Impact of vaginal microbiome communities on HIV antiretroviral-based pre-exposure prophylaxis (PrEP) drug metabolism

Despite the efficacy of antiretroviral-based pre-exposure prophylactics (PrEP) in men who have sex with men, studies in women have produced widely varying outcomes. Recent evidence demonstrates that vaginal microbial communities are associated with increased HIV acquisition risk and may impact PrEP efficacy. Here, we investigate the mechanisms underlying how vaginal bacteria alter PrEP drug levels and impact HIV infection rates ex vivo. Using cervicovaginal lavages (CVLs) from women with or without bacterial vaginosis (BV), we identified microbial metabolism of PrEP drugs in BV samples through LC-MS/MS analysis of soluble drug levels and metabolite formation in dual T-cell cultures. CVL samples were assessed for microbiome analysis using sequencing of bacterial 16S rRNA genes. We also observed non-Lactobacillus bacteria that are associated with BV may potentially impact PrEP efficacy through increased HIV infection rates in co-cultures containing Lactobacillus or BV bacteria, PrEP drugs, CEM-GFP cells, and HIV-1_LAI virus. Finally, we used these data to develop a novel predictive mathematical simulation modeling system to predict these drug interactions for future trials. These studies demonstrate how dysbiotic vaginal microbiota may impact PrEP drugs and provides evidence linking vaginal bacteria to PrEP efficacy in women.     

Intestinal Microbiota is Different in Women with Preterm Birth: Results from Terminal Restriction Fragment Length Polymorphism Analysis

Preterm birth is a leading cause of perinatal morbidity and mortality. Studies using a cultivation method or molecular identification have shown that bacterial vaginosis is one of the risk factors for preterm birth. However, an association between preterm birth and intestinal microbiota has not been reported using molecular techniques, although the vaginal microbiota changes during pregnancy. Our aim here was to clarify the difference in intestinal and vaginal microbiota between women with preterm birth and women without preterm labor. 16S ribosomal ribonucleic acid genes were amplified from fecal and vaginal DNA by polymerase chain reaction. Using terminal restriction fragment length polymorphism (T-RFLP), we compared the levels of operational taxonomic units of both intestinal and vaginal flora among three groups: pregnant women who delivered term babies without preterm labor (non-PTL group) (n = 20), those who had preterm labor but delivered term babies (PTL group) (n = 11), and those who had preterm birth (PTB group) (n = 10). Significantly low levels of Clostridium subcluster XVIII, Clostridium cluster IV, Clostridium subcluster XIVa, and Bacteroides, and a significantly high level of Lactobacillales were observed in the intestinal microbiota in the PTB group compared with those in the non-PTL group. The levels of Clostridium subcluster XVIII and Clostridium subcluster XIVa in the PTB group were significantly lower than those in the PTL group, and these levels in the PTL group were significantly lower than those in non-PTL group. However, there were no significant differences in vaginal microbiota among the three groups. Intestinal microbiota in the PTB group was found to differ from that in the non-PTL group using the T-RFLP method.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Lactobacilli Inactivate Chlamydia trachomatis through Lactic Acid but Not H2O2

Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H₂O₂, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H₂O₂ concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H₂O₂ production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H₂O₂, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection.     

Vaginal microbiome-hormonal contraceptive interactions associate with the mucosal proteome and HIV acquisition

Alterations to the mucosal environment of the female genital tract, such as genital inflammation, have been associated with increased HIV acquisition in women. As the microbiome and hormonal contraceptives can affect vaginal mucosal immunity, we hypothesized these components may interact in the context of HIV susceptibility. Using previously published microbiome data from 685 women in the CAPRISA-004 trial, we compared relative risk of HIV acquisition in this cohort who were using injectable depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (NET-EN), and combined oral contraceptives (COC). In women who were Lactobacillus-dominant, HIV acquisition was 3-fold higher in women using DMPA relative to women using NET-EN or COC (OR: 3.27; 95% CI: 1.24–11.24, P = 0.0305). This was not observed in non-Lactobacillus-dominant women (OR: 0.95, 95% CI: 0.44–2.15, P = 0.895) (interaction P = 0.0686). Higher serum MPA levels associated with increased molecular pathways of inflammation in the vaginal mucosal fluid of Lactobacillus-dominant women, but no differences were seen in non-Lactobacillus dominant women. This study provides data suggesting an interaction between the microbiome, hormonal contraceptives, and HIV susceptibility.     

A gastrointestinal nematode in pregnant and lactating mice alters maternal and neonatal microbiomes

The maternal microbiome is understood to be the principal source of the neonatal microbiome but the consequences of intestinal nematodes on pregnant and lactating mothers and implications for the neonatal microbiome are unknown. Using pregnant CD1 mice infected with Heligmosomoides bakeri, we investigated the microbiomes in maternal tissues (intestine, vagina, and milk) and in the neonatal stomach using MiSeq sequencing of bacterial 16S rRNA genes. Our first hypothesis was that maternal nematode infection altered the maternal intestinal, vaginal, and milk microbiomes and associated metabolic pathways. Maternal nematode infection was associated with increased beta-diversity and abundance of fermenting bacteria as well as Lactobacillus in the maternal caecum 2 days after parturition, together with down-regulated carbohydrate, amino acid and vitamin biosynthesis pathways. Maternal nematode infection did not alter the vaginal or milk microbiomes. Our second hypothesis was that maternal infection would shape colonization of the neonatal microbiome. Although the pup stomach microbiome was similar to that of the maternal vaginal microbiome, pups of infected dams had higher beta-diversity at day 2, and a dramatic expansion in the abundance of Lactobacillus between days 2 and 7 compared with pups nursing uninfected dams. Our third hypothesis that maternal nematode infection altered the composition of neonatal microbiomes was confirmed as we observed up-regulation of several putatively beneficial microbial pathways associated with synthesis of essential and branched-chain amino acids, vitamins, and short-chain fatty acids. We believe this is the first study to show that a nematode living in the maternal intestine is associated with altered composition and function of the neonatal microbiome.     

Characteristics of Vaginal Microbiome in Women with Pelvic Inflammatory Disease in Korea

Human vaginal microorganisms play an important role in maintaining good health throughout the human life cycle. An imbalance in the vaginal microbiota is associated with an increased risk of pelvic inflammatory disease (PID). This study aimed to characterize and compare vaginal microbial profiles of premenopausal Korean women with and without PID. 74 Korean premenopausal female vaginal samples were obtained; 33 were from healthy women (a control group) and 41 from PID patients. Vaginal fluid samples were collected from the vaginal wall and posterior cervix and then analyzed by 16S ribosomal ribonucleic acid (rRNA) gene-based amplicon sequencing. Results showed a significant difference between the vaginal microbial communities of the two groups (Jensen-Shannon, p = 0.014; Bray-Curtis, p = 0.009; Generalized UniFrac, p = 0.007; UniFrac, p = 0.008). Lactobacillus accounted for the highest percentage (61.0%) of the control group but was significantly decreased (34.9%) in PID patients; this was the most significant difference among all bacterial communities (p = 0.028, LDA effect size = 5.129). In addition, in the PID patient group, species diversity significantly increased (Simpson, p = 0.07) as the proportion of various pathogens increased evenly, resulting in a polymicrobial infection. Similarly, lactate, which constituted the highest percentage of the organic acids in the control group, was significantly decreased in the PID patient group (p = 0.04). The present study’s findings will help understand PID from the microbiome perspective and are expected to contribute to the development of more efficient PID diagnosis and treatment modalities.     

Life History Recorded in the Vagino-cervical Microbiome Along with Multi-omes

The vagina contains at least a billion microbial cells, dominated by lactobacilli. Here we perform metagenomic shotgun sequencing on cervical and fecal samples from a cohort of 516 Chinese women of reproductive age, as well as cervical, fecal, and salivary samples from a second cohort of 632 women. Factors such as pregnancy history, delivery history, cesarean section, and breastfeeding were all more important than menstrual cycle in shaping the microbiome, and such information would be necessary before trying to interpret differences between vagino-cervical microbiome data. Greater proportion of Bifidobacterium breve was seen with older age at sexual debut. The relative abundance of lactobacilli especially Lactobacillus crispatus was negatively associated with pregnancy history. Potential markers for lack of menstrual regularity, heavy flow, dysmenorrhea, and contraceptives were also identified. Lactobacilli were rare during breastfeeding or post-menopause. Other features such as mood fluctuations and facial speckles could potentially be predicted from the vagino-cervical microbiome. Gut and salivary microbiomes, plasma vitamins, metals, amino acids, and hormones showed associations with the vagino-cervical microbiome. Our results offer an unprecedented glimpse into the microbiota of the female reproductive tract and call for international collaborations to better understand its long-term health impact other than in the settings of infection or pre-term birth.     

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay

Background

Signature matching of nucleotide sequences in the V1 and V3 regions 16S rRNA genes using pyrosequencing technology is a powerful tool for typing vaginal Lactobacilli to the species level and has been used for investigating the vaginal microbial niche.

Methods

This study has characterized the normal cultivable vaginal Lactobacillus flora at varying estradiol levels in plasma; the study comprised 17 patients undergoing ovarian stimulation for In Vitro Fertilization (IVF) treatment. The vaginal status of each participant was initially assessed as normal according to Amsel and Nugent criteria.

Results

L. crispatus, L. gasseri and/or L. jensenii were present in 10 of the patients throughout the study period, and little variation among these three species was encountered in individual patients. The flora of three women was dominated by L. delbrüeckii, L. rhamnosus or L. vaginalis. One woman exhibited a dominance of L. iners. The flora of the remaining three women were initially dominated by L. rhamnosus or L. reuteri, but as their estrogen levels rose, their flora composition altered, to become dominated by one of the three species most common in a normal, healthy vagina.

Conclusion

Signature matching of nucleotide sequences in the V1 and V3 regions of 16S rRNA genes is a discriminative tool for the study of vaginal Lactobacilli and can be used to track the Lactobacillus flora under a variety of physiological conditions.     

Correlation of Gut Microbiome Between ASD Children and Mothers and Potential Biomarkers for Risk Assessment

Variation of maternal gut microbiota may increase the risk of autism spectrum disorders(ASDs) in offspring. Animal studies have indicated that maternal gut microbiota is related to neurodevelopmental abnormalities in mouse offspring, while it is unclear whether there is a correlation between gut microbiota of ASD children and their mothers. We examined the relationships between gut microbiome profiles of ASD children and those of their mothers, and evaluated the clinical discriminatory power of discovered bacterial biomarkers. Gut microbiome was profiled and evaluated by 16S ribosomal RNA gene sequencing in stool samples of 59 mother–child pairs of ASD children and 30 matched mother–child pairs of healthy children. Significant differences were observed in the gut microbiome composition between ASD and healthy children in our Chinese cohort. Several unique bacterial biomarkers, such as Alcaligenaceae and Acinetobacter, were identified. Mothers of ASD children had more Proteobacteria, Alphaproteobacteria, Moraxellaceae, and Acinetobacter than mothers of healthy children. There was a clear correlation between gut microbiome profiles of children and their mothers; however, children with ASD still had unique bacterial biomarkers, such as Alcaligenaceae, Enterobacteriaceae, and Clostridium. Candidate biomarkers discovered in this study had remarkable discriminatory power. The identified patterns of mother–child gut microbiome profiles may be important for assessing risks during the early stage and planning of personalized treatment and prevention of ASD via microbiota modulation.     

Focus: Microbiome: Unraveling the Dynamics of the Human Vaginal Microbiome

Four Lactobacillus species, namely L. crispatus, L. iners, L. gasseri, and L. jensenii, commonly dominate the vaginal communities of most reproductive-age women. It is unclear why these particular species, and not others, are so prevalent. Historically, estrogen-induced glycogen production by the vaginal epithelium has been proffered as being key to supporting the proliferation of vaginal lactobacilli. However, the ‘fly in the ointment’ (that has been largely ignored) is that the species of Lactobacillus commonly found in the human vagina cannot directly metabolize glycogen. It would appear that this riddle has been solved as studies have demonstrated that vaginal lactobacilli can metabolize the products of glycogen depolymerization by α-amylase, and fortunately, amylase activity is found in vaginal secretions. These amylases are presumed to be host-derived, but we suggest that other bacterial populations in vaginal communities could also be sources of amylase in addition to (or instead of) the host. Here we briefly review what is known about human vaginal bacterial communities and discuss how glycogen-derived resources and resource competition might shape the composition and structure of these communities.     

Diverse Vaginal Microbiomes in Reproductive-Age Women with Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is one of the most prevalent vaginal infectious diseases, and there are controversial reports regarding the diversity of the associated vaginal microbiota. We determined the vaginal microbial community in patients with VVC, bacterial vaginosis (BV), and mixed infection of VVC and BV using Illumina sequencing of 16S rRNA tags. Our results revealed for the first time the highly variable patterns of the vaginal microbiome from VVC patients. In general, the alpha-diversity results of species richness and evenness showed the following order: normal control < VVC only < mixed BV and VVC infection < BV only. The beta-diversity comparison of community structures also showed an intermediate composition of VVC between the control and BV samples. A detailed comparison showed that, although the control and BV communities had typical patterns, the vaginal microbiota of VVC is complex. The mixed BV and VVC infection group showed a unique pattern, with a relatively higher abundance of Lactobacillus than the BV group and higher abundance of Prevotella, Gardnerella, and Atopobium than the normal control. In contrast, the VVC-only group could not be described by any single profile, ranging from a community structure similar to the normal control (predominated with Lactobacillus) to BV-like community structures (abundant with Gardnerella and Atopobium). Treatment of VVC resulted in inconsistent changes of the vaginal microbiota, with four BV/VVC samples recovering to a higher Lactobacillus level, whereas many VVC-only patients did not. These results will be useful for future studies on the role of vaginal microbiota in VVC and related infectious diseases.     

Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.     

Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective

Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.

Materials and Methods

Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.

Results

Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.

Conclusions

Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.     

Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing Lactobacillus rhamnosus DR20

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 × 10⁹ lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10² cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.     

女性生殖系统微生物群落及其与早产相关性研究进展

女性生殖系统微生物群落与女性生殖健康密切相关.阴道微生物的菌群变化伴随着多种阴道疾病的发生并可能通过宫颈感染子宫,孕期子宫内的炎症反应会导致胎儿早产(Preterm Birth,PTB),PTB被定义为妊娠满28周但不足37周所发生的分娩现象,PTB婴儿死亡率很高并面临着多种健康疾病,除了明确病原微生物的感染,PTB还...     

The Vaginal Microbiota over an 8- to 10-Year Period in a Cohort of HIV-Infected and HIV-Uninfected Women

BackgroundWe identified predominant vaginal microbiota communities, changes over time, and how this varied by HIV status and other factors in a cohort of 64 women.MethodsBacterial 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.ResultsThe 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.ConclusionsThe 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.     

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.