Poster Abstracts - S618
Articles
The vaginal microenvironment prior to incident STI
Identification: Brotman, Rebecca
Credits: None available.
The vaginal microenvironment prior to incident STI
RM Brotman1, JL Borgogna2, CK Robinson1, MA Klebanoff3, CJ Yeoman2, J Ravel1, MD Shardell4
1Institute for Genome Sciences, University of Maryland; 2Montana State University; 3Ohio State University; 4National Institute on Aging
We conducted a nested case control study in the NIH's Longitudinal Study of Vaginal Flora (LSVF) to assess the vaginal microenvironment of 397 cases at the visit prior to an incident genital STI (Chlamydia trachomatis, Neisseria gonorrhea, or Trichomonas vaginalis) compared to 1,794 STI-negative controls. Vaginal microbiota, metabolites, and lactic acid isomers were assessed for factors that were associated with incident STI. Controls were matched to cases on age, race and follow-up time. Vaginal lavages and behavioral surveys were collected every three months for one year. Microbiota composition was characterized and bacterial community state types (CSTs) were assigned by hierarchical clustering. Conditional logistic regression with covariate adjustment (partner concurrency, number of sex partners, condom use) was used for modeling. P-values were computed using 1000 matching group-stratified permutations and 95% confidence intervals using 1000 bootstrap samples clustered by matching group. Women with a CST IV-A profile, low-Lactobacillus states dominated by high relative abundance of BVAB-1 and low G. vaginalis, had the highest odds of incident STI. CST-I (L. crispatus-dominated), CST-II (L. gasseri-dominated), CST-III-A, and CST-III-C (both L iners dominated with the latter having other Lactobacillus spp.) each had more than 50% lower odds of incident STI than women in CST IV-A (all p<0.01). CST-II had the lowest point estimate with a 72% reduction in the odds of STI outcome (p=.02). High concentration of the D-isomer of lactic acid was associated with lower STI risk, irrespective of L-isomer concentration (p<0.05). Combinations of D- and L-isomers of lactic acid that were associated with low STI risk included a disproportionately large number of participants in CST-I and CST III-A (p<0.01). Metabolomic analysis indicated multiple amino acids and higher concentrations of several biogenic amines were associated with higher STI risk (q-value<0.05). Microbial and chemical composition of the vaginal microenvironment may modulate STI risk. Metagenomic and immunologic data are currently being integrated into multi-omics models with pathogen-specific outcomes.
RM Brotman1, JL Borgogna2, CK Robinson1, MA Klebanoff3, CJ Yeoman2, J Ravel1, MD Shardell4
1Institute for Genome Sciences, University of Maryland; 2Montana State University; 3Ohio State University; 4National Institute on Aging
We conducted a nested case control study in the NIH's Longitudinal Study of Vaginal Flora (LSVF) to assess the vaginal microenvironment of 397 cases at the visit prior to an incident genital STI (Chlamydia trachomatis, Neisseria gonorrhea, or Trichomonas vaginalis) compared to 1,794 STI-negative controls. Vaginal microbiota, metabolites, and lactic acid isomers were assessed for factors that were associated with incident STI. Controls were matched to cases on age, race and follow-up time. Vaginal lavages and behavioral surveys were collected every three months for one year. Microbiota composition was characterized and bacterial community state types (CSTs) were assigned by hierarchical clustering. Conditional logistic regression with covariate adjustment (partner concurrency, number of sex partners, condom use) was used for modeling. P-values were computed using 1000 matching group-stratified permutations and 95% confidence intervals using 1000 bootstrap samples clustered by matching group. Women with a CST IV-A profile, low-Lactobacillus states dominated by high relative abundance of BVAB-1 and low G. vaginalis, had the highest odds of incident STI. CST-I (L. crispatus-dominated), CST-II (L. gasseri-dominated), CST-III-A, and CST-III-C (both L iners dominated with the latter having other Lactobacillus spp.) each had more than 50% lower odds of incident STI than women in CST IV-A (all p<0.01). CST-II had the lowest point estimate with a 72% reduction in the odds of STI outcome (p=.02). High concentration of the D-isomer of lactic acid was associated with lower STI risk, irrespective of L-isomer concentration (p<0.05). Combinations of D- and L-isomers of lactic acid that were associated with low STI risk included a disproportionately large number of participants in CST-I and CST III-A (p<0.01). Metabolomic analysis indicated multiple amino acids and higher concentrations of several biogenic amines were associated with higher STI risk (q-value<0.05). Microbial and chemical composition of the vaginal microenvironment may modulate STI risk. Metagenomic and immunologic data are currently being integrated into multi-omics models with pathogen-specific outcomes.
Measuring T cell responses to Chlamydia trachomatis in young South African women to characterise immune correlates in the context of HIV risk
Identification: Bunjun, Rubina
Credits: None available.
Measuring T cell responses to Chlamydia trachomatis in young South African women to characterise immune correlates in the context of HIV risk
Rubina Bunjun1,2*, Micaela Lurie1, Shaun Barnabas1,3, Smritee Dabee1,2, Frans Radebe4,6, Venessa Maseko4,6, Ranmini Kularatne4,6, Shameem Z. Jaumdally1, Hoyam Gamieldien1, Heather B. Jaspan1,5, Linda-Gail Bekker1,3, and Jo-Ann S. Passmore1,2,6
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa; 2DST-NRF CAPRISA Centers of Excellence in HIV prevention, University of Cape Town, South Africa; 3Desmond Tutu HIV Foundation, University of Cape Town, South Africa; 4National Institute for Communicable Diseases Sandringham, South Africa; 5Seattle Children's' Hospital, Seattle, USA; 6National Health Laboratory Services, South Africa; *Corresponding author
Chlamydia trachomatis (CT) is an extremely common bacterial sexually transmitted infection. Studies of South African adolescent girls reported a prevalence of >40% with more than 90% of these infections asymptomatic. Chlamydia infection is a major cause of poor reproductive health outcomes and contributes to HIV risk in young women by causing genital tract inflammation. There is still no vaccine against CT due to factors complicating vaccine development. Although Th1 responses play a role in immune control of CT, robust correlates of protection remain poorly defined. Testing immune correlates by studying T cell responses to chlamydia is challenging, with limited availability of commercial chlamydial antigens. Therefore, we aimed to generate CT antigens to investigate Chlamydia-specific CD4+ T cell memory responses in the context of CT clearance, genital inflammatory potential and HIV infectivity potential. CT serovar E, which was highly prevalent in South African women, was used to infect McCoy cells and blind passaged onto fresh monolayers sequentially until infection of four 6-well plates was achieved. CT elementary bodies were isolated by Renografin density gradient purification. PBMC from CT-infected young women were stimulated with heat killed or lysed CT. The cells were phenotyped and the production of IFN-γ, TNF-α and IL-17 measured by multiparameter flow cytometry. CT-specific CD4+ T cells primarily produced Th1 cytokines and expressed CCR6, associated with genital tract homing potential. These studies are vital in understanding and preventing chlamydia infections, with the ultimate goal of lowering HIV risk in young women.
Rubina Bunjun1,2*, Micaela Lurie1, Shaun Barnabas1,3, Smritee Dabee1,2, Frans Radebe4,6, Venessa Maseko4,6, Ranmini Kularatne4,6, Shameem Z. Jaumdally1, Hoyam Gamieldien1, Heather B. Jaspan1,5, Linda-Gail Bekker1,3, and Jo-Ann S. Passmore1,2,6
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa; 2DST-NRF CAPRISA Centers of Excellence in HIV prevention, University of Cape Town, South Africa; 3Desmond Tutu HIV Foundation, University of Cape Town, South Africa; 4National Institute for Communicable Diseases Sandringham, South Africa; 5Seattle Children's' Hospital, Seattle, USA; 6National Health Laboratory Services, South Africa; *Corresponding author
Chlamydia trachomatis (CT) is an extremely common bacterial sexually transmitted infection. Studies of South African adolescent girls reported a prevalence of >40% with more than 90% of these infections asymptomatic. Chlamydia infection is a major cause of poor reproductive health outcomes and contributes to HIV risk in young women by causing genital tract inflammation. There is still no vaccine against CT due to factors complicating vaccine development. Although Th1 responses play a role in immune control of CT, robust correlates of protection remain poorly defined. Testing immune correlates by studying T cell responses to chlamydia is challenging, with limited availability of commercial chlamydial antigens. Therefore, we aimed to generate CT antigens to investigate Chlamydia-specific CD4+ T cell memory responses in the context of CT clearance, genital inflammatory potential and HIV infectivity potential. CT serovar E, which was highly prevalent in South African women, was used to infect McCoy cells and blind passaged onto fresh monolayers sequentially until infection of four 6-well plates was achieved. CT elementary bodies were isolated by Renografin density gradient purification. PBMC from CT-infected young women were stimulated with heat killed or lysed CT. The cells were phenotyped and the production of IFN-γ, TNF-α and IL-17 measured by multiparameter flow cytometry. CT-specific CD4+ T cells primarily produced Th1 cytokines and expressed CCR6, associated with genital tract homing potential. These studies are vital in understanding and preventing chlamydia infections, with the ultimate goal of lowering HIV risk in young women.
Microbial Translocation from the Vaginal Microbiome and GI tract during Hyperacute SIVmac239 infection
Identification: Bussan, Hailey
Credits: None available.
Microbial Translocation from the Vaginal Microbiome and GI tract during Hyperacute SIVmac239 infection
Hailey Bussan1,3, Adam Ericsen4,5, Eric Peterson2, Laurel Stewart1, Matt Semler1, Gabrielle Barry2, Jens Eickhoff6, Dawn Dudley1, and David O'Connor1,2
1Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison; 2Wisconsin National Primate Research Center, University of Wisconsin-Madison; 3Medical Scientist Training Program, University of Wisconsin-Madison School of Medicine and Public Health; 4Yerkes National Primate Research Center; 5Emory University; 6Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison
Movement of microbial products from the GI tract is known to cause systemic immune activation during chronic human-immunodeficiency virus (HIV) and simian-immunodeficiency virus infection (SIV). Recently, we showed that microbial translocation occurs in the first days following intrarectal SIV infection. Previously, the origin of microbial translocation has only been tested and described from the GI tract. We hypothesized that since the vaginal microbiome has been implicated in risk acquisition of HIV and related inflammation, vaginal microbial products could also be implicated in hyperacute translocation. We sequenced and described microbial communities in plasma, stool, and vaginal swabs from eight MCM treated with dextran sodium sulfate, a chemical colitogen that causes intestinal epithelial damage, and infected with SIVmac239 intrarectally. Using 16S Illumina-based sequencing, we found plasma microbial diversity increased within two days of SIV infection with contributions of unique taxa from the stool and vaginal microbiome. We are in the process of repeating this study with DSS-naive macaques challenged intrarectally and intravaginally. This study suggests that the vaginal microbiome also contributes to acute translocation and immune activation even when individuals are infected intrarectally. Therapeutic methods aimed at preventing microbial translocation to reduce immune activation may need to target potential translocation from both the GI tract and vagina.
Hailey Bussan1,3, Adam Ericsen4,5, Eric Peterson2, Laurel Stewart1, Matt Semler1, Gabrielle Barry2, Jens Eickhoff6, Dawn Dudley1, and David O'Connor1,2
1Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison; 2Wisconsin National Primate Research Center, University of Wisconsin-Madison; 3Medical Scientist Training Program, University of Wisconsin-Madison School of Medicine and Public Health; 4Yerkes National Primate Research Center; 5Emory University; 6Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison
Movement of microbial products from the GI tract is known to cause systemic immune activation during chronic human-immunodeficiency virus (HIV) and simian-immunodeficiency virus infection (SIV). Recently, we showed that microbial translocation occurs in the first days following intrarectal SIV infection. Previously, the origin of microbial translocation has only been tested and described from the GI tract. We hypothesized that since the vaginal microbiome has been implicated in risk acquisition of HIV and related inflammation, vaginal microbial products could also be implicated in hyperacute translocation. We sequenced and described microbial communities in plasma, stool, and vaginal swabs from eight MCM treated with dextran sodium sulfate, a chemical colitogen that causes intestinal epithelial damage, and infected with SIVmac239 intrarectally. Using 16S Illumina-based sequencing, we found plasma microbial diversity increased within two days of SIV infection with contributions of unique taxa from the stool and vaginal microbiome. We are in the process of repeating this study with DSS-naive macaques challenged intrarectally and intravaginally. This study suggests that the vaginal microbiome also contributes to acute translocation and immune activation even when individuals are infected intrarectally. Therapeutic methods aimed at preventing microbial translocation to reduce immune activation may need to target potential translocation from both the GI tract and vagina.
The impact of vaginal microbial communities on immune cells essential for pathogen protection and epithelial integrity
Identification: Cheu, Ryan
Credits: None available.
The impact of vaginal microbial communities on immune cells essential for pathogen protection and epithelial integrity
Ryan K. Cheu1,2,3, Avid Mohammadi4, Tiffany Hensley-Mcbain1,2, Jennifer Manuzak1,2,3, Alexander S. Zevin1,2, Charlene Miller3, Mark Yudin5, Rupert Kaul4, Nichole R. Klatt1,2,3
1Department of Pharmaceutics, University of Washington, Seattle, WA, USA; 2Washington National Primate Research Center, University of Washington, Seattle, WA, USA; 3Department of Pediatrics, University of Miami Miller School of Medicine, Miami, FL, USA; 4Department of Immunology, University of Toronto, Toronto, Canada; 5Core Obstetrics & Gynecology, University of Toronto, Toronto, Canada
Background: Bacterial vaginosis (BV) is associated with an increased HIV transmission rate. Inflammatory cells such as neutrophils are critical for innate immune responses but can also contribute to barrier damage and inflammation. Currently, the role of neutrophils in HIV transmission and BV status is unknown. Here, we hypothesize that BV-associated bacteria (BVAB) increase HIV transmission rates by inducing activation of neutrophils and promoting accumulation of them within the female reproductive tract (FRT), resulting in epithelial barrier damage.
Methods: In order to elucidate the mechanisms for the negative outcomes of BV, we collected cervicovaginal cytobrushes from 11 women with BV and 10 women without BV. We used flow cytometry to assess phenotype and functionality of neutrophils and performed in vitro whole blood co-cultures with bacteria associated with BV (G. vaginalis), healthy commensals such as L. iners and L. crispatus, media alone (negative control) and lipopolysaccharide and peptidoglycan (positive controls). To determine the impact neutrophils have on epithelial integrity, we isolated neutrophils from healthy human whole blood to co-culture with FRT bacteria across a vaginal epithelial cellular monolayer and used a transepithelial electrical resistance (TEER) set-up to assess barrier damage.
Results: We demonstrated increased neutrophil activation (p=0.0022), prolonged lifespan (p=0.0022), and more total neutrophils (p=0.0022) in the cytobrushes from women with BV. Similarly, our co-culture experiments showed more neutrophil activation (p<0.0001), prolonged lifespan (p=0.008), and more total neutrophils (p<0.0001) in cultures with G. vaginalis compared with our negative controls and Lactobacillus cultures. Our 16S rRNA analysis identified two distinct community groups one dominated by Lactobacillus and the other a more diverse community primarily consisting of G. vaginalis. Our TEER assays demonstrated significant barrier damage in cultures with neutrophils and G. vaginalis when compared with neutrophils alone (p=0.001) or with neutrophils and Lactobacillus spp. (p=0.007).
Conclusions: Here, we demonstrate that BVAB induce neutrophil activation, delay apoptosis leading to accumulation within the FRT, and jeopardize epithelial integrity. This study provides potential mechanistic insights into how BV may lead to FRT inflammation and increased HIV transmission.
Ryan K. Cheu1,2,3, Avid Mohammadi4, Tiffany Hensley-Mcbain1,2, Jennifer Manuzak1,2,3, Alexander S. Zevin1,2, Charlene Miller3, Mark Yudin5, Rupert Kaul4, Nichole R. Klatt1,2,3
1Department of Pharmaceutics, University of Washington, Seattle, WA, USA; 2Washington National Primate Research Center, University of Washington, Seattle, WA, USA; 3Department of Pediatrics, University of Miami Miller School of Medicine, Miami, FL, USA; 4Department of Immunology, University of Toronto, Toronto, Canada; 5Core Obstetrics & Gynecology, University of Toronto, Toronto, Canada
Background: Bacterial vaginosis (BV) is associated with an increased HIV transmission rate. Inflammatory cells such as neutrophils are critical for innate immune responses but can also contribute to barrier damage and inflammation. Currently, the role of neutrophils in HIV transmission and BV status is unknown. Here, we hypothesize that BV-associated bacteria (BVAB) increase HIV transmission rates by inducing activation of neutrophils and promoting accumulation of them within the female reproductive tract (FRT), resulting in epithelial barrier damage.
Methods: In order to elucidate the mechanisms for the negative outcomes of BV, we collected cervicovaginal cytobrushes from 11 women with BV and 10 women without BV. We used flow cytometry to assess phenotype and functionality of neutrophils and performed in vitro whole blood co-cultures with bacteria associated with BV (G. vaginalis), healthy commensals such as L. iners and L. crispatus, media alone (negative control) and lipopolysaccharide and peptidoglycan (positive controls). To determine the impact neutrophils have on epithelial integrity, we isolated neutrophils from healthy human whole blood to co-culture with FRT bacteria across a vaginal epithelial cellular monolayer and used a transepithelial electrical resistance (TEER) set-up to assess barrier damage.
Results: We demonstrated increased neutrophil activation (p=0.0022), prolonged lifespan (p=0.0022), and more total neutrophils (p=0.0022) in the cytobrushes from women with BV. Similarly, our co-culture experiments showed more neutrophil activation (p<0.0001), prolonged lifespan (p=0.008), and more total neutrophils (p<0.0001) in cultures with G. vaginalis compared with our negative controls and Lactobacillus cultures. Our 16S rRNA analysis identified two distinct community groups one dominated by Lactobacillus and the other a more diverse community primarily consisting of G. vaginalis. Our TEER assays demonstrated significant barrier damage in cultures with neutrophils and G. vaginalis when compared with neutrophils alone (p=0.001) or with neutrophils and Lactobacillus spp. (p=0.007).
Conclusions: Here, we demonstrate that BVAB induce neutrophil activation, delay apoptosis leading to accumulation within the FRT, and jeopardize epithelial integrity. This study provides potential mechanistic insights into how BV may lead to FRT inflammation and increased HIV transmission.
Distinct cervical microbial diversity and community structures by human papillomavirus and cervical disease status in HIV positive women in South Africa
Identification: Denny, Lynette
Credits: None available.
Distinct cervical microbial diversity and community structures by human papilloma-virus and cervical disease status in HIV-positive women in South Africa
Annavajhala, Medini K^, Saidu, Rakiya+, Tergas, Ana^, Kuhn, Louise^, Denny, Lynette+, Uhlemann, Anne-Catrin^
^Columbia University, New York, + University of Cape Town
Background: Cervical cancer continues to cause significant morbidity and mortality in HIV+ women especially in resource-limited settings. Although HPV infection is a necessary cause of cervical cancer, other biomarkers which could distinguish which women with HPV infection are most likely to have cervical intraepithelial neoplasia (CIN) would be beneficial. The role of the cervical microbiome in progression of HPV-related disease, especially in HIV+ women, remains unclear. Here, we aimed to identify if cervical microbiota in HIV+ women with HPV16 predict CIN.
Methods: We enrolled HIV+ women aged 30 to 65 from Khayelitsha Day Hospital in Cape Town, South Africa. Exclusion criteria were history of anogenital cancer, treatment for cervical dysplasia, or hysterectomy. HIV and HPV testing and histological diagnosis based on biopsy and/or LEEP were performed to define three experimental categories: (1) HPV16+/CIN3 (n=17), (2) HPV16+/No CIN (n=15), and (3) negative for all high-risk HPV types with no CIN (n=19). Cervical swabs were collected during gynecological exams and stored in ThinPrep. We extracted DNA (Qiagen PowerViral) and performed 16S rRNA (V3V4) sequencing. Closed-reference operational taxonomic unit (OTU) picking against the Greengenes 97 database was performed using QIIME (minimum count cutoff: 2,500). Microbiome Chao alpha-diversity and UniFrac beta-diversity were calculated using phyloseq and differential abundance testing was performed using DESeq in R.
Results: After quality-filtering of sequencing data, we were able to analyze 42/51 cervical swabs (n=16 group 1 (HPV16+/CIN3), n=14 group 2 (HPV16+/No CIN), and n=12 group 3 (No hrHPV/No CIN)). We found that samples from women with no HPV had comparable alpha-diversity to those from women with HPV16 and CIN3. However, women with HPV16 and no CIN had significantly lower alpha-diversity (Chao index of 101 compared to 130 (group 3) and 142 (group 1), p<0.05). Overall microbial community structure was not significantly different across all three groups (UniFrac PERMANOVA, P=0.218). Compared to women without hrHPV, those with HPV16 and CIN3 had cervical communities significantly enriched in Lactobacillus iners and Akkermansia muciniphila (DESeq, p<0.05, FDR<0.05). Conversely, women without hrHPV had significantly higher levels of OTUs assigned to Prevotella, Fusobacterium, Megasphaera, Anaerococcus. Furthermore, HPV16-positive women with CIN3 were significantly enriched in L. iners and two OTUs within the Rs-045 family of the TM7 phylum. HPV16-positive women with no CIN were instead enriched in Prevotella.
Conclusions: Our results support distinct microbiota by HPV and CIN status in HIV+ women. Further studies are needed to evaluate their role in the pathogenesis of cervical cancer in HIV+HPV+ women. Our findings demonstrate the potential utility of the cervical microbiome and specific microbiota as adjunct diagnostic tools to improve screening.
Annavajhala, Medini K^, Saidu, Rakiya+, Tergas, Ana^, Kuhn, Louise^, Denny, Lynette+, Uhlemann, Anne-Catrin^
^Columbia University, New York, + University of Cape Town
Background: Cervical cancer continues to cause significant morbidity and mortality in HIV+ women especially in resource-limited settings. Although HPV infection is a necessary cause of cervical cancer, other biomarkers which could distinguish which women with HPV infection are most likely to have cervical intraepithelial neoplasia (CIN) would be beneficial. The role of the cervical microbiome in progression of HPV-related disease, especially in HIV+ women, remains unclear. Here, we aimed to identify if cervical microbiota in HIV+ women with HPV16 predict CIN.
Methods: We enrolled HIV+ women aged 30 to 65 from Khayelitsha Day Hospital in Cape Town, South Africa. Exclusion criteria were history of anogenital cancer, treatment for cervical dysplasia, or hysterectomy. HIV and HPV testing and histological diagnosis based on biopsy and/or LEEP were performed to define three experimental categories: (1) HPV16+/CIN3 (n=17), (2) HPV16+/No CIN (n=15), and (3) negative for all high-risk HPV types with no CIN (n=19). Cervical swabs were collected during gynecological exams and stored in ThinPrep. We extracted DNA (Qiagen PowerViral) and performed 16S rRNA (V3V4) sequencing. Closed-reference operational taxonomic unit (OTU) picking against the Greengenes 97 database was performed using QIIME (minimum count cutoff: 2,500). Microbiome Chao alpha-diversity and UniFrac beta-diversity were calculated using phyloseq and differential abundance testing was performed using DESeq in R.
Results: After quality-filtering of sequencing data, we were able to analyze 42/51 cervical swabs (n=16 group 1 (HPV16+/CIN3), n=14 group 2 (HPV16+/No CIN), and n=12 group 3 (No hrHPV/No CIN)). We found that samples from women with no HPV had comparable alpha-diversity to those from women with HPV16 and CIN3. However, women with HPV16 and no CIN had significantly lower alpha-diversity (Chao index of 101 compared to 130 (group 3) and 142 (group 1), p<0.05). Overall microbial community structure was not significantly different across all three groups (UniFrac PERMANOVA, P=0.218). Compared to women without hrHPV, those with HPV16 and CIN3 had cervical communities significantly enriched in Lactobacillus iners and Akkermansia muciniphila (DESeq, p<0.05, FDR<0.05). Conversely, women without hrHPV had significantly higher levels of OTUs assigned to Prevotella, Fusobacterium, Megasphaera, Anaerococcus. Furthermore, HPV16-positive women with CIN3 were significantly enriched in L. iners and two OTUs within the Rs-045 family of the TM7 phylum. HPV16-positive women with no CIN were instead enriched in Prevotella.
Conclusions: Our results support distinct microbiota by HPV and CIN status in HIV+ women. Further studies are needed to evaluate their role in the pathogenesis of cervical cancer in HIV+HPV+ women. Our findings demonstrate the potential utility of the cervical microbiome and specific microbiota as adjunct diagnostic tools to improve screening.
The metagenomics and culturomics as complementary approaches to better characterized the vaginal bacterial community associated with the bacterial vaginosis
Identification: Diop, Khoudia
Credits: None available.
The metagenomics and culturomics as complementary approaches to better characterized the vaginal bacterial community associated with the bacterial vaginosis
Khoudia Diop1, Ndeye Safietou Fall1, Cheikh Sokhna1, Didier Raoult1, and Florence Fenollar1*
1Aix Marseille Univ, IRD, AP-HM, IHU-Méditerranée Infection, Marseille, France
*Corresponding author: Prof. Florence Fenollar: florence.fenollar@univ-amu.fr
Over the last decades, increasing studies of the vaginal microbiota using advanced molecular and new OMICS techniques have revealed the impact of the vaginal microbiome on reproductive health. Indeed, the disruption of the vaginal bacterial community makes it prone to bacterial vaginosis and/or severe gynecological conditions, including preterm birth, pelvic inflammatory disease and also sexually transmitted diseases. Knowledge about normal and abnormal vaginal microbiota has become a little clearer in recent years. Culture techniques have made it possible to isolate and describe many bacterial species. Whereas molecular methods have highlighted the limits of culture by showing that the vagina is a complex ecosystem containing a wide range of non-cultured or difficult-to-identify bacteria. In this study, we analyzed vaginal samples of bacterial vaginosis patients and healthy women using the metagenomics and the “Culturomics” approaches to map the vaginal flora and better understand vaginosis condition with the aim to provide better treatment. Globally, we found a higher bacterial diversity in patients compared to control with the increase of species such as Gardnerella vaginalis, Atopobium vaginae as well as oxygen-sensitive prokaryotes including Gram-positive anaerobic cocci, and Prevotella spp. Notably, we found also an increased number of previously unknown species that we isolated for the first time. Combining the metagenomics and culturomics approaches has indeed allowed the identification of a complex of 10 bacterial species associated with bacterial vaginosis. However, there is an urgent need for more laboratory experimentations to better understand the factors underlaying their pathogenicity and virulence, to improve the current and future therapies against the bacterial vaginosis and prevent relapse and treatment failures.
Funding: This study was funded by the “Fondation Méditerranée Infection” and the French Government under the “Investissements d'avenir” program managed by the National Agency for Research (reference Méditerranée Infection 10-IAHU-03).
Khoudia Diop1, Ndeye Safietou Fall1, Cheikh Sokhna1, Didier Raoult1, and Florence Fenollar1*
1Aix Marseille Univ, IRD, AP-HM, IHU-Méditerranée Infection, Marseille, France
*Corresponding author: Prof. Florence Fenollar: florence.fenollar@univ-amu.fr
Over the last decades, increasing studies of the vaginal microbiota using advanced molecular and new OMICS techniques have revealed the impact of the vaginal microbiome on reproductive health. Indeed, the disruption of the vaginal bacterial community makes it prone to bacterial vaginosis and/or severe gynecological conditions, including preterm birth, pelvic inflammatory disease and also sexually transmitted diseases. Knowledge about normal and abnormal vaginal microbiota has become a little clearer in recent years. Culture techniques have made it possible to isolate and describe many bacterial species. Whereas molecular methods have highlighted the limits of culture by showing that the vagina is a complex ecosystem containing a wide range of non-cultured or difficult-to-identify bacteria. In this study, we analyzed vaginal samples of bacterial vaginosis patients and healthy women using the metagenomics and the “Culturomics” approaches to map the vaginal flora and better understand vaginosis condition with the aim to provide better treatment. Globally, we found a higher bacterial diversity in patients compared to control with the increase of species such as Gardnerella vaginalis, Atopobium vaginae as well as oxygen-sensitive prokaryotes including Gram-positive anaerobic cocci, and Prevotella spp. Notably, we found also an increased number of previously unknown species that we isolated for the first time. Combining the metagenomics and culturomics approaches has indeed allowed the identification of a complex of 10 bacterial species associated with bacterial vaginosis. However, there is an urgent need for more laboratory experimentations to better understand the factors underlaying their pathogenicity and virulence, to improve the current and future therapies against the bacterial vaginosis and prevent relapse and treatment failures.
Funding: This study was funded by the “Fondation Méditerranée Infection” and the French Government under the “Investissements d'avenir” program managed by the National Agency for Research (reference Méditerranée Infection 10-IAHU-03).
Identifying factors that influence the temporal stability of the vaginal microbiome
Identification: France, Michael
Credits: None available.
Identifying factors that influence the temporal stability of the vaginal microbiome
Michael France1, Lindsay Rutt1, Mike Humphrys1, Larry Forney2, Jacques Ravel1
1Institute of Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, United States 2Institute for Bioinformatics and Evolutionary Studies, Department of Biological Sciences, University of Idaho, Moscow Idaho
Longitudinal profiling of the human vaginal microbiome has demonstrated that the composition of these communities can be temporally variable. This dynamic behavior has been shown to differ among women: some have a relatively constant community composition over time (stable) while others experience a high degree of change over time (unstable). Shifts in community composition, particularly shifts to states that have low-Lactobacillus abundance, have been hypothesized to be associated with risk of adverse health outcomes including increased susceptibility to sexually transmitted infections. Unfortunately, the factors that drive changes in community composition of the vagina are not well understood. In this study, we used a systems biology approach to identify the factors that drive the stability of the vaginal microbiome. We leveraged a rich dataset derived from the daily sampling of over 100 women. Stability can be defined as a combination of a communities' ability to withstand perturbation (resistance) and its ability to rebound following perturbation (resilience). We developed a metric for community stability and classified the longitudinal profiles of these women into five categories: 1) stable Lactobacillus, 2) unstable Lactobacillus, 3) stable Non-Lactobacillus, 4) unstable Non-Lactobacillus, and 5) highly unstable. A multi-omics approach is being applied to compare and contrast these communities, in order to identify drivers of community stability. Here, we present new metrics to evaluate functional redundancy and diversity from metagenomic datasets, and their contribution to the resistance and resilience of vaginal microbial communities.
Michael France1, Lindsay Rutt1, Mike Humphrys1, Larry Forney2, Jacques Ravel1
1Institute of Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, United States 2Institute for Bioinformatics and Evolutionary Studies, Department of Biological Sciences, University of Idaho, Moscow Idaho
Longitudinal profiling of the human vaginal microbiome has demonstrated that the composition of these communities can be temporally variable. This dynamic behavior has been shown to differ among women: some have a relatively constant community composition over time (stable) while others experience a high degree of change over time (unstable). Shifts in community composition, particularly shifts to states that have low-Lactobacillus abundance, have been hypothesized to be associated with risk of adverse health outcomes including increased susceptibility to sexually transmitted infections. Unfortunately, the factors that drive changes in community composition of the vagina are not well understood. In this study, we used a systems biology approach to identify the factors that drive the stability of the vaginal microbiome. We leveraged a rich dataset derived from the daily sampling of over 100 women. Stability can be defined as a combination of a communities' ability to withstand perturbation (resistance) and its ability to rebound following perturbation (resilience). We developed a metric for community stability and classified the longitudinal profiles of these women into five categories: 1) stable Lactobacillus, 2) unstable Lactobacillus, 3) stable Non-Lactobacillus, 4) unstable Non-Lactobacillus, and 5) highly unstable. A multi-omics approach is being applied to compare and contrast these communities, in order to identify drivers of community stability. Here, we present new metrics to evaluate functional redundancy and diversity from metagenomic datasets, and their contribution to the resistance and resilience of vaginal microbial communities.
Extra-vaginal Bacterial Colonization and Risk for Bacterial Vaginosis (BV)
Identification: Fredricks, David
Credits: None available.
Extra-vaginal Bacterial Colonization and Risk for Bacterial Vaginosis (BV)
Fredricks DN1,2, Plantinga A1, Marrazzo JM3, Fiedler TL1, Wu M1, Srinivasan S1*
1Fred Hutchinson Cancer Research Center, 2University of Washington, Seattle, WA; 3University of Alabama, Birmingham, AL
Women who develop BV acquire anaerobic bacteria in the vagina that may be inoculated by exposure to sex partners. Alternatively, vaginal bacteria may emerge from endogenous reservoirs in women outside the vagina. A previous study in women who have sex with women demonstrated that colonization of extra-vaginal reservoirs with BV-associated anaerobes is a risk for subsequent BV. We sought to confirm or refute these findings in a population of women who have sex with men.
254 women were enrolled in a longitudinal study of the vaginal microbiota using vaginal swabs subjected to taxon-specific quantitative PCR (qPCR) assays. We analyzed data from 24 cases of incident or relapsing BV, compared to 48 control women who did not develop BV. BV was diagnosed by Gram stain of vaginal fluid with Nugent scoring. Samples were collected approximately one month prior to BV diagnosis in cases, and included swabs from the vagina, labia, mouth, and rectum. 16 taxon-specific qPCR assays were employed to measure presence and concentrations of key bacteria.
Both the presence and concentrations of Gardnerella vaginalis (p=0.011 and 0.008, respectively) and Sneathia spp. (p=0.005 and 0.002) in the rectum prior to diagnosis were positively associated with subsequent BV risk; Lactobacillus crispatus concentrations in rectal swabs were negatively associated with BV risk (p=0.006) using FDR adjusted p values. In an exploratory analysis, the presence of oral Dialister propionicifaciens imparted a relative risk of 23.3 for subsequent BV (p=0.002), while labial presence of BVAB2 (RR=6.24, p=0.027), Sneathia (RR=8.11, p=0.002), Megasphaera (RR=4.55, p=0.011), and Eggerthella (RR=3.36, p=0.017) were associated with increased risk for subsequent BV.
Previous findings were largely replicated in this population of women who have sex with men, suggesting that colonization of extra-vaginal sites with BV-associated bacteria is a consistent risk factor for BV regardless of the sexual partner preference. This knowledge can be used to identify women at high risk for BV, and may offer opportunities to reduce BV incidence by eradicating colonization of these extra-vaginal sites with bacteria linked to BV
Funded by NIH R01 AI061628
Fredricks DN1,2, Plantinga A1, Marrazzo JM3, Fiedler TL1, Wu M1, Srinivasan S1*
1Fred Hutchinson Cancer Research Center, 2University of Washington, Seattle, WA; 3University of Alabama, Birmingham, AL
Women who develop BV acquire anaerobic bacteria in the vagina that may be inoculated by exposure to sex partners. Alternatively, vaginal bacteria may emerge from endogenous reservoirs in women outside the vagina. A previous study in women who have sex with women demonstrated that colonization of extra-vaginal reservoirs with BV-associated anaerobes is a risk for subsequent BV. We sought to confirm or refute these findings in a population of women who have sex with men.
254 women were enrolled in a longitudinal study of the vaginal microbiota using vaginal swabs subjected to taxon-specific quantitative PCR (qPCR) assays. We analyzed data from 24 cases of incident or relapsing BV, compared to 48 control women who did not develop BV. BV was diagnosed by Gram stain of vaginal fluid with Nugent scoring. Samples were collected approximately one month prior to BV diagnosis in cases, and included swabs from the vagina, labia, mouth, and rectum. 16 taxon-specific qPCR assays were employed to measure presence and concentrations of key bacteria.
Both the presence and concentrations of Gardnerella vaginalis (p=0.011 and 0.008, respectively) and Sneathia spp. (p=0.005 and 0.002) in the rectum prior to diagnosis were positively associated with subsequent BV risk; Lactobacillus crispatus concentrations in rectal swabs were negatively associated with BV risk (p=0.006) using FDR adjusted p values. In an exploratory analysis, the presence of oral Dialister propionicifaciens imparted a relative risk of 23.3 for subsequent BV (p=0.002), while labial presence of BVAB2 (RR=6.24, p=0.027), Sneathia (RR=8.11, p=0.002), Megasphaera (RR=4.55, p=0.011), and Eggerthella (RR=3.36, p=0.017) were associated with increased risk for subsequent BV.
Previous findings were largely replicated in this population of women who have sex with men, suggesting that colonization of extra-vaginal sites with BV-associated bacteria is a consistent risk factor for BV regardless of the sexual partner preference. This knowledge can be used to identify women at high risk for BV, and may offer opportunities to reduce BV incidence by eradicating colonization of these extra-vaginal sites with bacteria linked to BV
Funded by NIH R01 AI061628
Antibiotic resistance versus biofilm production in Gardnerella vaginalis
Identification: Froissart, Rémy
Credits: None available.
Antibiotic resistance versus biofilm production in Gardnerella vaginalis
Valerie Masete1, Sylvain Godreuil2, Hélène Jean-Pierre2, Jo-Ann S. Passmore 1,3, Rémy Froissart4,*
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa; 2Centre Hospitalier Régional Universitaire de Montpellier, Hôpital Arnaud de Villeneuve, Département de Bactériologie-Virologie, Montpellier, France; 3National Health Laboratory Service, Cape Town, South Africa; 4UMR 5290 MIVEGEC, CNRS IRD Université Montpellier, France
*Corresponding author
Bacterial vaginosis (BV) is a common vaginal condition affecting reproductive-age women, especially in sub-Saharan Africa. Although BV is treatable with antibiotics, 50% of the women get recurrent BV within six months after treatment. While the etiology of BV is not well characterized, it is understood that Gardnerella vaginalis plays a critical role in BV by initiating the formation of the biofilm and by degrading vaginal mucus through the release of sialidase.
The aim of this study was thus to characterize the genotypic and phenotypic diversity (biofilm formation, susceptibility to four antibiotics using E-test method, and sialidase activity) of a large panel of vaginal G. vaginalis isolates. Using 109 G. vaginalis isolates, purified from vaginal samples of French women who were diagnosticated (through microscope observation of each fresh sample) BV-positive (75/109), BV-intermediate (20/109) or BV-negative (14/109). Of these, 90 isolates were successfully genotyped using their chaperonin-60 sequences, revealing the presence of four phylogenetic clades: 13/90 subgroup A, 17/90 subgroup B, 58/90 subgroup C and 2/90 subgroup D isolates. Sialidase activity was not detected in any of the subgroup A and D isolates but was detected at similar levels in subgroup B and C isolates. Isolates from all subgroups formed similar amounts of biofilm. Forty-five of the isolates were screened for antibiotic sensitivity: the majority (71%) were resistant to metronidazole, but sensitive to clindamycin (100%), moxifloxacin (91%) and augmentin (100%). In conclusion, G. vaginalis subgroup B and C isolates were the only ones that formed biofilms and had detectable sialidase activity, suggesting that G. vaginalis subgroups B and C are most likely to be involved in BV. These results contribute to our knowledge of BV and could be useful in future studies that aim to design better treatment strategies for BV.
Valerie Masete1, Sylvain Godreuil2, Hélène Jean-Pierre2, Jo-Ann S. Passmore 1,3, Rémy Froissart4,*
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa; 2Centre Hospitalier Régional Universitaire de Montpellier, Hôpital Arnaud de Villeneuve, Département de Bactériologie-Virologie, Montpellier, France; 3National Health Laboratory Service, Cape Town, South Africa; 4UMR 5290 MIVEGEC, CNRS IRD Université Montpellier, France
*Corresponding author
Bacterial vaginosis (BV) is a common vaginal condition affecting reproductive-age women, especially in sub-Saharan Africa. Although BV is treatable with antibiotics, 50% of the women get recurrent BV within six months after treatment. While the etiology of BV is not well characterized, it is understood that Gardnerella vaginalis plays a critical role in BV by initiating the formation of the biofilm and by degrading vaginal mucus through the release of sialidase.
The aim of this study was thus to characterize the genotypic and phenotypic diversity (biofilm formation, susceptibility to four antibiotics using E-test method, and sialidase activity) of a large panel of vaginal G. vaginalis isolates. Using 109 G. vaginalis isolates, purified from vaginal samples of French women who were diagnosticated (through microscope observation of each fresh sample) BV-positive (75/109), BV-intermediate (20/109) or BV-negative (14/109). Of these, 90 isolates were successfully genotyped using their chaperonin-60 sequences, revealing the presence of four phylogenetic clades: 13/90 subgroup A, 17/90 subgroup B, 58/90 subgroup C and 2/90 subgroup D isolates. Sialidase activity was not detected in any of the subgroup A and D isolates but was detected at similar levels in subgroup B and C isolates. Isolates from all subgroups formed similar amounts of biofilm. Forty-five of the isolates were screened for antibiotic sensitivity: the majority (71%) were resistant to metronidazole, but sensitive to clindamycin (100%), moxifloxacin (91%) and augmentin (100%). In conclusion, G. vaginalis subgroup B and C isolates were the only ones that formed biofilms and had detectable sialidase activity, suggesting that G. vaginalis subgroups B and C are most likely to be involved in BV. These results contribute to our knowledge of BV and could be useful in future studies that aim to design better treatment strategies for BV.
Impact of semen exposure on cytokine response and bacterial vaginosis in the female genital tract
Identification: Mngomezulu, Khanyisile
Credits: None available.
Impact of semen exposure on cytokine response and bacterial vaginosis in the female genital tract
Khanyisile Mngomezulu1, Cheryl Baxter1, Andile Mtshali1, Lenine Liebenberg1,2, Nigel Garrett1, Sinaye Ngcapu1,2
1Centre for the AIDS Programme of Research in South Africa (CAPRISA), Nelson Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa; 2Department of Medical Microbiology, University of KwaZulu-Natal, Durban, South Africa
Diverse microbial communities and inflammatory cytokine responses in the lower female genital tract (FGT) are closely associated with increased HIV risk, possibly through increasing mucosal HIV target cell frequency and T-cell activation. The presence of semen in the vagina during unprotected sex has been associated with short-term activation of mucosal immunity. Here, we investigated the extent to which partner semen impacts on cytokine and microbial profiles measured in 248 HIV-uninfected women at high risk for HIV infection. We assessed the semen exposure in SoftCup supernatants by quantifying prostate specific antigen (PSA) levels using ELISA. Luminex was used to measure 48 cytokines in SoftCup supernatants and the vaginal swabs were used for diagnosis of bacterial vaginosis by Nugent score. PSA, which denotes semen exposure within 48 hours prior to sampling, was detected in 19% (43/248) of SoftCup supernatants. Of the 43 PSA positive women, 70% (30/43) had self-reported condom use at their last sex act and 84% (36/43) had non-Lactobacillus dominant microbiota (Nugent score >7). In addition, PSA was significantly associated with prevalent bacterial vaginosis (Relative Risk (RR), 2.609; 95% Confidence Interval (CI), 1.104 - 6.165; p = 0.029), after adjusting for potential confounders such as age, STIs, current contraceptive use and condom use. Furthermore, women with detectable PSA had high median concentrations of MIP-1β (p=0.047) compared to those without PSA. These findings suggest that the presence of semen have a potential to alter the inflammatory response and microbial communities of the FGT, which may facilitate recruitment of HIV susceptible cells, resulting in increased susceptibility to HIV-1 infection.
Khanyisile Mngomezulu1, Cheryl Baxter1, Andile Mtshali1, Lenine Liebenberg1,2, Nigel Garrett1, Sinaye Ngcapu1,2
1Centre for the AIDS Programme of Research in South Africa (CAPRISA), Nelson Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa; 2Department of Medical Microbiology, University of KwaZulu-Natal, Durban, South Africa
Diverse microbial communities and inflammatory cytokine responses in the lower female genital tract (FGT) are closely associated with increased HIV risk, possibly through increasing mucosal HIV target cell frequency and T-cell activation. The presence of semen in the vagina during unprotected sex has been associated with short-term activation of mucosal immunity. Here, we investigated the extent to which partner semen impacts on cytokine and microbial profiles measured in 248 HIV-uninfected women at high risk for HIV infection. We assessed the semen exposure in SoftCup supernatants by quantifying prostate specific antigen (PSA) levels using ELISA. Luminex was used to measure 48 cytokines in SoftCup supernatants and the vaginal swabs were used for diagnosis of bacterial vaginosis by Nugent score. PSA, which denotes semen exposure within 48 hours prior to sampling, was detected in 19% (43/248) of SoftCup supernatants. Of the 43 PSA positive women, 70% (30/43) had self-reported condom use at their last sex act and 84% (36/43) had non-Lactobacillus dominant microbiota (Nugent score >7). In addition, PSA was significantly associated with prevalent bacterial vaginosis (Relative Risk (RR), 2.609; 95% Confidence Interval (CI), 1.104 - 6.165; p = 0.029), after adjusting for potential confounders such as age, STIs, current contraceptive use and condom use. Furthermore, women with detectable PSA had high median concentrations of MIP-1β (p=0.047) compared to those without PSA. These findings suggest that the presence of semen have a potential to alter the inflammatory response and microbial communities of the FGT, which may facilitate recruitment of HIV susceptible cells, resulting in increased susceptibility to HIV-1 infection.