Poster Abstracts - S618


Lactobacillus crispatus growth on glycogen is dependent on its type 1 pullulanase gene variant

Identification: Hertzberger, Rosanne

Credits: None available.


Lactobacillus crispatus growth on glycogen is dependent on its type 1 pullulanase gene variant
Rosanne Y. Hertzberger1,2, Alicia Brandt2,3, Charlotte vd Veer4, Jorne Swanenburg1,5, Remco Kort1,5
1VU University Amsterdam, Netherlands, 2REBLAB, Netherlands, 3University of Groningen, Netherlands, 4Public Health Service, Amsterdam, Netherlands, 5TNO, Zeist, Netherlands
Glycogen is an abundant carbohydrate in the vagina of reproductive-age women. Levels of free glycogen vary depending on the makeup of the vaginal microbiota: women with bacterial vaginosis, a common dysbiosis with higher pH and few lactobacilli, generally have lower glycogen levels. This finding has led to the general assumption that lactobacilli depend on this glycogen and use it as a carbon source for colonization and acidification. However, direct evidence for this mechanism has been lacking sofar.
We studied growth, enzymatic activity and metabolite production of a group of 19 Lactobacillus crispatus strains that were isolated from reproductive age women. 13 out of these 19 isolates were able to grow on glycogen whereas 6 did not use glycogen for growth. Starch was used as a proxy to study glycogen breakdown and starch metabolism activity was found in both supernatant as well as in the washed pellets after growth on glycogen. Several strains did not show activity after growth on glucose indicating differential regulation of this metabolic pathway.
A survey of the genomes to track down any carbohydrate active enzymes showed the presence of a putative cell surface associated type 1 pullulanase. The gene is predicted to encode a 140 kDa enzyme consisting of an N-terminal signal peptide, two carbohydrate binding modules, a carbohydrate active module belonging to the GH13_13 family and a C-terminal S-layer associated protein (SLAP) domain. The gene shows high similarity to the extracellular cell wall attached pullulanase previously found in a human gut isolate of Lactobacillus acidophilus.
Although a copy of the pullulanase gene was found in the genomes of all 19 L. crispatus strains, the six non-glycogen consuming strains all carried one of three different mutations in the N-terminal signal peptide sequence, expected to disrupt transcription of at least part of the gene.
Our results show for the first time that certain vaginal isolates of Lactobacillus crispatus are capable of metabolizing one of the most abundant vaginal carbohydrates. We identify a pullulanase that may be essential for this activity. These findings bring us further to understanding the basic mechanisms of Lactobacillus colonization and acidification of the human vagina.
This project is part of REBLAB, an Open Kitchen Science initiative. For more information see


The first nearly-complete genomes of BVAB1 derived from vaginal metagenomes

Identification: Holm, Johanna

Credits: None available.

The first nearly-complete genomes of BVAB1 derived from vaginal metagenomes
Johanna B. Holm, PhD, Michael France, PhD, Elias McComb, Bing Ma, PhD, Rebecca Brotman, MPH, PhD, Jacques Ravel, PhD
University of Maryland, MD, USA
Bacterial Vaginosis Associated Bacteria 1 (BVAB1) is an elusive bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales, and as its name suggests, is often associated with bacterial vaginosis (BV). In reproductive health BV is of concern due to the elevated risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae acquisition as well as preterm birth . BVAB1 has been shown to be associated with BV persistence after metronidazole treatment and increased vaginal inflammation, and to confer a higher risk of puerperal infections. To date BVAB1 has not been cultivated, which has made it difficult to understand its diseases associated features. We present here the first seven near-complete genomes of BVAB1, derived from metagenomes of cervicovaginal lavages. These genomes were reconstructed from 7 separate vaginal metagenomes using MegaHit and Anvi'o, and average nucleotide identity was calculated using FastANI. The genomes were 1.5-1.7 Mb long, with an average of 1,527 genes, CheckM-estimated genome completion of 98.2%, and >98% average nucleotide identity to each other. These genomes will be a valuable resource and will contribute to our understanding of the heterogenous etiologies of bacterial vaginosis.

New Insight into the Ecological Role for Host-associated Glycans in the Reproductive Tract

Identification: Hood-Pishchany, Marguerite

Credits: None available.

New Insight into the Ecological Role for Host-associated Glycans in the Reproductive Tract
Marguerite I. Hood Pishchany1, MD, PhD, Seth Rakoff-Nahoum1,2, MD, PhD
1Division of Infectious Diseases, 2Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115
The vaginal microbiome is emerging as central to reproductive tract health, impacting the risk of infection, preterm birth, and HIV acquisition. Despite this importance, we know relatively little about the ecological drivers that shape the female reproductive tract (FRT) microbiome.  Nutrient utilization is both critical for niche occupation and is the driver of competitive and cooperative interactions in microbial communities. The FRT is replete with host-associated glycans in the form of glycoproteins, epithelial glycogen stores and the breakdown products of these glycans. We hypothesized that host-associated glycans drive environment, microbe-microbe and host-microbe interactions in the FRT. To test this hypothesis, we developed model systems to dissect the ecology and niche occupation of the human FRT microbiome and have begun to comprehensively define the landscape of nutrient utilization of FRT bacteria.  Demonstrating the power of these in vitro models, we have defined the carbohydrate utilization profiles of approximately 60 unique FRT isolates. Strikingly, while utilization of mono- and disaccharides tends to segregate by species, utilization of more complex carbohydrates represents a major source of strain-level variation within a given species. We hypothesized that host-associated glycan utilization represents an adaptation to the vaginal environment, and tested the competitive fitness of our bacterial isolates in model, multispecies communities. In these model, polymicrobial communities, we found that bacterial isolates that encode the ability to degrade specific host polysaccharides, gained a competitive advantage over isolates of the same species that lacked these traits. In summary, we have developed robust, high-throughput methods for studying FRT isolates in both axenic and multispecies cultures. Using these methods, we have determined that glycan utilization traits vary at both the species and strain level among FRT bacteria. Importantly, strain-level variability in glycan utilization contributes to competitive fitness of these isolates in complex community, suggesting that these traits could influence community stability or persistence in vivo.

A Continuum of the Microbiome in the Urogenital Tract and Factors Influencing the Microbial Composition

Identification: Jia, Huijue

Credits: None available.


A Continuum of the Microbiome in the Urogenital Tract and Factors Influencing the Microbial Composition
Chen Chen1,2, Fei Li1,2,3, Zirong Wang2, Juanjuan Dai4,5, Xiaolei Song1,3, Lilan Hao1,2, Liju Song1,2, Weixia Wei4,5, Xiaowei Zhang1,2, Liping Zeng4,5, Hui Du4,5, Huiru Tang4,5, Na Liu6, Huanming Yang1,9, Jian Wang1,9, Lise Madsen1,7,10, Susanne Brix1,11, Karsten Kristiansen1,7, Xun Xu2,3, Junhua Li1,2,8, Ruifang Wu4,5, Huijue Jia1,2,8,12
1BGI-Shenzhen, Shenzhen 518083, China; 2China National GeneBank, BGI-Shenzhen, Shenzhen 518120, China; 3BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China; 4Peking University Shenzhen Hospital, Shenzhen 518036, China; 5Shenzhen Key Laboratory on Technology for Early Diagnosis of Major Gynecological diseases, Shenzhen, PR China; 6BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China; 7Laboratory of Genomics and Molecular Biomedicine, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark; 8Shenzhen Key Laboratory of Human Commensal Microorganisms and Health Research, BGI-Shenzhen, Shenzhen 518083, China; 9James D. Watson Institute of Genome Sciences, Hangzhou310000, China; 10Institute of Marine Research (IMR), Postboks 1870, Nordnes, N-5817, Bergen, Norway; 11Department of Biotechnology and Biomedicine, Technical University of Denmark, Soltofts Plads, 2800 Kongens. Lyngby, Denmark; 12Macau University of Science and Technology, Taipa, Macau 999078, China
The vaginal microbiota is typically dominated by Lactobacilli, and the existence of a microbiota beyond the vagina is often shadowed by infections during the course of pregnancy. In a relatively large cohort of reproductive age women who were operated for conditions not known to involve infection, we demonstrate a continuum of microbiota till around the ovaries. Fungal and viral sequences were also detected in the upper reproductive tract. Microbial composition and potential functions in the urine of the same individuals showed similarity to those in the reproductive tract, and the urine could possibly be used to detect diseases in the upper reproductive tract.
In a healthy young cohort, the proportion of individuals with a non-Lactobacilli type is generally consistent with previous reports, with new subtypes. We further explore associations between the human genome and the microbiome (M-GWAS), and the possibility of restoring a healthy reproductive tract microbiome through oral probiotics.


Prevalence and susceptibility of BV-associated Gardnerella vaginalis biotypes in a cohort of pregnant women attending antenatal clinics in the Western Cape

Identification: Jooste-Vraagom, Vanessa

Credits: None available.

Prevalence and susceptibility of BV-associated Gardnerella vaginalis biotypes in a cohort of pregnant women attending antenatal clinics in the Western Cape
Jooste-Vraagom V, Arendse M, Abrantes P, Africa CWJ.
Department of Medical Biosciences, University of the Western Cape, Robert Sobukwe Road, Bellville, Western Cape
Introduction: Bacterial vaginosis (BV) is characterized by a characteristic discharge, increased vaginal pH, squamous vaginal epithelia with granular borders, called 'clue cells'.and a reduction in Lactobacillus species.  
BV has repeatedly been associated with adverse pregnancy outcomes, including preterm delivery, late miscarriage and premature rupture of membranes, infection of the chorion, amnion, amniotic fluid and pelvic inflammatory disease
Objectives: This study sought to detect the prevalence of BV and the antimicrobial susceptibility of Gardnerella vaginalis in a cohort of pregnant women in the Western Cape.
Materials and Methods: Vaginal samples were collected without a speculum using a sterile cotton swab and transported to the laboratory in Amies media with charcoal (18114CST, Sterlin)
Vaginal swabs were processed within 2 hours of sampling and microscopically scored according to Nugent criteria (Nugent et al, 1991). The presence of G. vaginalis was determined by culture on enriched media incubated anaerobically for 48-72 Hours at 37°C. Susceptibility of G. vaginalis to metronidazole and clindamycin were determined using disc-diffusion.
Results: Gram stain analysis of vaginal swabs revealed that 32% of the study population harboured vaginal flora indicative of BV, with G. vaginalis resistance to metronidazole observed in 49% of BV cases.  
Discussion: Nugent scoring showed good specificity and sensitivity in diagnosing BV with metronidazole-resistant G. vaginalis biotype 7 most frequently detected.  
  1. Nugent R. P, Krohn M. A, Hiller S. L. reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. Journal of Clinical Microbiology. 1991; 29 (2): 297-301.


IL-33 induced by dysbiosis in vaginal microbiota contributes to impaired antiviral immunity against herpes simplex virus infection

Identification: Lee, Heung Kyu

Credits: None available.


IL-33 induced by dysbiosis in vaginal microbiota contributes to impaired antiviral immunity against herpes simplex virus infection
Ji Eun Oh1, Heung Kyu Lee1
1Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
Commensal microbiota are well known to play an important role in antiviral immunity by providing immune inductive signals; however, the consequence of dysbiosis on antiviral immunity remains unclear. We demonstrate that dysbiosis caused by oral antibiotic treatment directly impairs antiviral immunity following viral infection of the vaginal mucosa. Antibiotic-treated mice succumbed to mucosal herpes simplex virus type 2 infection more rapidly than water-fed mice, and also showed delayed viral clearance at the site of infection. However, innate immune responses including type I interferon and proinflammatory cytokine production at infection sites, as well as induction of virus-specific CD4 and CD8 T cell responses in draining lymph nodes, were not impaired in antibiotic-treated mice. By screening the factors controlling antiviral immunity, we found that interleukin-33, an alarmin released in response to tissue damage, was secreted from vaginal epithelium after the depletion of commensal microbiota. This cytokine suppresses local antiviral immunity by blocking the migration of effector T cells to the vaginal tissue, thereby inhibiting the production of interferon-γ, a critical cytokine for antiviral defense, at local infection sites. These findings provide insight into the mechanisms of homeostasis maintained by commensal bacteria, and reveal a deleterious consequence of dysbiosis in antiviral immune defense.


Developing A Large Collection of Vaginal Microbiota Assays Using TaqManTM Chemistry on High Throughput Nano-Microfluidic OpenArrayTM

Identification: Li, Kelly

Credits: None available.

Developing A Large Collection of Vaginal Microbiota Assays Using TaqManTM Chemistry on High Throughput Nano-Microfluidic OpenArrayTM
Kelly Li, Sunali Patel, Nicole Fantin, Ioanna Pagani, Nitin Puri, Evan Diamond, Kamini Varma 
Genetic Analysis R&D, Life Science Division, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080 
Imbalance of vaginal flora has been implicated in vaginal infections, like bacterial vaginosis (BV), vulvovaginal candidiasis (VC) and Trichomonas vaginitis.  These infections are associated in pre-term birth, infertility, and increased risk of sexually transmitted diseases (STD).  Studying vaginal microbiota, their dynamics and imbalance provides insights into women health. Current technologies for vaginal microbial studies are costly, lack sensitivity/specificity, or require lengthy workflows. Conventional qPCR-based detection is powerful, but lacks of target throughput. To address these unmet needs, we have developed a large collection of TaqManTM microbiota assays by leveraging TaqManTM chemistry and high throughput OpenArrayTM technology. We targeted over 30 healthy or pathogenic species in vaginal microbiota, including bacteria, fungi, protozoa and viruses. We identified unique gene targets that are species-specific and designed TaqManTM assays using proprietary design pipeline. These assays were evaluated with synthetic and ATCC genomic DNA (gDNA) controls on OpenArrayTM plate, a microscope slide with 3,072 through-holes. The assays were evaluated with each of their controls. The accuracy revealed that all the assays display expected on-target Ct values with their corresponding controls. For specificity, each assay was tested against all the rest of synthetic controls as well as inclusivity and exclusivity panels of ATCC gDNA samples. No significant cross-reactivity was observed. Linearity and analytical sensitivity studies revealed that at least 5 log linear dynamic range (with R2 >0.99) with limit of detection (LOD) down to ~100 copies for all and even lower for some of the analytes. In summary, we developed a large collection of TaqManTM vaginal microbiota assays.  We have demonstrated excellent assay performance of accuracy, sensitivity, specificity, and reproducibility. In conjunction with OpenArrayTM platform, the application enables researchers to study vaginal microbiota in a single reaction with a simple workflow, fast turnaround time, and high throughput yet flexible sample/target combinations. 
For Research Use Only.
Not for use in diagnostic procedures

Immunoregulatory properties of clinical Lactobacillus isolates from South African women at high risk of HIV acquisition

Identification: Manhanzva, Monalisa

Credits: None available.

Immunoregulatory properties of clinical Lactobacillus isolates from South African women at high risk of HIV acquisition
Monalisa T. Manhanzva1, Andrea Abrahams1, Hoyam Gamieldien1, Heather Jaspan1,2, Shameem Jaumdally1, Shaun L. Barnabas1, Smritee Dabee1, Lindi Masson1,3*
1Institute of Infectious Disease and Molecular Medicine (IDM), University of Cape Town, Cape Town, South Africa; 2Seattle Children's Research Institute, University of Washington, Seattle, Washington, USA; 3Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa
Background: Female genital tract (FGT) inflammation increases HIV infection susceptibility. Bacterial vaginosis (BV), characterised by depletion of Lactobacillus species and an overgrowth of BV-associated bacteria, is associated with genital tract inflammation. Lactobacillus species may protect against HIV partly by reducing inflammation in the FGT. We aimed to evaluate immunoregulatory properties of vaginal Lactobacillus isolates from South African women.
Methods: Eighty lactobacilli isolated from cervicovaginal fluid collected from BV positive and negative women were identified to species level by MALDI-TOF biotyping. We measured production of cytokines (IL-6, IL-8, IL-1α, IL-1β, IP-10, MIP-3α, MIP-1α, MIP-1β, IL-1RA) by vaginal epithelial cells in response to each Lactobacillus isolate, as well as Gardnerella vaginalis, in the presence or absence of lactobacilli using Luminex. We evaluated Lactobacillus growth rates, adhesion, culture pH, D-lactate and L-lactate production.
Results: Lactobacilli from BV positive women were more inflammatory than those from BV negative women with respect to IL-6, IL-8, IL-1, IL-1, MIP-1 and MIP-1 production. All lactobacilli suppressed IL-6 [adjusted (adj.) p=0.0003] and IL-8 (adj. p=0.0170) responses to G. vaginalis. Overall, lactobacilli from BV negative women suppressed inflammatory cytokine responses to G. vaginalis to a greater degree than isolates from BV positive women. Lactobacilli adhesion correlated negatively with IL-6, IL-8, MIP-1, IL-1 and IL-1RA, while IL-6 concentrations were inversely associated with D-lactate production.
Conclusion: Vaginal lactobacilli suppress inflammatory responses to G. vaginalis, and may decrease HIV acquisition risk by reducing inflammation in the FGT. Lactobacilli from BV positive women were more inflammatory compared with lactobacilli from BV negative women.

Iqela Lokutya – The Masi food club to engage adolescents in food literacy and functional food promotion for vaginal health

Identification: Martin, Tamlyn

Credits: None available.

Iqela Lokutya - The Masi food club to engage adolescents in food literacy and functional food promotion for vaginal health
Tamlyn S. Martin1, Felicity Hartley1, Marjanne Sennekal2, Marieke Theron2, Ramla Tanko1,3, Anna K. Coussens3,4, Smritee Dabee1,3, Dante Robbertze5, Katherine Gill5, Heather B. Jaspan3,6, Linda-Gail Bekker3,5, Virginia MacKenny7, Jo-Ann S. Passmore1,3,8,9
1Division of Medical Virology, University of Cape Town, Cape Town, South Africa; Human Nutrition, 2Department of Human Biology, University of Cape Town, Cape Town, South Africa; 3Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 4Walter and Eliza Hall Insstitute of Medical research, Parkville, Australia; 5Desmond Tutu HIV Foundation, University of Cape Town, Cape Town, South Africa; 6Seattle Childrens Hospital, Seattle, USA; 7Michaelis School of Fine Art, University of Cape Town, Cape Town, South Africa; 8SAMRC-UCT Gynaecological Cancer Research Centre, University of Cape Town, Cape Town, South Africa; 9National Health Laboratory Service, Cape Town, South Africa
In a setting of high HIV and human papillomavirus (HPV) prevalence, adolescent and young people living in South Africa are at high risk for infectious diseases of the mucosa. Modifiable biomedical risk factors, including compromised mucosal barrier function resulting from vitamin D deficiency and microbial dysbiosis, likely play a key role. Functional foods, including probiotic-rich and fermented products, offer both mucosal and systemic health benefits and are increasingly being implemented for gastrointestinal disorders.  Using a multi-disciplinary approach, we have started the “Iqela Lokutya” (isiXhosa - Food Club) to engage adolescents from socioeconomically-deprived communities around Cape Town in ecological and microbial principles of functional (probiotic-rich) food preparation, including relational aesthetics-based methodology.  To date, more than 100 adolescent males and females from Masiphumelele have participated in “Iqela Lokutya”, run as a weekly program at the Desmond Tutu HIV Foundation Youth Centre. At baseline, we found that the majority of participants (57%) were vitamin D3 deficient (median ~47 nmol/L), with 8% being severely deficient (<30nmol/L), assessed by liquid chromatography-tandem mass spectrometry (to differentiate vitamin D2 and D3) in plasma. A food frequency questionnaire demonstrated that high sugar products accounted for the majority of food items were consumed per week in this age group followed by high-fat snacks. In contrast, foods rich in probiotics (such as yoghurts and other fermented dairy, traditional beer); and foods rich in vitamin D (including oily fish, fortified margarines, breakfast cereals, eggs, mushrooms, cream, cheese and mayonnaise, beef, yogurt or fermented dairy) were rarely consumed. We propose to use Food club as the spring board to introduce both nutritional, ecological, aesthetic, and microbiological aspects of food preparation to integrate science engagement (microbes and fermentation), entrepreneurship, to bridge the disciplines of medicine, engineering, art and science. The ultimate aim of this new initiative is to improve vitamin D status, food literacy and reproductive health in young women in South Africa.

SIV susceptibility in the female genital tract of adolescent versus adult pigtail macaques

Identification: Miller, Charlene

Credits: None available.

SIV susceptibility in the female genital tract of adolescent versus adult pigtail macaques
Charlene Miller1, Mariluz Arainga3, Ernesto Coronado2, Tiffany Hensley-McBain1, Jennifer Manuzak1,2, Alicia Berard4, Adam Burgener4, Thomas J Hope5, Francois Villinger3, Nichole R Klatt1
1Department of Pediatrics, University of Miami, Miami, FL, USA
2Washington National Primate research Center, University of Washington, Seattle, WA, USA
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA, USA
4Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
5Department of Cell & Molecular Biology, Northwestern University, Chicago, IL, USA
In southern Africa, young women aged 15-24 contribute to nearly 30% of all new HIV infections. Although social, high-risk behavioral, access to healthcare services, and economic factors all contribute to increased HIV susceptibility in young women, biological factors are thought to play a significant role. Here, we use the pigtail macaque (PTM) model to investigate signatures of susceptibility in the female genital tract (FGT) of adolescent versus adults, in hopes to develop an adolescent NHP model to test altered susceptibility to HIV infection.
Comprehensive FGT samples were collected across the menstrual cycle over a two-month period in 10 adult and 10 adolescent PTMs, followed by multiple low-dose (LD) intravaginal SIV challenges. Flow cytometry was performed on vaginal biopsies to assess frequency and phenotype of HIV target cells. Inflammatory markers were measured from cervicovaginal cytobrush supernatant via Luminex assay. 16S sequencing was performed on vaginal swabs throughout the sex cycle leading up to SIV challenges.
Our model identified no significant differences in SIV susceptibility to LD intravaginal challenges between adolescent and adults PTMs. No significant differences in HIV target cells were detected in the vaginal tissue of adolescent and adult PTMs. Although, significantly more HLA-DR+ CD20+ B cells in the vagina were detected in adolescent PTMs when compared to adults (p= 0.0435). In contrast, significantly more CD8+ T cells were detected in adult PTMs (p= 0.0369). Several inflammatory markers assessed from cervicovaginal cytobrush supernatant were higher in the adult PTMs compared to adolescents such as, sCD40L, MCP-1, IL-12p40 and TNFa (p=0.0220, p=0.0041, p=0.0789, p=0.0789 respectively). Significant microbial community differences were detected between adolescent and adult PTMs.
Our PTM model did not reveal the enhanced HIV susceptibility that is seen in adolescent females, which is supported by our negative data of any mucosal characteristics of increased SIV susceptibility in adolescent PTMs. A validated SIV NHP model of HIV infection in adolescents is currently absent and is critically needed due to the ethical challenges of enrolling women under 18 years of age in HIV clinical trials. Further validation in the FGT of NHPs is critical in better understanding vaginal transmission and increased susceptibility in adolescent females.