV infection. We observed four CSTs: CST IV-B with a low relative abundance of Lactobacillus spp. in 50% of participants; CST III (dominated by L. iners) in 39·2%; CST I (dominated by L. crispatus) in 7·9%; and CST VI (dominated by proteobacteria) in 2·9% of participants. LEfSe analysis suggested an association between prevalent hrHPV infection and a decreased abundance of Lactobacillus sp. with increased abundance of anaerobes particularly of the genera Prevotella and Leptotrichia in HIV-negative women (P < 0·05). These results are hypothesis generating and further studies are required.
Epidemiology and Infection 06/2015; -1:1-15. DOI:10.1017/S0950268815000965 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
We identified predominant vaginal microbiota communities, changes over time, and how this varied by HIV status and other factors in a cohort of 64 women.
Bacterial DNA was extracted from reposited cervicovaginal lavage samples collected annually over an 8–10 year period from Chicago Women’s Interagency HIV Study participants: 22 HIV-negative, 22 HIV-positive with stable infection, 20 HIV-positive with progressive infection. The vaginal microbiota was defined by pyrosequencing of the V1/V2 region of the 16S rRNA gene. Scheduled visits included Bacterial vaginsosis (BV) screening; clinically detected cases were referred for treatment. Hierarchical clustering identified bacterial community state types (CST). Multinomial mixed effects modeling determined trends over time in CST, by HIV status and other factors.
The median follow-up time was 8.1 years (range 5.5–15.3). Six CSTs were identified. The mean relative abundance (RA) of Lactobacillus spp. by CST (with median number of bacterial taxa) was: CST-1–25.7% (10), CST-2–27.1% (11), CST-3–34.6% (9), CST-4–46.8% (9), CST-5–57.9% (4), CST-6–69.4% (2). The two CSTs representing the highest RA of Lactobacillus and lowest diversity increased with each additional year of follow-up (CST-5, adjusted odds ratio (aOR) = 1.62 [95% CI: 1.34–1.94]; CST-6, aOR = 1.57 [95 CI: 1.31–1.89]), while the two CSTs representing lowest RA of Lactobacillus and higher diversity decreased with each additional year (CST-1, aOR = 0.89 [95% CI: 0.80–1.00]; CST-2, aOR = 0.86 [95% CI: 0.75–0.99]). There was no association between HIV status and CST at baseline or over time. CSTs representing lower RA of Lactobacillus were associated with current cigarette smoking.
The vaginal microbial community significantly improved over time in this cohort of women with HIV and at high risk for HIV who had regular detection and treatment referral for BV.
PLoS ONE 02/2015; 10(2):e0116894. DOI:10.1371/journal.pone.0116894 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Intravaginal practices (IVP) are common among African women and are associated with HIV acquisition. A behavioral intervention to reduce IVP is a potential new HIV risk-reduction strategy. Fifty-eight HIV-1-uninfected Kenyan women reporting IVP and 42 women who denied IVP were followed for 3 months. Women using IVP attended a skill-building, theory-based group intervention occurring weekly for 3 weeks to encourage IVP cessation. Vaginal swabs at each visit were used to detect yeast, to detect bacterial vaginosis, and to characterize the vaginal microbiota. Intravaginal insertion of soapy water (59%) and lemon juice (45%) was most common among 58 IVP women. The group-counseling intervention led to a decrease in IVP from 95% (54/58) at baseline to 0% (0/39) at month 3 (p=0.001). After 3 months of cessation, there was a reduction in yeast on vaginal wet preparation (22% to 7%, p=0.011). Women in the IVP group were more likely to have a Lactobacillus iners-dominated vaginal microbiota at baseline compared to controls [odds ratio (OR), 6.4, p=0.006] without significant change in the microbiota after IVP cessation. The group counseling intervention was effective in reducing IVP for 3 months. Reducing IVP may be important in itself, as well as to support effective use of vaginal microbicides, to prevent HIV acquisition.
AIDS Research and Human Retroviruses 09/2014; 30(11). DOI:10.1089/aid.2013.0251 · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Smoking has been identified in observational studies as a risk factor for bacterial vaginosis (BV), a condition defined in part by decimation of Lactobacillus spp. The anti-estrogenic effect of smoking and trace amounts of benzo[a]pyrene diol epoxide (BPDE) may predispose women to BV. BPDE increases bacteriophage induction in Lactobacillus spp. and is found in the vaginal secretions of smokers. We compared the vaginal microbiota between smokers and non-smokers and followed microbiota changes in a smoking cessation pilot study.
In 2010–2011, 20 smokers and 20 non-smokers were recruited to a cross-sectional study (Phase A) and 9 smokers were enrolled and followed for a 12-week smoking cessation program (Phase B). Phase B included weekly behavioral counseling and nicotine patches to encourage smoking cessation. In both phases, participants self-collected mid-vaginal swabs (daily, Phase B) and completed behavioral surveys. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes (V1-V3 regions). Vaginal smears were assigned Nugent Gram stain scores. Smoking status was evaluated (weekly, Phase B) using the semi-quantitative NicAlert® saliva cotinine test and carbon monoxide (CO) exhalation.
In phase A, there was a significant trend for increasing saliva cotinine and CO exhalation with elevated Nugent scores (P value <0.005). Vaginal microbiota clustered into three community state types (CSTs); two dominated by Lactobacillus (L. iners, L. crispatus), and one lacking significant numbers of Lactobacillus spp. and characterized by anaerobes (termed CST-IV). Women who were observed in the low-Lactobacillus CST-IV state were 25-fold more likely to be smokers than those dominated by L. crispatus (aOR: 25.61, 95 % CI: 1.03-636.61). Four women completed Phase B. One of three who entered smoking cessation with high Nugent scores demonstrated a switch from CST-IV to a L.iners-dominated profile with a concomitant drop in Nugent scores which coincided with completion of nicotine patches. The other two women fluctuated between CST-IV and L. iners-dominated CSTs. The fourth woman had low Nugent scores with L. crispatus-dominated CSTs throughout.
Smokers had a lower proportion of vaginal Lactobacillus spp. compared to non-smokers. Smoking cessation should be investigated as an adjunct to reducing recurrent BV. Larger studies are needed to confirm these findings.
[Show abstract][Hide abstract] ABSTRACT: Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.
PLoS ONE 07/2014; 9(7):e102651. DOI:10.1371/journal.pone.0102651 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
We sought to describe the temporal relationship between vaginal microbiota and human papillomavirus (HPV) detection.
Thirty-two reproductive-age women self-collected midvaginal swabs twice weekly for 16 weeks (937 samples). Vaginal bacterial communities were characterized by pyrosequencing of barcoded 16S rRNA genes and clustered into 6 community state types (CSTs). Each swab was tested for 37 HPV types. The effects of CSTs on the rate of transition between HPV-negative and HPV-positive states were assessed using continuous-time Markov models.
Participants had an average of 29 samples, with HPV point prevalence between 58%-77%. CST was associated with changes in HPV status (P<.001). Lactobacillus gasseri-dominated CSTs had the fastest HPV remission rate, and a low Lactobacillus community with high proportions of the genera Atopobium (CST IV-B) had the slowest rate compared to L. crispatus-dominated CSTs (adjusted transition rate ratio [aTRR], 4.43, 95% confidence interval [CI], 1.11-17.7; aTRR, 0.33, 95% CI, .12-1.19, respectively). The rate ratio of incident HPV for low Lactobacillus CST IV-A was 1.86 (95% CI, .52-6.74).
Vaginal microbiota dominated by L. gasseri was associated with increased clearance of detectable HPV. Frequent longitudinal sampling is necessary for evaluation of the association between HPV detection and dynamic microbiota.
The Journal of Infectious Diseases 06/2014; 210(11). DOI:10.1093/infdis/jiu330 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
This study was undertaken to determine whether the vaginal microbiota of pregnant women who subsequently had a spontaneous preterm delivery is different from that of women who had a term delivery.
This was a nested case–control study of pregnant women who had a term delivery (controls) and those who had a spontaneous preterm delivery before 34 weeks of gestation (cases). Samples of vaginal fluid were collected longitudinally and stored at −70°C until assayed. A microbial survey using pyrosequencing of V1-V3 regions of 16S rRNA genes was performed. We tested the hypothesis of whether the relative abundance of individual microbial species (phylotypes) was different between women who had a term versus preterm delivery. A suite of bioinformatic and statistical tools, including linear mixed effects models and generalized estimating equations, was used. We show that: 1) the composition of the vaginal microbiota during normal pregnancy changed as a function of gestational age, with an increase in the relative abundance of four Lactobacillus spp., and decreased in anaerobe or strict-anaerobe microbial species as pregnancy progressed; 2) no bacterial taxa differed in relative abundance between women who had a spontaneous preterm delivery and those who delivered at term; and 3) no differences in the frequency of the vaginal community state types (CST I, III, IV-B) between women who delivered at term and those who delivered preterm were detected.
The bacterial taxa composition and abundance of vaginal microbial communities, characterized with 16S rRNA gene sequence-based techniques, were not different in pregnant women who subsequently delivered a preterm neonate versus those who delivered at term.
[Show abstract][Hide abstract] ABSTRACT: To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality.
Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp "heterogeneity spacer" to the index sequence that allows an equal proportion of samples to be sequenced out of phase.
Our approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.
[Show abstract][Hide abstract] ABSTRACT: This study was undertaken to characterize the vaginal microbiota throughout normal human pregnancy using sequence-based techniques. We compared the vaginal microbial composition of non-pregnant patients with a group of pregnant women who delivered at term.
A retrospective case-control longitudinal study was designed and included non-pregnant women (n = 32) and pregnant women who delivered at term (38 to 42 weeks) without complications (n = 22). Serial samples of vaginal fluid were collected from both non-pregnant and pregnant subjects. A 16S rRNA gene sequence-based survey was conducted using pyrosequencing to characterize the structure and stability of the vaginal microbiota. Linear mixed effects models and generalized estimating equations were used to identify the phylotypes whose relative abundance was different between the two study groups. The vaginal microbiota of normal pregnant women was different from that of non-pregnant women (higher abundance of Lactobacillus vaginalis, L. crispatus, L. gasseri and L. jensenii and lower abundance of 22 other phylotypes in pregnant women). Bacterial community state type (CST) IV-B or CST IV-A characterized by high relative abundance of species of genus Atopobium as well as the presence of Prevotella, Sneathia, Gardnerella, Ruminococcaceae, Parvimonas, Mobiluncus and other taxa previously shown to be associated with bacterial vaginosis were less frequent in normal pregnancy. The stability of the vaginal microbiota of pregnant women was higher than that of non-pregnant women; however, during normal pregnancy, bacterial communities do shift but almost exclusively from one CST dominated by Lactobacillus spp. to another CST dominated by Lactobacillus spp.
We report the first longitudinal study of the vaginal microbiota in normal pregnancy. Differences in the composition and stability of the microbial community between pregnant and non-pregnant women were observed. Lactobacillus spp. were the predominant members of the microbial community in normal pregnancy. These results can serve as the basis to study the relationship between the vaginal microbiome and adverse pregnancy outcomes.
[Show abstract][Hide abstract] ABSTRACT: Bacterial vaginosis (BV) is a common gynecologic diagnosis characterized by dysbiosis of the vaginal microbiota. It is often accompanied by vaginal symptoms such as odor and discharge, but can be asymptomatic. Despite over 50 years of research, the etiology of BV is not well understood, which is a major impediment to treatment and prevention of BV.
Here we report on the temporal dynamics of 25 vaginal communities over a 10 week period using samples collected daily from women who were diagnosed with symptomatic BV (15 women), asymptomatic BV (6 women), and women who did not have BV (4 women).
This unique resource of samples and data will contribute to a better understanding of the role that the vaginal microbes have in the natural history of BV and lead to improved diagnosis and treatment.
[Show abstract][Hide abstract] ABSTRACT: The vaginal microbiota helps protect the female genital tract from disease. We sought to describe the composition of the vaginal microbiota in premenopausal, perimenopausal, and postmenopausal women and to explore the association between the microbiota and vulvovaginal atrophy (VVA).
Eighty-seven women (aged 35-60 y) were classified as premenopausal (n = 30), perimenopausal (n = 29), or postmenopausal (n = 28) according to Stages of Reproductive Aging Workshop guidelines. Midvaginal bacterial community composition was characterized by 16S ribosomal RNA gene analysis.
Bacterial communities clustered into six community state types (CSTs), of which four were dominated by Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, or Lactobacillus jensenii, and two (CST IV-A and CST IV-B) had low relative abundance of Lactobacillus. CST IV-A was characterized by Streptococcus and Prevotella, whereas CST IV-B was characterized by Atopobium. There were significant associations between menopause stage and CST (P = 0.004) and between VVA and CST (P = 0.002). Perimenopausal women were more likely to be classified as CST IV-A or L. gasseri CST, whereas postmenopausal women were often classified as CST IV-A. CSTs dominated by L. crispatus and L. iners were more prevalent in premenopausal women. Nineteen participants had signs of mild or moderate VVA. Compared with women with no VVA, the vaginal microbiota of women with mild or moderate atrophy had 25-fold greater odds of being classified as CST IV-A versus L. crispatus CST (adjusted odds ratio, 25.89; 95% credible interval, 2.98-406.79).
A distinct bacterial community state (CST IV-A) with a low relative abundance of Lactobacillus is associated with VVA. Future studies recruiting a larger number of women are needed to replicate the findings. This study provides an impetus for future longitudinal studies designed to manage, modulate, and restore vaginal microbiota homeostasis, which would provide stronger evidence for a causal relationship with VVA and ultimately improve the treatment and prevention of atrophic vaginitis in menopause.
Menopause (New York, N.Y.) 09/2013; 21(5). DOI:10.1097/GME.0b013e3182a4690b · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background Chlamydia trachomatis (CT) genital infection is one of the most widespread sexually transmitted infections (STIs). It is thought that certain types of vaginal microbiota are better able to prevent STIs, however, very little is known about the relationships between species and genomic composition of the vaginal microbiome and risk of infection. Information about these can be used to advance prevention strategies for STIs. We sought to characterise the vaginal microbiome at the time of diagnosis and after treatment of CT infection.
Methods 101 women with CT genital infection were recruited to a longitudinal study in Baltimore, MD. Participants were treated at diagnosis and returned for clinical visits every 3 months for 9 months, at which times the vaginal microbiota was determined using 16S rRNA analysis. We further applied high-throughput metagenomics to characterise the genomic makeup of the vaginal microbiome.
Results The average age of participants was 19.3 (range 14–28) and 92% were African-American. During CT infection, comprehensive surveys of the vaginal microbiota demonstrated two distinct community state types characterised by: (1) a complex assemblage of strict anaerobes with low proportions of Lactobacillus spp. (60% of samples) or (2) a high relative abundance of Lactobacillus iners-dominated communities (37% of samples) . Metagenomic evaluation suggested unique characteristics of the genomes of the dominant species in these communities (i.e., L. iners, Gardnerella vaginalis). L. iners appeared more genetically diverse than other Lactobacillus species.
Conclusions Complex communities of strict anaerobes with low proportions of Lactobacillus or specific L. iners genome types represented a hallmark of the chlamydia -infected state in this population. Some L. iners may be better suited to adapt to diverse environments while others may be contributing factors to an at-risk microbiome. In the era of personalised medicine, future work will enhance our ability to intervene and establish a protective vaginal microbiome.
[Show abstract][Hide abstract] ABSTRACT: Background:
High systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation.
Fifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM), and their sum (total estrogens) were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, β-glucuronidase and β-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences), high] and compared to loge estrogens, β-glucuronidase and β-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with α=0.05.
In men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM) were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R≥0.50, P≤0.003). These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57-0.70, P=0.03-0.002). Estrone, but not other EM, in urine correlated significantly with functional activity of fecal β-glucuronidase (R=0.36, P=0.04). In contrast, fecal β-glucuronidase correlated inversely with fecal total estrogens, both conjugated and deconjugated (R≤-0.47, P≤0.01). Premenopausal female estrogen levels, which were collected across menstrual cycles and thus highly variable, were completely unrelated to fecal microbiome and enzyme parameters (P≥0.6).
Intestinal microbial richness and functions, including but not limited to β-glucuronidase, influence levels of non-ovarian estrogens via enterohepatic circulation. Thus, the gut microbial community likely affects the risk for estrogen-related conditions in older adults. Understanding how Clostridia taxa relate to systemic estrogens may identify targets for interventions.
Journal of Translational Medicine 12/2012; 10(1):253. DOI:10.1186/1479-5876-10-253 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Vaginal HIV microbicides offer great promise in preventing HIV transmission, but failures of phase 3 clinical trials, in which microbicide-treated subjects had an increased risk of HIV transmission, raised concerns about endpoints used to evaluate microbicide safety. A possible explanation for the increased transmission risk is that the agents shifted the vaginal bacterial community, resulting in loss of natural protection and enhanced HIV transmission susceptibility. We characterized vaginal microbiota, using pyrosequencing of bar-coded 16S rRNA gene fragments, in samples from 35 healthy, sexually abstinent female volunteer subjects (ages 18 to 50 years) with regular menses in a repeat phase 1 study of twice-daily application over 13.5 days of 1 of 3 gel products: a hydroxyethylcellulose (HEC)-based "universal" placebo (10 subjects), 6% cellulose sulfate (CS; 13 subjects), and 4% nonoxynol-9 (N-9; 12 subjects). We used mixed effects models inferred using Bayesian Markov chain Monte Carlo methods, which showed that treatment with active agents shifted the microbiota toward a community type lacking significant numbers of Lactobacillus spp. and dominated by strict anaerobes. This state of the vaginal microbiota was associated with a low or intermediate Nugent score and was not identical to bacterial vaginosis, an HIV transmission risk factor. The placebo arm contained a higher proportion of communities dominated by Lactobacillus spp., particularly L. crispatus, throughout treatment. The data suggest that molecular evaluation of microbicide effects on vaginal microbiota may be a critical endpoint that should be incorporated in early clinical assessment of microbicide candidates.
Despite large prevention efforts, HIV transmission and acquisition rates remain unacceptably high. In developing countries, transmission mainly occurs through heterosexual intercourse, where women are significantly more vulnerable to infection than men. Vaginal microbicides are considered to be one of the most promising female-controlled products, in that women themselves insert the microbicides into the vagina to prevent HIV transmission during sexual intercourse. The failure of several microbicides in clinical trials has raised questions concerning the low in vivo efficacy of such anti-HIV molecules. This study was designed to gain insights into the failures of two microbicides by testing the hypothesis that the microbicides negatively affect a critical line of defense against HIV, the vaginal microbiota. The results suggest that in the early assessment of candidate microbicides, culture-independent evaluation of their effect on the vaginal microbiota should be considered and may constitute a critical endpoint.