Poster Abstracts - S618


Vaginal microbiome and birth weight in a Zimbabwean cohort

Identification: Mugabe, Muchaneta

Credits: None available.

Vaginal microbiome and birth weight in a Zimbabwean cohort
Muchaneta Gudza-Mugabe1,2,6*, Enock Havyarimana1, Shameem Jaumdally1, Christina Balle1, Katie Lennard1, Andrew Tarupiwa2, Fortunate Mugabe4, Rooyen Mavenyengwa3 Lindi Masson1, Heather Jaspan1,5
1University of Cape Town, South Africa; 2National Microbiology reference laboratory, Zimbabwe; 3University of Zimbabwe; 4Harare Central Hospital, Zimbabwe; 5Seattle Children's Research Institute, USA; 6Letten Foundation Research Centre
Poor birth outcomes are a major cause of child morbidity and mortality, particularly in developing countries.  Birth weight is an objective measure of birth outcomes, encompassing both preterm and growth restricted pregnancies. This study investigated the relationship between the vaginal microbiome during pregnancy and low birth weight (LBW).
Vaginal swabs were collected from 420 pregnant Zimbabwean women between 13-35 weeks of gestation who were then followed until delivery.  Nugent score was used to determine bacterial vaginosis (BV) status. DNA was extracted and sequencing of the 16S rRNA gene V4 hypervariable region was performed using Illumina MiSeq. Cytokine analyses were performed using Luminex.
There were 233 singleton pregnancies with complete sequencing and birth outcome data, of which 41 (17.6%) resulted in the delivery of LBW infants (<2500g). Women who delivered LBW infants had significantly lower vaginal alpha diversity than women who experienced normal births and were less likely to have BV compared to term births (Median Shannon indices 0.67 vs 1.05; p=0.02). However, no significant differences in beta diversity of Bray Curtis distances were evident between women who delivered LBW versus normal infants (ADONIS p= 0.06). The vaginal microbiota of the cohort clustered into three community types using Fuzzy clustering (C1 and C2 were Lactobacillus iners and crispatus dominant, respectively, whereas C3 was Gardnerella vaginalis dominated). Women who delivered LBW infants clustered mainly with C1.  Vaginal concentrations of multiple inflammatory cytokines and growth factors were significantly lower in women with LBW infants. In a multivariate model, preterm delivery, community type, pregnancy-induced hypertension and growth factors were independently associated with risk for LBW, after adjusting for important confounders such as maternal age.
In this cohort of Zimbabwean women, multiple factors measured during pregnancy were found to be predictive of birth weight, including preterm delivery, pregnancy-induced hypertension, a vaginal microbiome dominated by L. iners and a deficit of cellular growth factors.

Overview of Vaginal Practices in Nigerian Women, effects on Microbiome and Cervical Cancer

Identification: Nwaokorie, Francisca

Credits: None available.

Overview of Vaginal Practices in Nigerian Women, effects on Microbiome and Cervical Cancer
Francisca Nwaokorie*1, Oliver Ezechi2, Folasade Ogunsola3
1Department of Medical Laboratory Science; Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Nigeria; 2Clinical Science Division, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria, 3Department of Medical Microbiology and Parasitology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Nigeria.
*Corresponding author
There is increasing evidence that vaginal microbiome can influence women's reproductive health.  The presence of certain species are associated with the maintenance of a healthy vagina.  Disruption of the microbiota, due to unprotected sexual activities, as well as intravaginal practices may put women at a higher risk of acquiring sexually transmitted infection including Human immunodeficiency Virus/Acquired immune Deficiency Syndrome (HIV/AIDS) and cervical cancer. Prevalent high-risk HPV infection have been identified in Nigerian women with or without HIV infection. However, the pattern of microbiome and its association with cervical cancers and HIV infection which is endemic in Nigeria is not fully established. Vaginal products and or vaginal practices are used in cleansing, tightening and or drying the vagina. To what extent this contributes to vaginal microbiome is not well understood. This review provide information on composition of cultivable and non culturable bacterial species found in Nigerian women in health and disease conditions. It also assessed factors including traditional practices that may lead to changes in microbiome of women of reproductive age. Such requires studies to see what maybe their roles in vaginal microbiome and possible relationship to high risk human papilloma virus.  

Non-human primate model for genital tract microbiome research

Identification: Obiero, Jael

Credits: None available.

Non-human primate model for genital tract microbiome research
Jael Obiero1,2, Peter G. Mwethera1
1Institute of Primate Research, Department of Reproductive Health & Biology; 2Jomo Kenyatta University of Agriculture & Technology, Department of Medical Microbiology
Background: The vaginal microbiome is believed to influence host health by providing protection from pathogens and influencing reproductive outcomes such as fertility and gestational length. Knowledge of the composition of vaginal microbial ecosystem is essential for understanding the etiology, prevention, and treatment of vaginal diseases. A baboon model has been used to provide detailed understanding of reproductive physiology and immunology applicable to women. However, little is known about the composition of its vaginal microbial ecosystem.
Methods: Gram-stain and Nugent scores were used for assessment of baboon vaginal microbial flora. Biochemical identification and analysis of isolates were performed using the Analytical Profile Index kits and identification software.
Results: Species of Lactobacilli, Staphylococci, Clostridia, Bacilli, Corynebacteria, Gram-negative rods, other Gram-positive rods, cocci and Candida, were isolated. Healthy vaginal microbiota consisted mainly of lactobacillus morphotypes. Animals with high Nugent scores had increased number of Gram-positive cocci and variable rods, with increased number of Gram-negative morphotypes.
Conclusion: The baboon vaginal microbiota is heterogeneous in terms of species composition and is typified by a scarcity of lactobacilli. Characterisation of baboon vaginal microbial communities, their interactions and impact on reproductive outcomes warrant investigation.

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.

Increase in Lactobacillus and reduction in anaerobes in coronal sulcus during initial weeks following circumcision

Identification: Garson, Kirsty

Credits: None available.


Increase in Lactobacillus and reduction in anaerobes in coronal sulcus during initial weeks following circumcision
Garson KL1, Lennard K1,2, Botha G1, Havyarimana E2, Blakney A3, Curry L2,4, Harryparsad R2, Olivier AJ2, Otwombe K5, Dietrich J5, Wilson D6, Gray C2, Jaspan H2,7,8, Mulder N1
1Computational Biology Division, University of Cape Town, South Africa; 2Immunology Division, University of Cape Town, South Africa; 3Department of Bioengineering, University of Washington, USA; 4Division of Forensic Medicine and Toxicology, University of Cape Town, South Africa; 5Perinatal HIV Research Unit, Faculty of Health Sciences, University of the Witwatersrand, South Africa; 6Department of Internal Medicine, Edendale Hospital, University of KwaZulu-Natal, South Africa; 7Seattle Children's Research Institute, USA; 8Departments of Pediatrics and Global Health, University of Washington, USA
Higher abundances of various anaerobic bacteria present on the uncircumcised penis have been linked to increased risk of HIV acquisition. Male circumcision lowers the abundance of these taxa and reduces this risk. Many of these taxa are also linked to increased risk of HIV acquisition in the female genital tract, while Lactobacillus species are known to have a protective role.
To investigate how these key taxa adapt in the early stages after circumcision, coronal sulcus swabs were collected from 14-24 year-old South African males prior to, and at 2, 12 and 24 weeks after circumcision.
The relative abundance of various anaerobic genera were significantly reduced by 12 weeks post-circumcision. In some cases, such as Peptoniphilus, Anaerococcus and Prevotella, the decline was moderate and consistent over time, while changes in relative abundance of Porphyromonas and Peptostreptococcus were more erratic. Mobiluncus relative abundance showed a sudden decrease at 2 weeks, remaining relatively constant thereafter. Conversely, Lactobacillus had significantly increased in relative abundance at 12 weeks. The abundance of Peptoniphilus and Lactobacillus were among the most characteristic features of pre- and post-circumcision composition.
Numerous taxa of interest present in the coronal sulcus show marked changes in the weeks following circumcision, which varied by genus. Further insight could enhance methods to reduce HIV susceptibility in the genital tract.
Funding was provided by the South African National Research Foundation and the European & Developing Countries Clinical Trials Partnership.


Subclinical vaginal infections and adverse pregnancy outcome

Identification: Hansen, Anna

Credits: None available.

Subclinical vaginal infections and adverse pregnancy outcome
Hansen AS1, Maimburg RD1, Lindahl C1, Uldbjerg N1, Jensen JS2.
1Department of Obstetrics and Gynecology, Aarhus University Hospital, Aarhus, Denmark
2Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark

Aim: We aimed to investigate if a) certain subclasses of bacterial vaginosis and b) vaginal colonization with the Chlamydia-like bacterium, Waddlia chondrophila are associated with increased risk of spontaneous abortion or spontaneous preterm birth.
Background: Bacterial vaginosis (BV) has been associated with preterm birth as well as early miscarriage. However, only a minority of women with BV experience these outcomes. A better understanding of differences in the microbial composition may identify subclasses of BV that impart a higher risk than others.
W. chondrophila is an intracellular bacterium of the Chlamydiales class. It has been associated with abortion in bovines and may be involved in the etiology of spontaneous abortion in humans.

Materials and methods: An ongoing prospective cohort study including two cohorts; a) 1400 unselected pregnant women enrolled at 12 weeks of gestation (GA) and b) 330 pregnant women admitted with symptoms like bleeding and pain in early pregnancy, enrolled at a median of 7+5 weeks of GA. At inclusion, we obtained vaginal swabs and vaginal smears from all participants. The vaginal smears were Gram stained and classified according to the Nugent criteria for BV. The vaginal swabs will be examined for selected BV associated bacteria using species-specific quantitative PCR assays. Waddlia chondrophila will be identified using PCR. Pregnancy outcomes were collected from the electronic birth files. We used Fisher's exact test and chi-squared test for statistical analysis.
Preliminary results: Among the unselected pregnant women, 5% (57/1148) had BV (Nugent score ≥ 7) and 10% (114/1148) had intermediate scores (Nugent score 4-6). Within this cohort, 4.6% (56/1218) gave birth before 37+0 weeks of GA. This figure was 2.1% (3/144) among those with BV and 5.3% (46/871) among women without BV (OR 0.38; 95% CI 0.12 - 1.24).
Among the 330 women with symptoms in early pregnancy, 4.6% (3/65) had BV and 17% (11/65) women had intermediate scores. In this cohort, 62% (185/298) had a spontaneous abortion. This figure was 57% (8/14) among those with BV and 49% (25/51) among those without BV (OR 1.38; 95% CI 0.42 - 4.5)

Results from PCR analysis for BV associated bacteria and Waddlia chondrophila are pending and will be presented at the conference.
Funding sources: Danish Medical Research Council and Aarhus University

Lessons learnt from the first South African Health Products Regulatory Authority (SAHPRA)-approved probiotics trial to improve cure of bacterial vaginosis (BV) in a region with high BV and HIV rates

Identification: Happel, Anna-Ursula

Credits: None available.

Lessons learnt from the first South African Health Products Regulatory Authority (SAHPRA)-approved probiotics trial to improve cure of bacterial vaginosis (BV) in a region with high BV and HIV rates
Anna-Ursula Happel1, Ravesh Singh2,3, Nireshni Naidoo2,3, Koleka Mlisana2,3, Heather Jaspan1,4, Shaun Barnabas1, Jo-Ann Passmore*,1,2,5
*Corresponding author
1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa; 2National Health Laboratory Service, Cape Town, South Africa; 3Department of Medical Virology, University of KwaZulu Natal, South Africa; 4Seattle Children's Hospital, USA; 5SAMRC Gynaecological Cancer Research Centre, South Africa
BV is associated with genital inflammation, increased HIV risk and adverse reproductive outcomes. The standard of care for BV is antibiotics, although recurrence is high. In combination with antibiotics, biotherapeutics may improve efficacy and durability of BV treatment. No randomized trial comparing antibiotic treatment of BV to adjunctive vaginal probiotics has been conducted in South Africa, and no probiotic trial has yet been approved by SAHPRA.
A single-blind, randomized controlled trial in STI- BV+ women with abnormal vaginal discharge compared metronidazole (n=20) to metronidazole with a commercially available oral/vaginal probiotic, available over-the-counter (OTC) in South Africa (n=30). The primary endpoint was BV cure (assesed by Nugent Score) at one-month. Among screened women, the BV prevalence was 56.5 %, while 25.7% of BV+women also had a STI. Vaginal discharge was a poor predictor for BV. To date, 23/50 have completed the trial. Preliminary results showed a high rate of STI
aquistion during the study (20% incidence). The probiotic was well-accepted and no study-product related adverse events were reported. An interim analysis suggests no beneficial effects of the probiotic on BV cure and genital inflammation compared to metronidazole alone, although BV recurrence was lower. Similar concentrations of beneficial Lactobacillus spp. and a similar decrease in BV-associated spp. was found in both groups.
Although explicitly marketed for vaginal health, the probiotic tested did not contain Lactobacillus spp. typically found in the vagina. This product provided little benefit to improving BV cure compared to metronidazole alone, although recurrence was lower. Importantly, this first to be registered by SAHPRA trial has laid the path for furture probiotic testing in South Africa.

Metaproteomics Characterization of the Vaginal Microbiome in Pregnant Women

Identification: Hassan, Zaneera

Credits: None available.

Metaproteomics Characterization of the Vaginal Microbiome in Pregnant Women
Zaneera H. Hassan1, Myrna G. Serrano2, Jennifer M. Fettweis2-3, Kimberly K. Jefferson4, Gregory A. Buck2,4, Adam M. Hawkridge1,5
1Department of Pharmaceutics, 2Center for the Study of Biological Complexity, 3Department of Obstetrics and Gynecology, 4Department of Microbiology and Immunology, 5Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University (VCU), Richmond, VA
African American women have a disproportionately higher risk for pregnancy related complications such as preterm birth1, yet the cause(s) of this disparity is (are) currently unknown. The impact of the vaginal microbiome on pregnancy is poorly understood, but striking differences have been observed in African American women relative to other racial groups1. To better understand the complex interplay of the microbiome and pregnancy, we used high-performance mass spectrometry (LC-MS/MS) to map the vaginal metaproteomes in ten pregnant African American women participating in the VCU Multi-'Omic Microbiome Study-Pregnancy Initiative (MOMS-PI).  The LC-MS/MS analysis resulted in the identification of 1,331 proteins, ~67% identified as human while the remaining ~33% were bacterial. A total of 29 genera were matched to the bacterial proteins identified, with the most dominant organisms being from the genera Lactobacillus and Gardnerella. Label-free quantification of these proteins compared to microbial profiling by 16S ribosomal RNA (rRNA) sequencing show good agreement in many of the samples analyzed. This positive correlation, more apparent for abundant organisms than for less dominant taxa, is promising in our efforts to integrate these 'omics datasets. Our long-term goal is to implement quantitative LC-MS/MS method to characterize, stratify, and uncover complex microbiome-host pathways interactions in full-term and preterm phenotypes that could be used for early diagnosis and treatment.
  1. Fettweis, J. M., Brooks, J. P., Serrano, M. G., Sheth, N. U., Girerd, P. H., Edwards, D. J., Buck, G. A. (2014). Differences in vaginal microbiome in African American women versus women of European ancestry. Microbiology, 160(Pt_10), 2272-2282.

This work was supported by funds from the Global Alliance to Prevent Prematurity and Stillbirth, an initiative of Seattle Children's, the Bill & Melinda Gates Foundation, and the National Institutes of Health.

Strain-level variation in the microbiome of the female genital tract

Identification: Hayward, Matthew

Credits: None available.

Strain-level variation in the microbiome of the female genital tract
Matthew R. Hayward1, Seth M. Bloom1,2, Nomfuneko A. Mafunda1, Jiawu Xu1, Brittany A. Bowman1, Christina Gosmann1, Bjorn Corleis1, Mara Farcasanu1, Justin K. Rice1, Curtis Huttenhower2,3, Caroline M. Mitchell2 and Douglas S. Kwon1,2
1Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3The Broad Institute of MIT and Harvard, Cambridge, USA
The microbes colonising the female genital tract (FGT) influence multiple health outcomes including preterm birth, HIV acquisition and baseline inflammation. Most studies have profiled the microbiome through bacterial 16S rRNA gene sequencing; though informative, 16S-based taxonomic classification is limited to species-level resolution. Within-species genetic differences can be vast, with some species-level pangenomes (all the unique genes seen for a species) exceeding the size of any single strain's genome by orders of magnitude. Furthermore, 16S sequencing provides no functional information; placing a limit on our mechanistic understanding of disease associations.
To better characterize strain-level associations with major reproductive stages (pre-menopause, pregnancy, and post-menopause), BV and geography we sequenced 1000 primary bacterial isolates and generated shotgun metagenomes for 500 samples from North America and South Africa. We combined these data with >1800 curated reference genomes and >1000 publicly available metagenomic sequences. By integrating species-specific pangenomes and shotgun metagenomes we were able to assemble strain-level gene profiles. Profiles were partitioned using centroid based clustering to form strain-level groups containing similar gene complements. Functional annotation of the genes in these groups was used to identify potential strain-level phenotypes which differ between the FGT microbiome under different conditions.
We show that most FGT species possess a small core genome (~1000 genes) with an extensive pangenome (6000 to 30,000 genes) that varies between strains and encodes diverse metabolic functions. We show that some women are colonised by sub-species complexes (multiple distinct strains of the same species). We determine which strains form these complexes and relate them to primary isolates. These findings open avenues for mechanistic studies between the FGT microbiome and the reproductive health of the host.

Site-Specific Cytotoxicity and Epithelial Host Defense Mechanisms to Sneathia amnii Infection in the Female Reproductive Tract

Identification: Herbst-Kralovetz, Melissa

Credits: None available.

Site-Specific Cytotoxicity and Epithelial Host Defense Mechanisms to Sneathia amnii Infection in the Female Reproductive Tract
Paweł Łaniewski1, Adriana Tonachio1, Melissa Herbst-Kralovetz1,2,3
1Departments of Basic Medical Sciences and 2Obstetrics and Gynecology, College of Medicine-Phoenix, University of Arizona, Phoenix, AZ, USA; 3UA Cancer Center, University of Arizona, Tucson/Phoenix, AZ, USA
Sneathia species are emerging bacterial pathogens found in the lower and upper female reproductive tract (FRT). In recent clinical studies, we and others identified Sneathia spp. to be associated with serious gynecologic and obstetric sequelae, including cervical dysplasia/cancer, pelvic inflammatory disease and preterm birth. However, there is a fundamental gap that exists in understanding the functional impact of these bacteria throughout the FRT microenvironment that may contribute to disease and poor health outcomes. To determine the epithelial host response to Sneathia infection, we utilized two human three-dimensional (3-D) FRT epithelial cell models and a vaginal isolate Sneathia amnii strain Sn35. We used a reductionist approach and infected 3-D aggregates with S. amnii with a range of multiplicities of infection under anaerobic conditions. Morphological changes and host-bacteria interactions were visualized using scanning electron microscopy. Level of cytotoxicity was measured by lactate dehydrogenase assay. Acidic mucins were selectively stained by Alcian blue. Changes in gene expression were determined by real time quantitative PCR and gene expression arrays. S. amnii infection caused site-specific cytotoxicity compared to other bacterial vaginosis-associated bacteria. The 3-D endometrial aggregates infected with S. amnii exhibited a high level of cytotoxicity, which was not observed in the endocervical aggregates. Similarly, membrane blebbing, an indicator of apoptosis, was observed in 3-D endometrial cells following S. amnii infection. Bacteria frequently interacted simultaneously with multiple epithelial cells and localized in folds and crevices of 3-D aggregates. S. amnii also induced acidic mucin production in both endocervical and endometrial epithelial cells. Expression of proinflammatory cytokine genes were induced following S. amnii infection. Additionally, we showed that S. amnii infection altered expression of genes involved in regulating apoptosis and angiogenesis. These data suggest that S. amnii is a potential microbial driver of pathophysiological changes in the FRT that may contribute to obstetric and gynecological sequelae.