Congzhou Liu

Temporal Variability of Human Vaginal Bacteria and Relationship with Bacterial Vaginosis

Background Little is known about short-term bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal bacteria in healthy women and the immediate response to antibiotic treatment in women with bacterial vaginosis (BV). Methodology/Principal Findings Twenty-two women assessed for BV using Amsels criteria were evaluated daily for 7 or 14 days, then at 2, 3 and 4 weeks, using a panel of 11 bacterium-specific quantitative PCR assays. Participants with BV were treated with 5 days of intravaginal metronidazole. Participants without BV had vaginal biotas dominated by lactobacilli, whose levels fluctuated with menses. With onset of menstruation, quantities of Lactobacillus jensenii and Lactobacillus crispatus decreased and were found to be inversely related to Gardnerella vaginalis concentrations (p<0.001). Women with BV had a variety of fastidious bacteria whose concentrations dropped below detection thresholds 1–5 days after starting metronidazole. Recurrent BV was characterized by initial profound decreases of BV-associated bacteria after treatment followed by subsequent increases at relapse. Conclusions/Significance The microbiota of the human vagina can be highly dynamic. Healthy women are colonized with Lactobacillus species, but levels can change dramatically over a month. Marked increases in G. vaginalis were observed during menses. Participants with BV have diverse communities of fastidious bacteria that are depleted by vaginal metronidazole therapy. Women with recurrent BV initially respond to antibiotic treatment with steep declines in bacterial concentrations, but these bacteria later reemerge, suggesting that antibiotic resistance in these bacteria is not an important factor mediating BV recurrence.

Extravaginal Reservoirs of Vaginal Bacteria as Risk Factors for Incident Bacterial Vaginosis

BACKGROUND Bacterial vaginosis (BV) represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. Although antibiotics relieve symptoms and temporarily eradicate BV-associated bacteria (BVAB), BV usually recurs. We investigated the role of extravaginal BVAB reservoirs in recurrence. METHODS Risks for BV acquisition over the course of 1 year were defined. DNA in vaginal, anal, and oral swab samples from enrollment was subjected to quantitative polymerase chain reaction assays targeting 16S ribosomal RNA genes of Gardnerella vaginalis, Lactobacillus crispatus, BVAB1, BVAB2, BVAB3, Megasphaera spp., Lactobacillus jensenii, and Leptotrichia/Sneathia spp. A case-control approach analyzed BVAB detection at enrollment for case patients (BV acquisition) versus controls (none). RESULTS Of 239 women enrolled without BV, 199 were seen in follow-up, and 40 experienced BV; 15 had all samples for analysis. Detection of G. vaginalis in oral cavity or anal samples and Leptotrichia/Sneathia spp. in anal samples was more common at enrollment among case patients, who also had higher concentrations of these bacteria and Megasphaera relative to 30 controls at each site. In contrast, L. crispatus was detected more frequently in anal samples among controls. CONCLUSIONS Women who acquire BV are more likely have previous colonization of extravaginal reservoirs with some BVAB, and less likely to have L. crispatus, suggesting that BVAB may be acquired vaginally from extravaginal reservoirs.

More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the “Bacteroides morphotype,” whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.

Early Pregnancy Changes in Bacterial Vaginosis-associated Bacteria and Preterm Delivery

BACKGROUND We evaluated the importance of measuring early vaginal levels of eight bacterial vaginosis (BV)-associated bacteria, at two points in pregnancy, and the risk of spontaneous preterm delivery (SPTD) among pregnant women and the subgroup of pregnant women with a history of preterm delivery (PTD). METHODS This prospective cohort study enrolled women at five urban obstetric practices at Temple University Hospital in Philadelphia PA. Women with singleton pregnancies less than 16 weeks gestation self-collected vaginal swabs at two points in pregnancy, prior to 16 weeks gestation and between 20-24 weeks gestation, to measure the presence and level of eight BV-associated bacteria. Women were followed-up for gestational age at delivery via medical records. RESULTS Among women reporting a prior PTD, women with higher levels of Leptotrichia/Sneathia species, BVAB1 and Mobiluncus spp., prior to 16 weeks gestation, were significantly more likely to experience a SPTD. In addition, pregnant women with a prior PTD and increasing levels of Leptotrichia/Sneathia species (aOR: 9.1, 95% CI 1.9, 42.9), BVAB1 (aOR: 16.4, 95% CI 4.3, 62.7) or Megasphaera phylotype 1 (aOR: 6.2, 95% CI 1.9, 20.6), through 24 weeks gestation, were significantly more likely to experience an SPTD. Among the overall group of pregnant women, the levels of BV-associated bacteria were not related to SPTD. CONCLUSION Among the group of women reporting a prior PTD, increasing levels of BVAB1, Leptotrichia/Sneathia species, and Megasphaera phylotype 1, through mid-pregnancy were related to an increased risk of SPTD.

Phase I Randomized Safety Study of Twice Daily Dosing of Acidform Vaginal Gel: Candidate Antimicrobial Contraceptive

Background Acidform gel, an acid-buffering product that inactivates spermatozoa, may be an effective topical non-hormonal contraceptive. This study was designed to evaluate the safety of vaginal dosing and effects of Acidform on mucosal immune mediators, antimicrobial properties of genital secretions, and vaginal microbiota. Methods Thirty-six sexually abstinent U.S. women were randomized to apply Acidform or hydroxyethylcellulose (HEC) placebo gel twice daily for 14 consecutive days. Safety was assessed by symptoms and pelvic examination. The impact of gel on mucosal immunity was assessed by quantifying cytokines, chemokines, antimicrobial proteins and antimicrobial activity of genital secretions collected by cervicovaginal lavage (CVL) at screening, 2 hours after gel application, and on days 7, 14 and 21. Vaginal microbiota was characterized at enrollment and day 14 using species-specific quantitative PCR assays. Results The median vaginal and cervical pH was significantly lower 2 hours after application of Acidform and was associated with an increase in the bactericidal activity of CVL against E. coli. However, 65% of women who received Acidform had at least one local adverse event compared with 11% who received placebo (p = 0.002). While there was no increase in inflammatory cytokines or chemokines, CVL concentrations of lactoferrin and interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory protein, were significantly lower following Acidform compared to HEC placebo gel application. There were no significant changes in Lactobacillus crispatus or Lactobacillus jensenii in either group but there was a decrease in Gardnerella vaginalis in the Acidform group (p = 0.08). Conclusions Acidform gel may augment mucosal defense as evidenced by an increase in bactericidal activity of genital secretions against E. coli and a decrease in Gardnerella vaginalis colonization. However, Acidform was associated with more irritation than placebo and lower levels of antimicrobial (lactoferrin) and anti-inflammatory (IL-1ra) proteins. These findings indicate the need for additional safety studies of this candidate non-hormonal contraceptive. Trial Registration ClinicalTrials.gov NCT00850837

Associations between vaginal bacteria and levels of vaginal defensins in pregnant women

OBJECTIVE We evaluated vaginal defensin concentrations and levels of bacterial vaginosis-associated bacterial species in pregnant women. STUDY DESIGN Self-collected vaginal swabs from 2 visits during pregnancy were tested with quantitative polymerase chain reaction for 9 bacterial species. Beta defensins 2-3 and alpha defensins 1-3 were measured by enzyme-linked immunosorbent assay. RESULTS Our 126 participants were primarily African American (60%), had a mean gestational age at enrollment of 10 ± 3 weeks and at follow-up visit of 25 ± 6 weeks. At enrollment, the prevalence of bacterial vaginosis was 74% (94/126 women), which decreased to 60% (75/126 specimens) at follow-up visit. At enrollment, beta defensin 3 concentrations were significantly lower in women with bacterial vaginosis (2.64 ± 0.91 vs 3.25 ± 0.99 log(10) pg/mL; P = .003). Higher concentrations of Atopobium vaginae, bacterial vaginosis-associated bacteria1 and 2 were associated with significantly lower concentrations of beta defensin 3 (P < .01). CONCLUSION Bacterial vaginosis was associated with lower vaginal concentrations of beta defensin 3, but not beta defensin 2 or alpha defensins 1-3, in pregnant women.

Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women

BackgroundThe presence of hydrogen peroxide (H2O2) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H2O2-producers.MethodsSamples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR.ResultsCulture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45).ConclusionsAmong HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.

Stool Microbiota at Neutrophil Recovery Is Predictive for Severe Acute Graft vs Host Disease After Hematopoietic Cell Transplantation

Low alpha diversity in stool microbiota at neutrophil recovery associates with increased severe graft-versus-host disease (GVHD), greater abundance of oral Actinobacteria or Firmicutes, and decreased abundance of intestinal Lachnospiraceae in stool microbiota at neutrophil recovery associated with increased severe GVHD.

Vaginal microbiota and genitourinary menopausal symptoms: a cross-sectional analysis

Objective: To examine associations between the composition of the vaginal microbiota and genitourinary menopausal symptoms, serum estrogen, and vaginal glycogen. Methods: For this cross-sectional study, 88 women aged 40 to 62 years, enrolled in a hot flash treatment trial, provided vaginal swabs and a blood sample at enrollment. Bacterial communities were characterized using 16S rRNA PCR and deep sequencing targeting the V3-V4 region. Quantities of Lactobacillus crispatus and Lactobacillus iners were measured using qPCR. Self-reported genitourinary symptoms included: presence and severity of individual symptoms and identification of most bothersome symptom. Glycogen was measured fluorometrically in swab eluate. Serum estradiol (E2) and estrone (E1) were measured by liquid chromatography/mass spectrometry. Associations between bacteria, symptoms, glycogen, and serum estrogens were tested by linear regression or Wilcoxon signed-rank test, adjusted for multiple comparisons. Comparisons between groups used Kruskall-Wallis or Fishers exact test. Results: Of the 88 women, 33 (38%) had a majority of Lactobacillus species, whereas 58 (66%) had any Lactobacillus detected. Over half (53%) reported at least one vulvovaginal symptom (most commonly dryness), but symptoms were not associated with the presence of Lactobacillus species. Women with Lactobacillus-dominant communities had higher unconjugated serum estrone, but no difference in vaginal glycogen levels, compared with those with non-Lactobacillus-dominant communities. Higher serum E2 and E1 were not associated with either higher vaginal glycogen or detection of individual genera. Conclusions: Presence of Lactobacillus-dominant vaginal microbiota was not associated with fewer vulvovaginal symptoms. Serum estrone was higher in women with Lactobacillus dominance, but vaginal-free glycogen was not associated with composition of the vaginal microbiota.

Associations between improvement in genitourinary symptoms of menopause and changes in the vaginal ecosystem

Objective: The aim of the study was to identify associations between improvement in genitourinary symptoms of menopause (GSM) and vaginal microbiota, vaginal glycogen, and serum estrogen. Methods: Thirty postmenopausal women enrolled in a hot flash treatment trial (oral estradiol vs venlafaxine vs placebo) who reported GSM and provided vaginal swabs at 0, 4, and 8 weeks were studied. Bacterial communities were characterized using deep sequencing targeting the 16S rRNA gene V3-V4 region. Participants selected a most bothersome genitourinary symptom (dryness, discharge, pain, itch/burn, or inability to have sex) and rated severity on a 10-point scale at baseline and 8 weeks. Vaginal glycogen and serum estradiol and estrone were measured at enrollment and 8 weeks. Comparisons according to improvement in most bothersome symptom (MBS) were made using &khgr;2, Wilcoxon signed-rank test, or Hotellings t test. Results: Of 30 participants, 21 (70%) had improvement in MBS over the 8-week study and 9 (30%) had no improvement or worsening of MBS. A higher proportion of women receiving estradiol or venlafaxine reported improvement in MBS (88%, 78%) compared with placebo (54%; P = 0.28). MBS improvement was associated with Lactobacillus-dominant vaginal microbiota at enrollment (57% vs 22%, P = 0.08). Vaginal glycogen, serum estradiol, and estrone significantly increased in women whose MBS improved. Conclusions: A larger proportion of women whose MBS improved had a Lactobacillus dominant microbiota at enrollment than those who had no improvement during the trial, though this difference was not statistically significant. Larger trials are needed to determine whether vaginal microbiota modify or mediate treatment responses in women with GSM.