Poster Abstracts - S618
Articles
Hormonal Contraception is Associated with Lactobacillus iners Dominated Cervicovaginal Microbiota in Reproductive-Age Black South African Women
Identification: Onywera, Harris
Credits: None available.
Hormonal Contraception is Associated with Lactobacillus iners-Dominated Cervicovaginal Microbiota in Reproductive-Age Black South African Women
Harris Onywera1,2, Anna-Lise Williamson1,2,3, Zizipho Z. A. Mbulawa1,2,3,4, David Coetzee5, Tracy L. Meiring1,2*
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 2Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, South Africa; 3UCT-MRC Clinical Gynaecological Cancer Research Centre, University of Cape Town, Cape Town, South Africa; 4Center for HIV & STIs, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa; 5Center for Infectious Disease Epidemiology and Research, School of Public Health and Family Medicine, University of Cape Town, South Africa
*Corresponding author
Cervicovaginal microbiotas (CVMs) have a profound influence on the women's reproductive health. A CVM dominated by Lactobacillus spp. is thought to be a biomarker for cervicovaginal health. Complex and undesirable imbalances in the CVMs predisposes women to bacterial vaginosis (BV), the most common vaginal syndrome among reproductive-age women. BV has been associated with infertility and increased susceptibility to infections such as genital human papillomavirus (HPV), which is causally associated with cervical cancer. Despite the high health burden of HPV in Africa and evidence that a majority of women of African ancestry lack Lactobacillus-dominated CVMs, the CVMs of African women remain understudied. Here, the CVMs of Black South African women with and without HPV were characterized and associated with the participants' metadata. The CVMs of 62 reproductive-age women were profiled from cervical DNA by using Ion Torrent sequences from the V4 hypervariable region 16S rRNA gene. The CVMs were analyzed using QIIME, UPARSE, and metagenomeSeq tools. Associations of CVMs with participants' categorical and continuous variables were computed by Chi-square/Fisher's exact and Kruskal-Wallis tests, respectively. Twenty three women (37.1%) were HPV-positive. Twenty five women (40.3%) were on hormonal contraception. The CVMs clustered into three discrete community state types (CSTs): CST I (n=24, 38.7%) and CST II (n=3, 4.8%) that were dominated by Lactobacillus iners and an unclassified Lactobacillus species, correspondingly; and CST III (n=35, 56.5%) that was enriched with an array of heterogeneous BV-associated bacterial taxa, predominantly Gardnerella, Prevotella, Sneathia, and Shuttleworthia. CST III was associated with BV (p=0.001). Neither CST nor bacterial diversity was associated with HPV infection. Women in CST I were more likely to be on hormonal contraception, especially progestin-based, compared to women in CST III (odds ratio: 5.2 [95% CI 1.6-17.2]; p=0.006). The majority of the women had CVMs not dominated by Lactobacillus. Additional studies are needed to examine whether these CVMs represent abnormal, intermediate or variant states of health. Our findings on the association of hormonal contraception with L. iners-dominated CVMs warrants further investigation and may have implications on personalized microbiota-based diagnostics and probiotics that promote reproductive health.
Harris Onywera1,2, Anna-Lise Williamson1,2,3, Zizipho Z. A. Mbulawa1,2,3,4, David Coetzee5, Tracy L. Meiring1,2*
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 2Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, South Africa; 3UCT-MRC Clinical Gynaecological Cancer Research Centre, University of Cape Town, Cape Town, South Africa; 4Center for HIV & STIs, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa; 5Center for Infectious Disease Epidemiology and Research, School of Public Health and Family Medicine, University of Cape Town, South Africa
*Corresponding author
Cervicovaginal microbiotas (CVMs) have a profound influence on the women's reproductive health. A CVM dominated by Lactobacillus spp. is thought to be a biomarker for cervicovaginal health. Complex and undesirable imbalances in the CVMs predisposes women to bacterial vaginosis (BV), the most common vaginal syndrome among reproductive-age women. BV has been associated with infertility and increased susceptibility to infections such as genital human papillomavirus (HPV), which is causally associated with cervical cancer. Despite the high health burden of HPV in Africa and evidence that a majority of women of African ancestry lack Lactobacillus-dominated CVMs, the CVMs of African women remain understudied. Here, the CVMs of Black South African women with and without HPV were characterized and associated with the participants' metadata. The CVMs of 62 reproductive-age women were profiled from cervical DNA by using Ion Torrent sequences from the V4 hypervariable region 16S rRNA gene. The CVMs were analyzed using QIIME, UPARSE, and metagenomeSeq tools. Associations of CVMs with participants' categorical and continuous variables were computed by Chi-square/Fisher's exact and Kruskal-Wallis tests, respectively. Twenty three women (37.1%) were HPV-positive. Twenty five women (40.3%) were on hormonal contraception. The CVMs clustered into three discrete community state types (CSTs): CST I (n=24, 38.7%) and CST II (n=3, 4.8%) that were dominated by Lactobacillus iners and an unclassified Lactobacillus species, correspondingly; and CST III (n=35, 56.5%) that was enriched with an array of heterogeneous BV-associated bacterial taxa, predominantly Gardnerella, Prevotella, Sneathia, and Shuttleworthia. CST III was associated with BV (p=0.001). Neither CST nor bacterial diversity was associated with HPV infection. Women in CST I were more likely to be on hormonal contraception, especially progestin-based, compared to women in CST III (odds ratio: 5.2 [95% CI 1.6-17.2]; p=0.006). The majority of the women had CVMs not dominated by Lactobacillus. Additional studies are needed to examine whether these CVMs represent abnormal, intermediate or variant states of health. Our findings on the association of hormonal contraception with L. iners-dominated CVMs warrants further investigation and may have implications on personalized microbiota-based diagnostics and probiotics that promote reproductive health.
Sexual behaviours impact the vaginal microbiota of women who have sex with women
Identification: Plummer, Erica
Credits: None available.
Sexual behaviours impact the vaginal microbiota of women who have sex with women
Plummer EL1, Vodstrcil LA1,2, Murray GL3, Tabrizi SN3, Garland SM3, Fairley CK1, Tan A3, Law M4, Hocking JS2, Bulach DM5, Philip G5, Bradshaw CS1,2
1Central Clinical School, Monash University; Melbourne Sexual Health Centre, Alfred Health; 2Melbourne School of Population & Global Health, The University of Melbourne; 3The Royal Women's Hospital; 4Kirby Institute, UNSW Australia; 5Melbourne Bioinformatics, The University of Melbourne
We investigated the impact of sexual behaviors on the vaginal microbiota (VM) of women-who-have-sex-with-women (WSW) participating in a 2 year cohort study. Women self-collected high vaginal swabs and completed a behavioral questionnaire every 3 months for 24 months or until incident bacterial vaginosis (BV). We characterized the VM using 16S-rRNA gene sequencing of the V3V4 region. Community state types (CSTs) were identified using hierarchical clustering. Bacterial diversity was calculated using the Shannon diversity index and instability of the VM was assessed using change of CST and Bray-Curtis dissimilarity between consecutive longitudinal specimens. The impact of behaviors on diversity and instability of the VM was determined using multivariate regression models. Linear discriminant analysis effect size was used to identify bacteria associated with exposure to a new sexual partner. 360 specimens from 100 women were included in analyses. The VM clustered into 5 CSTs: 3 dominated by Lactobacillus spp. (CST1 L. crispatus; CST2 L. crispatus and L. iners; CST3 L. iners), one abundant in Gardnerella vaginalis and one of mixed bacteria. Exposure to a new sexual partner increased bacterial diversity (Adj coef=0.33,95%CI:0.11,0.54) and instability of the VM, both in terms of change of CST (AOR=2.69,95%CI:1.37,5.28) and increased Bray-Curtis dissimilarity (Adj coef=0.22,95%CI:0.12,0.32). Sex with a new partner increased the abundance of bacteria often seen in BV including G. vaginalis, Megasphaera and BVAB1 (p<0.05). Conversely, no sex/sex in established ongoing relationships was associated with a favorable vaginal microbiota abundant in L. crispatus. Sex with a new partner markedly reshapes the VM of WSW by increasing the diversity and abundance of potentially pathogenic bacteria.
Funding: National Health and Medical Research Council (NHMRC) Program Grant #1071269 and NHMRC Project Grant #1020457
Plummer EL1, Vodstrcil LA1,2, Murray GL3, Tabrizi SN3, Garland SM3, Fairley CK1, Tan A3, Law M4, Hocking JS2, Bulach DM5, Philip G5, Bradshaw CS1,2
1Central Clinical School, Monash University; Melbourne Sexual Health Centre, Alfred Health; 2Melbourne School of Population & Global Health, The University of Melbourne; 3The Royal Women's Hospital; 4Kirby Institute, UNSW Australia; 5Melbourne Bioinformatics, The University of Melbourne
We investigated the impact of sexual behaviors on the vaginal microbiota (VM) of women-who-have-sex-with-women (WSW) participating in a 2 year cohort study. Women self-collected high vaginal swabs and completed a behavioral questionnaire every 3 months for 24 months or until incident bacterial vaginosis (BV). We characterized the VM using 16S-rRNA gene sequencing of the V3V4 region. Community state types (CSTs) were identified using hierarchical clustering. Bacterial diversity was calculated using the Shannon diversity index and instability of the VM was assessed using change of CST and Bray-Curtis dissimilarity between consecutive longitudinal specimens. The impact of behaviors on diversity and instability of the VM was determined using multivariate regression models. Linear discriminant analysis effect size was used to identify bacteria associated with exposure to a new sexual partner. 360 specimens from 100 women were included in analyses. The VM clustered into 5 CSTs: 3 dominated by Lactobacillus spp. (CST1 L. crispatus; CST2 L. crispatus and L. iners; CST3 L. iners), one abundant in Gardnerella vaginalis and one of mixed bacteria. Exposure to a new sexual partner increased bacterial diversity (Adj coef=0.33,95%CI:0.11,0.54) and instability of the VM, both in terms of change of CST (AOR=2.69,95%CI:1.37,5.28) and increased Bray-Curtis dissimilarity (Adj coef=0.22,95%CI:0.12,0.32). Sex with a new partner increased the abundance of bacteria often seen in BV including G. vaginalis, Megasphaera and BVAB1 (p<0.05). Conversely, no sex/sex in established ongoing relationships was associated with a favorable vaginal microbiota abundant in L. crispatus. Sex with a new partner markedly reshapes the VM of WSW by increasing the diversity and abundance of potentially pathogenic bacteria.
Funding: National Health and Medical Research Council (NHMRC) Program Grant #1071269 and NHMRC Project Grant #1020457
Vaginal Microbiome of premenopausal Indian women: A comparison of healthy and dysbiotic flora
Identification: Pramanick, Rinku
Credits: None available.
Vaginal Microbiome of premenopausal Indian women: A comparison of healthy and dysbiotic flora
Rinku Pramanick1, Niranjan Mayadeo2, Himangi Warke2 and Clara Aranha1
1 ICMR-National Institute for Research in Reproductive Health, Parel, Mumbai
2 Department of Obstetrics and Gynecology, Seth G.S. Medical College & KEM Hospital, Parel, Mumbai
Presenting author email: rinku.pramanick@gmail.com
Corresponding author email: aranhac@nirrh.res.in
The vaginal microbiome plays a critical role in determining the progression of female genital tract infections. Since the vaginal microbiome varies with geography and ethnicities, deciphering the lacking information on Indian women is needed. We aimed to decode the vaginal microbial architecture of women with healthy and dysbiotic flora. To achieve this, we sequenced 16S rRNA V3-V4 region (HiSeq Illumina) of DNA extracted from vaginal swabs collected from women with bacterial vaginosis (BV) (n=10) and healthy controls (n=10) of 18-45yrs. The abundance profile of 20 samples had 876 OTUs from 33 families and 45 genera. Rarefaction analysis showed higher number of species in normal flora compared to BV. Alpha diversity as measured by Shannon diversity revealed normal flora had less diverse communities compared to BV. Beta diversity comparison using Bray Curtis indicated distinct microbial communities between normal and BV flora. The most abundant Phylum that showed significant difference between normal and BV samples were Firmicutes (p=0.0004) and Actinobacteria (p=0.009) where Firmicutes were abundant in normal flora. A significant difference was observed for relative proportions of Lactobacillus, Gardnerella, Aerococcus, Brevibacterium between the groups. Lactobacillus abundance decreased significantly in BV flora (14.57%), as compared to normal flora (83.25%) whereas the abundance of Bifidobacterium was observed increased from 12.0% in normal flora to 45.25% in BV. Besides, increased levels of Klebsiella, Sneathia, Coriobacteriaceae and Prevotella was noted in BV. L.crispatus was exclusively present in normal flora whereas L.iners was detected from both the groups with 80% and 100% prevalence in normal and BV flora respectively.
The study provides insights into the vaginal microbiome structure of Indian women that will enable us to unravel the microbial biomarkers and explore the prospective candidates for restoring the vaginal microbiota.
Rinku Pramanick1, Niranjan Mayadeo2, Himangi Warke2 and Clara Aranha1
1 ICMR-National Institute for Research in Reproductive Health, Parel, Mumbai
2 Department of Obstetrics and Gynecology, Seth G.S. Medical College & KEM Hospital, Parel, Mumbai
Presenting author email: rinku.pramanick@gmail.com
Corresponding author email: aranhac@nirrh.res.in
The vaginal microbiome plays a critical role in determining the progression of female genital tract infections. Since the vaginal microbiome varies with geography and ethnicities, deciphering the lacking information on Indian women is needed. We aimed to decode the vaginal microbial architecture of women with healthy and dysbiotic flora. To achieve this, we sequenced 16S rRNA V3-V4 region (HiSeq Illumina) of DNA extracted from vaginal swabs collected from women with bacterial vaginosis (BV) (n=10) and healthy controls (n=10) of 18-45yrs. The abundance profile of 20 samples had 876 OTUs from 33 families and 45 genera. Rarefaction analysis showed higher number of species in normal flora compared to BV. Alpha diversity as measured by Shannon diversity revealed normal flora had less diverse communities compared to BV. Beta diversity comparison using Bray Curtis indicated distinct microbial communities between normal and BV flora. The most abundant Phylum that showed significant difference between normal and BV samples were Firmicutes (p=0.0004) and Actinobacteria (p=0.009) where Firmicutes were abundant in normal flora. A significant difference was observed for relative proportions of Lactobacillus, Gardnerella, Aerococcus, Brevibacterium between the groups. Lactobacillus abundance decreased significantly in BV flora (14.57%), as compared to normal flora (83.25%) whereas the abundance of Bifidobacterium was observed increased from 12.0% in normal flora to 45.25% in BV. Besides, increased levels of Klebsiella, Sneathia, Coriobacteriaceae and Prevotella was noted in BV. L.crispatus was exclusively present in normal flora whereas L.iners was detected from both the groups with 80% and 100% prevalence in normal and BV flora respectively.
The study provides insights into the vaginal microbiome structure of Indian women that will enable us to unravel the microbial biomarkers and explore the prospective candidates for restoring the vaginal microbiota.
The vaginal microbiota of HIV –infected pregnant women: associations with local inflammation and gestational age at delivery
Identification: Short, Charlotte
Credits: None available.
The vaginal microbiota of HIV -infected pregnant women: associations with local inflammation and gestational age at delivery
C Short1, R Quinlan1, R Brown1, Y Lee1, R Shattock1, P Bennett1, G Taylor1, D MacIntyre1 and London HIV Pregnancy Research Group
1Imperial College London
HIV + women experience high rates of PTB, in spite this, there is little data on their vaginal microbiota during pregnancy. We sought to describe the microbiome in a group of HIV+ pregnant women and explore associations with local cytokine environment and gestational age at delivery (GA).
A prospective observational multi-site study. Vaginal and cervicovaginal fluid (CVF) were obtained using a swab and menstrual softcup during the 2nd trimester from HIV+ and HIV- pregnant women (Exclusion criteria: <350 cells/mm3, multiple or in-vitro pregnancy and injecting drug user). MiSeq sequencing of 16S rRNA gene amplicons was used to characterize the vaginal microbiome. Multiplex assays were used to measure CVF cytokine concentrations. Multivariate modeling was performed to explore associations with bacterial genus/species, CVF cytokine concentrations and clinical data.
HIV+ women (n=53) had a median age of 35, 85% were Black and 14% had PTB. HIV- women (n=30) had a median age of 33 and 50% were Caucasian. HIV+ women delivering at term had higher abundance of Gardnerella (18% versus 3% p=0.003) and Prevotella genera (4% versus 0.1% p=0.002) and lower proportions of Lactobacillus species (70% versus 93% p=0.009) compared to HIV- women. The predominant vaginal community state type (CST) of HIV+ pregnant women was III (L.iners dominant) 55% (n=29), 26% (14) were CST IV (high diversity, anaerobic), 13% (7) were CST I and 2% (1) were CST II. Amongst HIV+ women, PTB was associated with increased proportions of Gardnerella spp. (p<0.0001), Prevotella spp. (p<0.0001), Aerococcus spp. (p=0.015) and Megasphaera spp. (p=0.03) compared with term birth. All PTBs were in women assigned to CST III and IV. The proportion of read counts of CST IV associated anaerobes were positively correlated with CVF IFNγ, IL-1β, IL12p70 and TNFα. IL-1β was positively associated with bacterial diversity and richness. No associations between cytokine concentrations and GA were observed.
Diverse vaginal bacterial communities in HIV+ women are associated with PTB. The associated local proinflammatory cytokine profile may reflect the pathogenic contribution of these organisms to the early trigger of labor.
Funding: Wellcome Trust
Impacts of microbial derived short chain fatty acids in regulating immune activation at the female reproductive tract via epigenetic mechanisms
Identification: Siddik, Abu Bakar
Credits: None available.
Impacts of microbial derived short chain fatty acids in regulating immune activation at the female reproductive tract via epigenetic mechanisms
Abu Bakar Siddik1, Chih-Yu Chen2, Chris Grant2, Garrett Westmacott2, T. Blake Ball1,2, Ruey-Chyi Su1,2,*
1University of Manitoba, Medical Microbiology & Infectious Diseases; 2National HIV and Retrovirology Laboratory, JC Wilt Infectious Diseases Research Centre, Winnipeg, Manitoba, Canada
*Corresponding author.
Epithelia are physical barriers, as well as key regulators of mucosal immunity. Soluble mucosal factors such as short chain fatty acids (SCFAs) in the vaginal fluids (VF) can alter the epigenetic regulation of immunologic genes via inhibition of cellular histone deacetylase (HDAC) activities. The role of HDAC is to deacetylase histone which consequently wrapped the DNA tightly with the histone core. So the inhibition of HDAC cause keeping DNA in open conformation which eventually become favourable for gene expression. This study explores the impact of SCFAs in regulating the transcription of immunologic genes in epithelial cells derived from the female reproductive tract (FRT). To define the effects of sodium butyrate (NaB), one of the vaginal SCFAs, on the expression of anti- and pro-inflammatory mediators, and to identify cellular proteins that are specifically affected by exposure to NaB. The vaginal epithelial cell line 'Vk2' was pre-treated with NaB at non-toxic concentrations prior to stimulation with Toll-like-receptor (TLR) agonists. Cellular RNA was quantitated with RT-qPCR, and cellular proteins were analyzed for post-translational modifications (i.e. acetylation, methylation, and phosphorylation) using proteomic profiling. NaB (5mM) significantly enhanced transcription of the pro-inflammatory cytokines IL-6 and TNFα, and only TNFα transcript levels rose in response to TLR-1, -2, -3, -5, and -6 stimulation. A change in the profile of phosphorylated proteins was also observed following NaB-treatment in the absence of TLR stimulation. Proteins that lost phosphorylation in NaB-treated Vk2 cells were implicated in cell-cell adhesion and mRNA splicing. This suggests that changes in cytokine/chemokine expression may be affected at transcription or post-transcription. In addition, NaB may affect epithelial barrier function by altering protein phosphorylation.
Abu Bakar Siddik1, Chih-Yu Chen2, Chris Grant2, Garrett Westmacott2, T. Blake Ball1,2, Ruey-Chyi Su1,2,*
1University of Manitoba, Medical Microbiology & Infectious Diseases; 2National HIV and Retrovirology Laboratory, JC Wilt Infectious Diseases Research Centre, Winnipeg, Manitoba, Canada
*Corresponding author.
Epithelia are physical barriers, as well as key regulators of mucosal immunity. Soluble mucosal factors such as short chain fatty acids (SCFAs) in the vaginal fluids (VF) can alter the epigenetic regulation of immunologic genes via inhibition of cellular histone deacetylase (HDAC) activities. The role of HDAC is to deacetylase histone which consequently wrapped the DNA tightly with the histone core. So the inhibition of HDAC cause keeping DNA in open conformation which eventually become favourable for gene expression. This study explores the impact of SCFAs in regulating the transcription of immunologic genes in epithelial cells derived from the female reproductive tract (FRT). To define the effects of sodium butyrate (NaB), one of the vaginal SCFAs, on the expression of anti- and pro-inflammatory mediators, and to identify cellular proteins that are specifically affected by exposure to NaB. The vaginal epithelial cell line 'Vk2' was pre-treated with NaB at non-toxic concentrations prior to stimulation with Toll-like-receptor (TLR) agonists. Cellular RNA was quantitated with RT-qPCR, and cellular proteins were analyzed for post-translational modifications (i.e. acetylation, methylation, and phosphorylation) using proteomic profiling. NaB (5mM) significantly enhanced transcription of the pro-inflammatory cytokines IL-6 and TNFα, and only TNFα transcript levels rose in response to TLR-1, -2, -3, -5, and -6 stimulation. A change in the profile of phosphorylated proteins was also observed following NaB-treatment in the absence of TLR stimulation. Proteins that lost phosphorylation in NaB-treated Vk2 cells were implicated in cell-cell adhesion and mRNA splicing. This suggests that changes in cytokine/chemokine expression may be affected at transcription or post-transcription. In addition, NaB may affect epithelial barrier function by altering protein phosphorylation.
Morphotypic variation of vaginal clue cells broadens understanding of vaginal biofilms
Identification: Sycuro, Laura
Credits: None available.
Morphotypic variation of vaginal clue cells broadens understanding of vaginal biofilms
Laura K. Sycuro*,1,2, Shaelen Konschuh1, George Kuo2, and David N. Fredricks2,3
1International Microbiome Centre, University of Calgary; 2Fred Hutchinson Cancer Research Center; 3University of Washington
*Corresponding author
Bacterial vaginosis (BV) is a dysbiotic state of the vaginal microbiota that affects 10-60% of premenopausal women globally. BV increases a woman's risk of acquiring sexually transmitted infections (STI) and experiencing pregnancy complications such as preterm birth. One of the pivotal problems in the clinical management of BV is its high rate of recurrence. A primary hypothesis for why BV recurs is the presence of an adherent vaginal biofilm that enables microbes to survive exposure to antimicrobials. Here we report a longitudinal sub-study aimed at describing the morphotypic appearance of vaginal epithelial cells coated in adherent biofilm (clue cells) from diagnosis and treatment through 3-6 months of follow-up. Of 85 mostly African American (58%) and White (34%) women enrolled through the Public Health Seattle and King County Sexually Transmitted Diseases Clinic, 38 (45%) completed all 6 months of follow-up and 60% experienced BV recurrence. Most participants (73/74) who were BV-positive at the baseline visit had vaginal clue cells observed in their vaginal fluid. Freshly collected cervicovaginal lavage fluid from the study's 374 patient visits was microscopically observed, Gram stained, and fixed for downstream analysis. Using microscopy and molecular probing we observed that in addition to the established clue cell morphotype consisting predominantly of Gardnerella vaginalis and Atopobium vaginae, there was a second, although rare morphotype dominated by curved rod bacteria. We confirmed the identity of the abundantly adherent bacteria in some samples was Mobiluncus mulieris, and subsequent in vitro studies suggested this species' ability to form adherent biofilms is strain-variable. To our knowledge, this study is the first to report bacteria other than G. vaginalis and A. vaginae can dominate the vaginal biofilm and contribute a distinct morphotypic variation of vaginal clue cells. Efforts to identify the other curved rod bacteria contributing to the 'fuzzy' appearance of vaginal clue cells are on-going, as is a systematic assessment of how variable clue cell morphotypes are linked to patient outcomes.
Laura K. Sycuro*,1,2, Shaelen Konschuh1, George Kuo2, and David N. Fredricks2,3
1International Microbiome Centre, University of Calgary; 2Fred Hutchinson Cancer Research Center; 3University of Washington
*Corresponding author
Bacterial vaginosis (BV) is a dysbiotic state of the vaginal microbiota that affects 10-60% of premenopausal women globally. BV increases a woman's risk of acquiring sexually transmitted infections (STI) and experiencing pregnancy complications such as preterm birth. One of the pivotal problems in the clinical management of BV is its high rate of recurrence. A primary hypothesis for why BV recurs is the presence of an adherent vaginal biofilm that enables microbes to survive exposure to antimicrobials. Here we report a longitudinal sub-study aimed at describing the morphotypic appearance of vaginal epithelial cells coated in adherent biofilm (clue cells) from diagnosis and treatment through 3-6 months of follow-up. Of 85 mostly African American (58%) and White (34%) women enrolled through the Public Health Seattle and King County Sexually Transmitted Diseases Clinic, 38 (45%) completed all 6 months of follow-up and 60% experienced BV recurrence. Most participants (73/74) who were BV-positive at the baseline visit had vaginal clue cells observed in their vaginal fluid. Freshly collected cervicovaginal lavage fluid from the study's 374 patient visits was microscopically observed, Gram stained, and fixed for downstream analysis. Using microscopy and molecular probing we observed that in addition to the established clue cell morphotype consisting predominantly of Gardnerella vaginalis and Atopobium vaginae, there was a second, although rare morphotype dominated by curved rod bacteria. We confirmed the identity of the abundantly adherent bacteria in some samples was Mobiluncus mulieris, and subsequent in vitro studies suggested this species' ability to form adherent biofilms is strain-variable. To our knowledge, this study is the first to report bacteria other than G. vaginalis and A. vaginae can dominate the vaginal biofilm and contribute a distinct morphotypic variation of vaginal clue cells. Efforts to identify the other curved rod bacteria contributing to the 'fuzzy' appearance of vaginal clue cells are on-going, as is a systematic assessment of how variable clue cell morphotypes are linked to patient outcomes.
Role of Bacterial Vaginosis associated bacterial sialidase in HIV infection
Identification: Traviss, Riley
Credits: None available.
Role of Bacterial Vaginosis associated bacterial sialidase in HIV infection
Riley Traviss and Zenda Woodman
University of Cape Town, Integrative Biomedical Sciences, Cape Town, South Africa
Studies suggest that women with Bacterial Vaginosis (BV) are at a higher risk of being infected by HIV-1 than those with a microbiome dominated by Lactobacillus spp.. A potential mechanism is the secretion of sialidase by BV-associated bacteria (BVAB) that modify either host cell and/or viral N-glycans, facilitating binding of Envelope (Env) to CD4 and/or CCR5 and thus enhance viral entry. When TZM-bl cells were infected with pseudovirus in the presence of sialidase, infection increased 2-fold and this effect was reduced upon addition of N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid (DMM), an inhibitor of sialidase. Therefore, the addition of bacterial sialidase enhanced HIV infection. When TZM-bl cells were infected with pseudovirus in the presence of brain heart infusion (BHI) medium used to culture the BVAB, Gardnerella vaginalis (GV), abiotic GV culture medium inhibited pseudovirus entry without affecting TZM-bl cell viability. This suggested that either sialidase was not secreted by the GV strain and/or another factor was influencing pseudovirus infection. When the abiotic BHI medium was adjusted to pH 7.48, the inhibitory effect of GV was reduced 2-fold and when the abiotic BHI medium was boiled, GV culture no longer inhibited pseudovirus entry. Therefore, GV secreted a heat labile factor that inhibited pseudovirus infection without being toxic to TZM-bl cells. GV secretes Vaginolysin, a member of the cholesterol-dependent cytolysin family believed to contribute to BV pathogenesis. As the MTT test indicated that TZM-bl cells remained viable during incubation with abiotic GV culture medium, they are unlikely to have lysed. Although we cannot discount the effect of Vaginolysin in our assay, it is possible that anaerobic culture of GV in BHI might stimulate the secretion of a compound that inhibits HIV-1 infection in vivo and that other mitigating factors such as inflammation could negate the protective effect.
Riley Traviss and Zenda Woodman
University of Cape Town, Integrative Biomedical Sciences, Cape Town, South Africa
Studies suggest that women with Bacterial Vaginosis (BV) are at a higher risk of being infected by HIV-1 than those with a microbiome dominated by Lactobacillus spp.. A potential mechanism is the secretion of sialidase by BV-associated bacteria (BVAB) that modify either host cell and/or viral N-glycans, facilitating binding of Envelope (Env) to CD4 and/or CCR5 and thus enhance viral entry. When TZM-bl cells were infected with pseudovirus in the presence of sialidase, infection increased 2-fold and this effect was reduced upon addition of N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid (DMM), an inhibitor of sialidase. Therefore, the addition of bacterial sialidase enhanced HIV infection. When TZM-bl cells were infected with pseudovirus in the presence of brain heart infusion (BHI) medium used to culture the BVAB, Gardnerella vaginalis (GV), abiotic GV culture medium inhibited pseudovirus entry without affecting TZM-bl cell viability. This suggested that either sialidase was not secreted by the GV strain and/or another factor was influencing pseudovirus infection. When the abiotic BHI medium was adjusted to pH 7.48, the inhibitory effect of GV was reduced 2-fold and when the abiotic BHI medium was boiled, GV culture no longer inhibited pseudovirus entry. Therefore, GV secreted a heat labile factor that inhibited pseudovirus infection without being toxic to TZM-bl cells. GV secretes Vaginolysin, a member of the cholesterol-dependent cytolysin family believed to contribute to BV pathogenesis. As the MTT test indicated that TZM-bl cells remained viable during incubation with abiotic GV culture medium, they are unlikely to have lysed. Although we cannot discount the effect of Vaginolysin in our assay, it is possible that anaerobic culture of GV in BHI might stimulate the secretion of a compound that inhibits HIV-1 infection in vivo and that other mitigating factors such as inflammation could negate the protective effect.
Acceptability of Vaginal Probiotics Use To Prevent Bacterial Vaginosis Recurrence In High-Risk Rwandan Women
Identification: van de Wijgert, Janneke
Credits: None available.
Acceptability of Vaginal Probiotics Use To Prevent Bacterial Vaginosis Recurrence In High-Risk Rwandan Women
Verwijs MC1, Agaba SK2, Uwineza M2, Umulisa MM2, van de Wijgert JHHM2,3
1Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom; 2Rinda Ubuzima, Kigali, Rwanda. 3Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands
Aim: Bacterial vaginosis (BV) in Rwandan women at risk of HIV and sexually transmitted infections is common. We conducted a pilot study to determine the feasibility, acceptability, and preliminary efficacy of three interventions to prevent BV recurrence.
Methods: 68 women who completed oral metronidazole treatment for BV and/or Trichomonas vaginalis were randomised to four groups (N=17 each): no product, or intermittent use of oral metronidazole, or one of two vaginal probiotics. Participants inserted the first dose under direct observation and used products for two months. They were counselled to practice safer sex, cease vaginal practices, and encourage male partner penile hygiene. Data were collected in face-to-face and in-depth interviews, daily diaries, and focus group discussions.
Results: Most women (93%) were sex workers and the BV prevalence (by Nugent) at baseline was 83.6%. None had ever heard of or used probiotics. Women were able to insert correctly without practice (100%), inserted before going to sleep (100%), while lying down (94%), and reported that inserting became easier over time (100%). Most women (90%) reported to have used >80% of the required doses (Fisher's exact p=0.371 between groups), and 60% of women had perfect adherence. Most frequently reported reasons of non-adherence were 'simply forgetting' (n=11), being away from home and forgetting the product (n=2), side-effects (n=2), and having menses (n=2). Qualitative data suggested that women believed the products were helpful, but that male partners were not always supportive. 51% of the women at baseline reported to wash inside the vagina but this decreased to 19% during follow-up (McNemar's p<0.001). There were no changes in sexual behaviours (e.g. increased condom use) over time.
Conclusion: Vaginal probiotic use is feasible in this setting, and acceptability and adherence were high. Targeted counselling is needed to stimulate safer sex, and acceptability of investigational products by loved ones.
Verwijs MC1, Agaba SK2, Uwineza M2, Umulisa MM2, van de Wijgert JHHM2,3
1Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom; 2Rinda Ubuzima, Kigali, Rwanda. 3Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands
Aim: Bacterial vaginosis (BV) in Rwandan women at risk of HIV and sexually transmitted infections is common. We conducted a pilot study to determine the feasibility, acceptability, and preliminary efficacy of three interventions to prevent BV recurrence.
Methods: 68 women who completed oral metronidazole treatment for BV and/or Trichomonas vaginalis were randomised to four groups (N=17 each): no product, or intermittent use of oral metronidazole, or one of two vaginal probiotics. Participants inserted the first dose under direct observation and used products for two months. They were counselled to practice safer sex, cease vaginal practices, and encourage male partner penile hygiene. Data were collected in face-to-face and in-depth interviews, daily diaries, and focus group discussions.
Results: Most women (93%) were sex workers and the BV prevalence (by Nugent) at baseline was 83.6%. None had ever heard of or used probiotics. Women were able to insert correctly without practice (100%), inserted before going to sleep (100%), while lying down (94%), and reported that inserting became easier over time (100%). Most women (90%) reported to have used >80% of the required doses (Fisher's exact p=0.371 between groups), and 60% of women had perfect adherence. Most frequently reported reasons of non-adherence were 'simply forgetting' (n=11), being away from home and forgetting the product (n=2), side-effects (n=2), and having menses (n=2). Qualitative data suggested that women believed the products were helpful, but that male partners were not always supportive. 51% of the women at baseline reported to wash inside the vagina but this decreased to 19% during follow-up (McNemar's p<0.001). There were no changes in sexual behaviours (e.g. increased condom use) over time.
Conclusion: Vaginal probiotic use is feasible in this setting, and acceptability and adherence were high. Targeted counselling is needed to stimulate safer sex, and acceptability of investigational products by loved ones.
Effects of an over-the-counter lactic-acid containing intra-vaginal douching product on the vaginal microbiota
Identification: van der Veer, Charlotte
Credits: None available.
Effects of an over-the-counter lactic-acid containing intra-vaginal douching product on the vaginal microbiota
Van der Veer C1, Bruisten SM1,2, van Houdt R3 Matser A1, van de Wijgert J4, de Vries HJC1,2,5, van der Helm JJ1
1Public Health Service, GGD, Department Infectious diseases, Amsterdam, the Netherlands;
2Amsterdam Infection & Immunity Institute, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands; 3VU University Medical Center, Department of Medical Microbiology and Infection prevention, Amsterdam, the Netherlands; 4University of Liverpool, Institute of Infection and Global Health, Liverpool, United Kingdom; 5Academic Medical Centers, Department of Dermatology, Amsterdam, the Netherlands
Background: Lactic-acid containing intra-vaginal douches are marketed as vaginal hygiene products that support optimal vaginal pH balance. We report the effect of Etos® intra-vaginal douche on the vaginal microbiota (VM).
Methods: Through advertisements 25 healthy women were recruited, aged 18-36 years, in 2015-2017. Participants were followed over 3 menstrual cycles and instructed to douche thrice weekly during cycle 2. Participants completed questionnaires at baseline, kept daily diaries to report douching, menses and sex, self-collected vaginal swabs almost daily and measured vaginal pH mid-cycle. We assessed the VM by 16SrRNA gene sequencing and tested for Candida albicans by PCR at four time-points.
Results: Participants had a median age of 24 years [IQR: 22-29], were mostly Dutch-Caucasian (88%), and 60% used combined oral contraceptives. VM was assessed for a median of 44 vaginal swabs [IQR: 41-50] per participant. At baseline, 21 participants had Lactobacillus-dominated VM (Lactobacillus crispatus (n=14), L. iners (n=6), or diverse Lactobacillus species (n=1)) and 4 participants had VM consisting of diverse anaerobes (dysbiosis). In multinomial logistic regression models, having dysbiosis was more likely in the second and third cycle, compared to the first cycle, after adjusting for menses (OR=1.4 (95% CI: 0.9-2.1) and OR=1.7 (95% CI: 0.9-3.1), respectively), though not significantly so (p=0.376). Douching did not affect vaginal pH (p=0.943). Menses increased the odds for having dysbiosis 1.7 fold (95% CI: 1.0-2.8), while douching during menses increased the odds 2.6 fold (95% CI: 1.0-6.5), compared to not menstruating (p=0.099). Participants were more likely to test positive for C. albicans after cycle two, compared to cycle one (OR = 3.0 (95% CI: 1.2 - 7.2); p=0.017).
Conclusion: Etos® lactic-acid intra-vaginal douche did not significantly affect the VM composition or vaginal pH, but increased odds for vaginal dysbiosis was observed, especially when douching during menses. Furthermore, douching may promote C. albicans infections.
Modulation of dendritic cell immune responses by Envelope, and not functional fitness, might be important for subtype C HIV-1 transmission
Identification: Woodman, Zenda
Credits: None available.
Modulation of dendritic cell immune responses by Envelope, and not functional fitness, might be important for subtype C HIV-1 transmission
Evelyn Ngwa Lumngwena1,2, Bahiah Meyer3, Riley Traviss3 and Zenda Woodman3
1Institute for Medical Research and Medicinal Plants Research, MINRESI, Cameroon; 2Faculty of Health Sciences, University of Cape Town, South Africa
HIV-1 Envelope (Env) plays a major role in the competitive ability of the virus and a number of studies have investigated the role that it might play in transmission. One such study identified subtype B transmission motifs: a Histidine at position 12 (H12) of the signal peptide and the absence of a potential N-glycosylation site (PNG) at position 413 (N413), suggested to enhance viral fitness and/or facilitate escape from immune responses. We determined how Env might contribute to the selective advantage of subtype C transmitted founders (TF) by: 1) comparing four TF Env to matched chronic infection (CI) variants, and 2) characterising the impact of H12 and N413 on Env processing and phenotype. All nine subtype C Envs carried a Glutamic acid at position 12 (Q12) and a PNG at 413 representative of the population frequency, suggesting that subtype B TFs might have evolved by a pathway distinct from other subtypes. When a subtype C TF Env was mutated, pseudovirus (PSV) infectivity of constructs Q12H and Q12A decreased significantly, suggesting that H12 was not linked to enhanced infectivity as reported for subtype B TF. On the contrary, deletion of N413 significantly increased PSV entry efficiency, suggesting that even though the presence of a PNG at this site had a fitness cost, it was conserved in subtype C variants, including TFs. Furthermore, the processing and phenotype of TF Envs and PSV entry efficiency did not differ significantly from matched CI variants supporting the suggestion that Env functional fitness was not important for subtype C transmission. However, TFs were better able to stimulate monocyte derived dendritic cells (MDDCs) to secrete higher levels of IL-10, TNF-α, IL-6, IL-8 and MIP1β than their matched CI counterparts. This suggests that subtype C TFs might have the ability to induce immune responses in the female genital tract that favour transmission. Overall, HIV-1 subtype C transmission might not be dependent on Env function but rely more on its ability to induce dendritic cell-mediated immune responses that promote HIV survival within the female genital tract.
Evelyn Ngwa Lumngwena1,2, Bahiah Meyer3, Riley Traviss3 and Zenda Woodman3
1Institute for Medical Research and Medicinal Plants Research, MINRESI, Cameroon; 2Faculty of Health Sciences, University of Cape Town, South Africa
HIV-1 Envelope (Env) plays a major role in the competitive ability of the virus and a number of studies have investigated the role that it might play in transmission. One such study identified subtype B transmission motifs: a Histidine at position 12 (H12) of the signal peptide and the absence of a potential N-glycosylation site (PNG) at position 413 (N413), suggested to enhance viral fitness and/or facilitate escape from immune responses. We determined how Env might contribute to the selective advantage of subtype C transmitted founders (TF) by: 1) comparing four TF Env to matched chronic infection (CI) variants, and 2) characterising the impact of H12 and N413 on Env processing and phenotype. All nine subtype C Envs carried a Glutamic acid at position 12 (Q12) and a PNG at 413 representative of the population frequency, suggesting that subtype B TFs might have evolved by a pathway distinct from other subtypes. When a subtype C TF Env was mutated, pseudovirus (PSV) infectivity of constructs Q12H and Q12A decreased significantly, suggesting that H12 was not linked to enhanced infectivity as reported for subtype B TF. On the contrary, deletion of N413 significantly increased PSV entry efficiency, suggesting that even though the presence of a PNG at this site had a fitness cost, it was conserved in subtype C variants, including TFs. Furthermore, the processing and phenotype of TF Envs and PSV entry efficiency did not differ significantly from matched CI variants supporting the suggestion that Env functional fitness was not important for subtype C transmission. However, TFs were better able to stimulate monocyte derived dendritic cells (MDDCs) to secrete higher levels of IL-10, TNF-α, IL-6, IL-8 and MIP1β than their matched CI counterparts. This suggests that subtype C TFs might have the ability to induce immune responses in the female genital tract that favour transmission. Overall, HIV-1 subtype C transmission might not be dependent on Env function but rely more on its ability to induce dendritic cell-mediated immune responses that promote HIV survival within the female genital tract.