Having IMPACTT 3:
Advancing Microbiome Research
July 10-12, 2023
Selected Abstracts
Selected Abstracts for Oral and Poster Presentation
See below for the abstracts selected for oral and poster presentations for our Having IMPACTT 3: Advancing Microbiome Research Symposium on July 10-12, 2023 at the Malcolm Hotel in Canmore, AB, Canada.
Presenters
Oral Presenters
Session 1: Microbiome Beyond Bacteria (Monday, July 10, 2023)
Diana Rojas
Aviva Rotter-Maskowitz
Session 3: Microbiome Across Life Stages (Tuesday, July 11, 2023)
Dr. Amee Manges
Dr. Van Ortega
Session 4: Microbiome & Nutrition (Tuesday, July 11, 2023)
Dr. Thierry Mallevaey
Erin Strachan
Session 5: Omics & Bioinformatics (Wednesday, July 12, 2023)
Ben Grodner
Dr. John Parkinson
Flash Talk & Poster Presenters
Flash Talk & Poster Session #1 (Monday, July 10, 2023)
Monica Alvaro Fuss (Poster 1)
Dale Archer (Poster 2)
Ben Fisher (Poster 3)
Mackenzie Gutierrez (Poster 4)
Dr. Natasha Haskey (Poster 5)
Courtney Hoskinson (Poster 6)
Flash Talk & Poster Session #2 (Tuesday, July 11, 2023)
Eszter Pal (Poster 45)
Kenny Shin (Poster 46)
Dr. Matthew Sorbara (Poster 47)
Gillian Tanabe (Poster 48)
Dr. Carolyn Thomson (Poster 49)
Dr. Weilan Wang (Poster 50)
Poster Presenters
Poster Session #1 (Monday, July 10, 2023)
Spencer Abbott (Poster 7)
Dr. Sima Afsharnezhad (Poster 8)
Dr. Shokouh Ahmadi (Poster 9)
Nazanin Arjomand-Fard (Poster 10)
Jérémie Auger (Poster 12)
Abimbola Badejo (Poster 13)
Sahar Bagheri (Poster 14)
Delaney Barth (Poster 15)
Dr. Evandro Beraldi (Poster 16)
Dr. Dominique Bihan (Poster 17)
Dr. Kirsty Brown (Poster 18)
Dr. Larissa Celiberto (Poster 19)
Catherine Chan (Poster 20)
Diana Changirwa (Poster 21)
Leah D’Aloisio (Poster 22)
Darlene Dai (Poster 23)
Dr. Marcela Davoli-Ferreira (Poster 24)
Dr. Vanessa DeClercq (Poster 25)
Dr. Dhwani Desai (Poster 26)
Pedro Dimitriu (Poster 27)
Liz-Audrey Djomnang (Poster 28)
Peter Dobranowski (Poster 29)
Breenna Dobson (Poster 30)
Toluwatise Ehindero (Poster 31)
Dr. Han Fang (Poster 32)
Zhi Yi Fang (Poster 33)
Bradley Fitzgerald (Poster 34)
Dr. Fabien Franco (Poster 35)
Dr. Thaís Glatthardt (Poster 36)
Dr. Simon Hirota (Poster 37)
Dr. Aline Ignacio (Poster 38)
Maira Jiménez-Sanchez (Poster 39)
Wasif Khan (Poster 41)
Dr. Yeganeh Khaniani (Poster 42)
Reihane Khorasaniha (Poster 43)
Pardis Kiani (Poster 44)
Poster Session #2 (Tuesday, July 11, 2023)
Dr. Larisa Kovtonyuk (Poster 51)
Samuel Lawal (Poster 52)
Dr. Karen Lithgow (Poster 53)
Dana Lowry (Poster 54)
Yao Lu (Poster 55)
Madeline Mellet (Poster 56)
Emily Mercer (Poster 57)
Dr. Selene Meza-Perez (Poster 58)
Dr. Chunlong Mu (Poster 59)
Kevin Muirhead (Poster 60)
Hana Olof (Poster 61)
Dr. Mona Parizadeh (Poster 62)
Dr. John Parkinson (Poster 63)
Dr. Sarah Piché-Choquette (Poster 64)
Erika Prando Munhoz (Poster 65)
Dr. Charlène Roussel (Poster 66)
Mahana Sabachvili (Poster 67)
Maricarmen Salas-López (Poster 68)
Jared Schlechte (Poster 69)
Shrushti Shah (Poster 70)
Shrushti Shah (Poster 71)
Sara Shama (Poster 72)
Dr. Saif Sikdar (Poster 73)
Isla Skalosky (Poster 74)
Sara Stickley (Poster 75)
Dr. Lena Takayasu (Poster 76)
Dr. Marcel van de Wouw (Poster 77)
Erik van Tilburg Bernardes (Poster 78)
Dr. Jennifer Vena (Poster 79)
Sasanka Weerasingha (Poster 80)
Logan Wisteard (Poster 81)
Anthony Wong (Poster 82)
Dr. Robyn Wright (Poster 83)
Wendy Zhang (Poster 84)
Dr. Reda Albadawy (Poster 85)
Abstracts
Oral Presenters
Diana Rojas (Session 1: Microbiome Beyond Bacteria)
Graduate Student, CINVESTAV (Mexico)
Characterization of the archaeal community bound by immunoglobulins in colostrum and newborn feces
Diana Laura Rojas-Guerrero, Karina Corona-Cervantes, Carmen Josefina Juárez-Castelán, Alberto PiñaEscobedo, Bulmaro Cisneros-Vega, Leopoldo Santos-Argumedo, Tito Ramírez-Lozada, Gustavo AcostaAltamirano, Mónica Sierra-Martínez, Paola Berenice Zárate-Segura, Jaime García-Mena
Archaea, the most recently discovered prokaryote lineage, were thought to inhabit only extreme environments until they were isolated from human feces in the early 80s. For instance, the presence of methanogenic archaea has been demonstrated in human milk, meconium, and neonatal stool obtained from healthy donors, suggesting they are pioneer colonizers of the human gut. However, we do not know their mechanisms of colonization. In the case of bacteria, the better-known human-associated prokaryotes, they are coated by maternal immunoglobulins in human colostrum and are vertically transmitted to the newborn during breastfeeding. These bacteria might train the newborn’s developing immune system to tolerate specific symbionts that will initiate the colonization in the gut. Our objective was to describe the taxonomic composition of archaea differentially bound by immunoglobulin A (subclass 1 and 2), G, and M, in human colostrum and neonatal stool. With this purpose, we used monoclonal antibodies to label the immunoglobulin-coated archaea, isolating them as complexes using magnetically activated cell sorting and carrying 16S rRNA amplicon sequencing. We found that members of the genera Methanosarcina and Methanoculleus were the most abundant and densely coated archaeal taxa in colostrum and neonatal stool, whereas some taxa appeared to escape immunoglobulin-coating in colostrum but not in the neonatal stool. Interestingly, non-classified archaea are bound by IgM and IgG in colostrum but by IgA1 and IgA2 in stool samples, which suggests differential recognition patterns in colostrum that may shift under the environmental conditions in the newborn’s gut. Additionally, functional metagenome prediction shows that while archaea in colostrum are associated to anaerobic respiration (i.e. methanogenesis), nucleotide and amino acid biosynthesis are the most abundant pathways in newborn feces. Acknowledgments. Work supported by CONACyT 163235 INFR-2011-01 and CONACyT FORDECYT-PRONACES/6669/2020_Programa Presupuestario F003-Ciencia de Frontera 2019.
Aviva Rotter-Maskowitz (Session 1: Microbiome Beyond Bacteria)
PhD Student, Weizmann Institute of Science (Israel)
Bacteria Mediated ALKi Chemoresistance in EML4-ALK fusion NSCLC
Aviva Rotter Maskowitz,Deborah Nejman Dan Raz, Ravid Straussman
Targeted therapies have revolutionized the management of non-small cell lung cancer (NSCLC). For ALK-rearranged NSCLC, several ALK inhibitors have been developed. Despite these advances, partial response and acquired resistance to ALK inhibition therapy is still the rule. The tumor microenvironment (TME) is an essential factor in modulating the response of cancer cells to anti-cancer therapy. We have shown previously that the TME harbors various bacteria and that these bacterial populations are tumor-type specific. To assess bacterial-mediated resistance in ALK mutated NSCLC, we performed a large in vitro screen that tested the response of an EML4-ALK fusion NSCLC cell line to the ALK inhibitor, crizotinib in the presence of pre-conditioned media (PCM) of 50 bacterial species (prioritized by their presence in human tumors). One bacterium robustly abrogated the therapeutic effect of crizotinib. An additional 124 bacterial species have been tested and demonstrated that this resistance to crizotinib is genus-specific. Furthermore, this resistance phenotype is seen using multiple ALKi drugs, such as alectinib and lorlatinib, as well as in multiple ALK-rearranged cancer cell lines. The addition of bacterial PCM did not affect ALK phosphorylation levels. However, pAKT and pERK levels are increased when bacterial PCM is added to ALKi-treated EML4-ALK cells, indicating the activation of a bypass pathway. We continue to investigate and uncover the mechanisms whereby the tumour microbiome affects the response to anti-cancer drugs in the expectation that this will lead to improved patient care.
Dr. Amee Manges (Session 3: Microbiome Across Life Stages)
Professor & Division Head, University of British Columbia (Canada)
The gut microbiome and early-life growth in a population with high prevalence of stunting
Ruairi C. Robertson, Thaddeus J. Edens, Lynnea Carr, Kuda Mutasa, Ethan K. Gough, Ceri Evans, Hyun Min Geum, Iman Baharmand, Sandeep K. Gill, Robert Ntozini, Laura E Smith, Bernard Chasekwa, Florence D. Majo, Naume V. Tavengwa, Batsirai Mutasa, Freddy Francis, Joice Tome, Rebecca J. Stoltzfus, Jean H. Humphrey, Andrew J. Prendergast, Amee R. Manges
Stunting affects one-in-five children globally and is associated with greater infectious morbidity, mortality and neurodevelopmental deficits. Recent evidence suggests that the early-life gut microbiome affects child growth through immune, metabolic and endocrine pathways. Using whole metagenomic sequencing, we map the assembly of the gut microbiome in 335 children from rural Zimbabwe from 1–18 months of age who were enrolled in the Sanitation, Hygiene, Infant Nutrition Efficacy Trial (SHINE; NCT01824940), a randomized trial of improved water, sanitation and hygiene (WASH) and infant and young child feeding (IYCF). Here, we show that the early-life gut microbiome undergoes programmed assembly that is unresponsive to the randomized interventions intended to improve linear growth. However, maternal HIV infection is associated with over-diversification and over-maturity of the early-life gut microbiome in their uninfected children, in addition to reduced abundance of Bifidobacterium species. Using machine learning models (XGBoost), we show that taxonomic microbiome features are poorly predictive of child growth, however functional metagenomic features, particularly B-vitamin and nucleotide biosynthesis pathways, moderately predict both attained linear and ponderal growth and growth velocity. New approaches targeting the gut microbiome in early childhood may complement efforts to combat child undernutrition.
Dr. Van Ortega (Session 3: Microbiome Across Life Stages)
Postdoctoral Fellow, University of Calgary (Canada)
The influence of early-life gut microbiome disruption on HPA axis dysregulation and allergic asthma
Van A. Ortega, Colin Mackenzie, Fernando A. Vicentini, Muhammad Hafeez, Shirin Moossavi, Mona Parizadeh, Erik van Tilburg Bernardes, Veronika Kuchařová, Emily Mercer, Kristen Kalbfleisch, Jumana Samara, Keith A. Sharkey, Marie-Claire Arrieta
Asthma is a major inflammatory disease of the bronchial airways that affects 1 in 8 Canadian children. While the underlying reasons are not fully understood, neonatal disruption of the gut microbiome (via stress and/or antibiotics) during postnatal-developmental periods are believed to be triggers for asthma development in adult life. We explored the influence of early-life microbiome disruption on gut-to-brain signaling pathways involved in programming the hypothalamic-pituitary-adrenal (HPA) stress axis by measuring brain microglia influence on stress-axis development/regulation and ovalbumin-induced airway inflammation (i.e. asthma) susceptibility in adult mice that were treated as pups with two antibiotic cocktails (augmentin-clavulanate or broad spectrum), while also inducing maternal separation stress for 21-days post-birth to alter microbiome communities. We observed microbial alterations from the combined or individual effects of antibiotics and chronic stress at both 21 and 80-days post-birth. These included increased alpha-diversity and relative abundances of Staphylococcus, Streptococcus, Klebsiella and Escherichia in stressed mice treated with broad spectrum at 21-days post-birth, with stress alone significantly altering microbiome alpha-diversity at 21 days. While many of the changes in relative abundance at 21 days were maintained into adulthood at 80-days post-birth, the high alpha diversity in the broad spectrum antibiotic group at 21 days, dropped to the lowest by 80 days, showing lasting effects from early-life antibiotic exposure. Early-life stress and/or antibiotic exposure also resulted in disrupted levels of glucocorticoid hormones (end-products of HPA stress activation) at 21 days, higher proportions of activated microglia at weaning age, with reduced immune responses to lipopolysaccharide, and altered dendritic and macrophage splenocyte populations. Early-life exposure to stress and/or augmentin-clavulanate also increased the susceptibility of adult mice to allergic asthma at 80 days. These results demonstrate that the microbiome is a major factor regulating normal development of the neuroendocrine stress axis, which can increase asthma susceptibility via neuroimmune mechanisms.
Dr. Thierry Mallevaey (Session 4: Microbiome & Nutrition)
Associate Professor, University of Toronto (Canada)
Microbiota – iNKT cell interactions: a one-way street?
Meggie Kuypers, Qiaochu Lin, Tijana Despot, Carolina de Amat Herbozo, Dana Philpott and Thierry Mallevaey
Invariant Natural Killer T (iNKT) cells are unconventional T cells that bridge innate and adaptive immune responses, and exert rapid helper and cytotoxic responses following activation. iNKT cells are very conserved in mammalian species, but their prevalence and responses are heterogenous in humans. These cells are thought to contribute to intestinal homeostasis. However, the role they play in the development or control of intestinal inflammation is not fully understood. iNKT cells are specialized in the recognition of glycollipid antigens presented by the major histocompatibility class Ib molecule CD1d. Such lipid antigens have been isolated from several pathogenic and commensal microbial species. iNKT cells are also activated indirectly, following the detection of microbial-associated molecular patterns (MAMPs). Here, we show that iNKT cell homeostasis and cytokine secretion is affected in germ-free mice. More subtle manipulations of microbiota composition through fecal transplant had minimum impact on iNKT cell tissue distribution, effector differentiation and cytokine secretion. However, we show that the transcriptional programs of iNKT cells at steady state and following activation in vivo are modulated by the microbiota. Finally, iNKT cell responses are severely blunted in pet store outbred mice, as well as in C57BL/6 mice reconstituted with the microbiota of pet store mice, compared to specific pathogen-free mice. Conversely, we show that the presence of iNKT cells or their specific activation has little impact on microbiota composition. Our findings revealed that microbiota composition impacts iNKT cell biology, and may provide a rationale to understand the heterogenous iNKT cell responses observed in clinical settings.
Erin Strachan (Session 4: Microbiome & Nutrition)
MSc Student, University of Alberta (Canada)
Effects of Maternal Dysbiosis on Type 1 Diabetes Incidence in Offspring
Erin Strachan, Tingting Ju, Paulo José Basso, Ben Willing, Xavier Clemente Casares, Sue Tsai
There is a growing appreciation for the role of gastrointestinal (GI) health in promoting whole body wellbeing. Key features of a healthy gut include a balanced microbiota and their interaction with host immune defense mechanisms such as the production of Immunoglobulin A (IgA). Synergistically, the microbiota and IgA work to preserve the integrity of the gut barrier and regulate immune responses both locally and systemically. During infancy, both the microbiota and immune system undergo rapid development and are strongly influenced by maternal factors, conveyed largely via events in the neonatal GI tract. When these events fail to occur at appropriate times, the long term detrimental effects resulting from suboptimal immune development can contribute to subsequent development of pathologies such as Type 1 Diabetes (T1D), where the erroneous activation of autoreactive lymphocytes leads to the destruction of the pancreatic beta cells. Interestingly, despite the well-documented importance of maternal factors on infant immune development, whether maternal immune dysregulation and dysbiosis can perpetuate the same in offspring remains largely unknown. To gain an understanding of how these maternal factors impact infant disease development, we use IgA-deficiency induced maternal dysbiosis in Non-Obese Diabetic (NOD) dams to study T1D development in their offspring. We found that maternal dysbiosis arising from IgA deficiency results in numerous neonatal changes in IgA-sufficient offspring, including increased GI immune cell numbers and cytokine production, increased gut inflammation, altered gut microbiome composition and a modified GI metabolomic profile. In adulthood, IgA-sufficient offspring born to IgA-deficient dams show lessened insulitis and a lower incidence of T1D overall as compared to those reared by IgA-sufficient dams. Cross-fostering experiments indicate this protective effect is mediated post-natally, leading us to hypothesize that maternal microbiome transfer plays a significant role. We are currently exploring various characteristics of breast milk from IgA-deficient dams as well as conducting fecal microbiota transfer experiments to explore how this protective effect may be mediated.
Ben Grodner (Session 5: Omics & Bioinformatics)
PhD Student, Cornell University (USA)
Spatial Mapping of Mobile DNA Elements in Complex Microbiomes at Single Cell Resolution
Benjamin Grodner, Hao Shi, Owen Farchione, Albert Vill, Peter Diebold, Ioannis Ntekas, Warren Zipfel, Ilana Brito, Iwijn De Vlaminck
Bacteria in the human microbiome harbor a variety of mobile genetic elements (MGEs), often carried on phage and plasmids, which transfer between cells and radically enhance the genetic adaptability of the community. The distribution of MGEs in complex microbiomes is thought to be heterogeneous. Yet, there are few substantive tools to map the spatial location of MGEs in microbiomes, and it remains difficult to associate MGEs with their host taxa using metagenomic sequencing data alone. We previously developed High Phylogenetic Resolution Fluorescence In Situ Hybridization (HiPR-FISH), a method for multiplexed mapping of bacterial taxonomic identity. In this study, we combine HiPR-FISH with single-molecule DNA FISH to concurrently map taxa and MGEs in taxonomically diverse, spatially structured microbiomes. We applied this method to spatially map antimicrobial resistance (AMR) plasmids and bacteriophages and found that MGEs and their host taxa are spatially clustered in plaque biofilms. Our findings suggest that regions within plaque biofilms exist where there is extensive short range (~10μm) MGE transfer, specifically due to dense clusters of MGE host taxa, but long range (~100μm) movement of an MGE between clusters of MGE host taxa is limited by intervening non-MGE taxa. We discovered two genera that were highly enriched in regions between clusters of an AMR plasmid, suggesting that these non-MGE taxa spatially restrict the spread of the AMR plasmid-carrying species. Phages infected some dense clusters of host taxa, but other spatially separate clusters of host taxa were uninfected, which may indicate that spatially isolated islands of host bacteria limit the spread of a phage in a biofilm. We expect that the combination of HiPR-FISH and MGE mapping will advance the study of MGE ecology and address critical questions regarding antimicrobial resistance and phage therapy.
Dr. John Parkinson (Session 4: Omics & Bioinformatics + Poster 63)
Senior Scientist, SickKids
Chick-MaTron: in silico modelling of complex microbial communities in the poultry gut
Irina Utkina, John Parkinson
Metagenomics and metatranscriptomics represent powerful technologies to functionally interrogate microbial communities. The challenge is how best to interpret these complex datasets. To meet this challenge, computational modeling is emerging as a complementary approach, capable of informing on the molecular interactions that drive microbial dynamics. Of particular relevance are genome-scale metabolic reconstructions and constraints based methods, such as flux balance analysis, that yield insights into metabolic interactions. To demonstrate the potential of computational modeling to augment meta’omics datasets, we present Chick-MaTron, an integrated model of the chicken gut. Adopting the BacArena modeling framework, Chick-MaTron captures metabolic interactions between 29 taxa across six interconnected gut compartments (gizzard, duodenum, jejunum, ileum, cecum and colon). Regular chicken feed is initially fed into the gizzard and breakdown products are absorbed by the host and/or transit to downstream compartments. Our interest in modeling the poultry gut is driven by the need to promote healthy gut microbial communities as the industry transitions away from the use of antibiotics used in feed formulations. The development of the Chick-MaTron modeling environment allows us to investigate the impact of dietary pertubations by simulating thousands of different dietary additives under a range of different concentrations. In addition to changes in community composition, Chick-MaTron is capable of predicting the production of key metabolites such as short chain fatty acids, considered to promote gut health. Our initial results reveal cellulose and cobalt have the greatest impact on community composition, as well as the production of butyrate and proprionate. Poultry trials are currently being initiated to validate model predictions. While here our focus in on the poultry microbiome, we expect similar approaches will readily translate to studies investigating perturbations in the mammalian gut.
Flash Talk & Poster Presenters
Monica Alvaro Fuss (Poster 1)
Graduate Student, Dalhousie University (Canada)
Microgravity and the microbiome: exploring the effects of head-down tilt bedrest and exercise on gut and oral microbiota
Monica Alvaro Fuss, Vanessa DeClercq, Susan Howlett, Joanna M. Blodgett, Olga Theou, Morgan Langille, Robert Beiko
The physiologic decline and increase in frailty exhibited by astronauts due to microgravity highly resembles the effects of ageing and physical inactivity, although at a much faster rate. Frailty can be measured as an accumulation of health deficits across several systems and is a strong predictor of mortality and other adverse outcomes in older adults, but can be mitigated with proper nutrition, exercise, and other wellness incentives. The human microbiome, defined as the collective network of microbes associated with a human host, is becoming increasingly associated with many aspects of human health and disease, and is thought to play a role in the pathogenesis of frailty. In this study, 22 healthy and physically active participants aged 55-65 were subjected to a 14-day head down tilt bedrest (HDBR) protocol, often used to model the physiologic changes of spaceflight and ageing, with half of the participants undertaking a structured exercise program in bed. Stool and saliva samples were collected before, during and after the bedrest period and subjected to 16S rRNA sequencing to measure and track changes to the gut and oral microbiomes respectively. Our results suggest that alpha diversity of the gut microbiome decreased significantly throughout the bedrest period but was maintained in the exercise group, and there were no changes in alpha diversity of the oral microbiome during this time. We found no significant associations between beta diversity clustering and exercise and sex grouping, but differential abundance of several taxa, including genera belonging to the Lachnospiraceae and Ruminococcaeae families, was significantly correlated with the interaction between days of bedrest and exercise group. Further analysis will incorporate various frailty indexes as well as immunological profiles collected throughout the study, providing key insights into frailty in ageing populations, and helping protect astronauts in the advent of longer space exploration missions.
Dale Archer (Poster 2)
PhD Student, University of Alberta (Canada)
The Effects of VANISH Species of Bacteria in a Mouse Model of Multiple Sclerosis
Archer D., Perez-Muñoz M.E., Tollenaar S., Veniamin S., Hotte N., Cheng C.C., Power C., Krishnamoorthy G., Barreda D.R., and Walter J.
Lifestyle factors in industrialized nations have been associated with an increased incidence of autoimmune diseases, including multiple sclerosis (MS), and the loss of microbial gut symbionts. However, it is not known whether and how bacteria that have become underrepresented in industrialized humans (VANISH species) can modulate immune-mediated pathological processes. The objective of this work is to determine the mechanisms by which VANISH species, including Limosilactobacillus reuteri and Helicobacter species, modulate disease severity and pathology in a TCR transgenic, spontaneous, relapsing-remitting EAE (RR-EAE) mouse model of MS. Treatment of RR-EAE mice with L. reuteri R2lc, which produces a potent agonist of the aryl hydrocarbon receptor (AhR), resulted in severe disease with high mortality (60%). Conversely, treatment with an isogenic mutant, that does not produce the wild-type strain’s dominant AhR agonist, resulted in mild disease with low mortality (14%) (Mantel-Cox test comparing survival curves, L. reuteri R2lc vs the mutant: P = 0.0684). The Helicobacter species showed contrasting effects with H. macacae promoting a mild disease course with low mortality (20%), while H. pylori promoted severe disease with high mortality (75%) (Mantel-Cox test, H. macacae vs H. pylori: P = 0.0154). Furthermore, H. pylori promoted an expansion of immune cell populations known to drive CNS inflammation, including microglia and macrophages in the brain and activated splenic T cell populations. H. pylori-treated mice also had lower levels of splenic B cell populations that have been shown to have regulatory properties in MS and EAE, including plasma cells and plasmablasts. This work shows that VANISH species of bacteria have variable effects in a mouse model of MS that range from protective to detrimental. These findings provide critical information to inform strategies for the development of microbial-based treatments for MS patients.
Ben Fisher (Poster 3)
Graduate Student, Dalhousie University (Canada)
The Relationship between Blood and Tumour Microbes in Patients with Cancer
Benjamin R. Fisher, Robyn J. Wright, Vanessa DeClercq, Morgan G.I. Langille
Research has shown an association between the human microbiome and several types of cancer, such as breast, colorectal and lung cancer. Recent work suggests microbes reside in tumours, with each tumour type having a unique microbiome. Furthermore, some research suggests that microbes reside in the blood, and that blood-based microbial analysis may be useful for predicting or detecting cancer. More investigation is needed to understand the relationship between tumour and blood microbiomes in cancer and how blood microbial communities change with cancer. Using computational methodologies, the objectives of this project are to: 1) determine composition and diversity of microbes residing in blood samples of patients with specific types of cancer; 2) characterize composition and diversity of microbes residing in specific tumour types; 3) determine if there are common microbes that reside within the blood and tumours of the same individuals; 4) evaluate predictability of microbial blood markers for specific cancers. Data to address these objectives were obtained from the BC Cancer Personalized Onco-Genomics (POG) program. Blood and tumour samples from nearly 1000 patients within the POG program have been shotgun sequenced. Sequencing data is being processed to identify microbial reads within samples that have high human-derived sequence content. Microbial reads have been detected in both blood and tumour samples. The number of microbial taxa identified (alpha diversity) is greater in tumour samples than blood. Blood and tumour samples have similar microbial community composition (beta diversity) in patients with lung cancer, and the abundances of several microbial taxa are similar between patients. Future work will focus on creating metagenomically assembled genomes and functionally annotating genes within the tumour and blood microbiomes. This study will provide fundamental knowledge about the role of the blood and tumour microbiome in specific types of cancer and identify potential targets for early diagnostics or therapeutic strategies.
Mackenzie Gutierrez (Poster 4)
MD/PhD Student, University of Calgary (Canada)
Gut mycobiome core species causally modulate metabolic health in mice
Mackenzie W. Gutierrez, Erik van Tilburg Bernardes, Kristen Kalbfleisch, Madeline Day, Thaís Glatthardt, Emily Mercer, Faye Chleilat, Raylene A. Reimer, Marie-Claire Arrieta
The gut microbiome is linked to metabolic diseases including obesity; however, the role of fungi remains understudied. We recently identified core species of the infant gut mycobiome that correlated with infant body mass index. In this study, we aimed to determine the causal contribution of early-life colonization with these core fungal species in obesity development. Gnotobiotic mice were colonized from birth with 12 mouse-derived bacteria (Oligo-MM12) alone or in combination with either Rhodotorula mucilaginosa, Malassezia restricta, or Candida albicans. Mice were weaned onto standard diet (SD) or high-fat-high-sucrose diet (HFHS) and evaluated at 12 weeks for obesity, metabolic disease development and characterization of the white adipose tissue (WAT) immune landscape. Colonization with each species resulted in bacterial microbiome compositional and functional differences under both diets. R. mucilaginosa colonization led to increased adiposity in mice fed SD, poor glycemic control and elevated plasma triglycerides and LDL-cholesterol on HFHS. This was accompanied by increased populations of obesity-associated macrophages in WAT. M. restricta colonization also resulted in increased adiposity on SD, though without any indications of metabolic disease and a reduction in several immune cell populations in WAT. In contrast, C. albicans colonization resulted in leanness, resistance to diet-induced obesity and enhanced WAT inflammation on both diets, primarily characterized as a type1/17 response. The increased adiposity phenotypes occurring under typical WAT inflammation (R. mucilaginosa), or reduced inflammation (M. restricta) indicate that gut fungi can induce expansion of adiposity under distinct immune landscapes. Meanwhile, the combined leanness and elevated WAT inflammation in C. albicans colonized mice suggests immune-mediated dysfunction of energy storage. This work revealed that three common fungal colonizers have diverse and striking causal influences on obesity and metabolic inflammation and prompts for the inclusion of fungi in microbiome studies on host metabolism.
Dr. Natasha Haskey (Poster 5)
Postdoctoral Fellow, University of British Columbia Okanagan (Canada)
The Crohn’s Disease Therapeutic Diet Intervention (CD-TDI) for reducing inflammation in mild-moderately active Crohn’s disease: A preliminary analysis
Natasha Haskey, Andreina Bruno, Hena R. Ramay, Munazza Yousuf, Lorian M. Taylor, Christina Ohland, Kathy D. McCoy, Christopher Ma, Subrata Ghosh, Simona Veniamin, Levinus A. Dieleman, Maitreyi Rama
Recent studies have demonstrated a relationship between diet and disease remission in Crohn’s disease (CD); however, there is a lack of well-defined relationships between dietary components and disease relapse. There is a need for adjunct therapies, including diet, that can alter the disease course. The CD Therapeutic Dietary Intervention (CD-TDI) is a novel dietary approach incorporating best practices and current evidence. We assessed its therapeutic potential to reduce inflammation and influence the bacteriome in patients with mild-to-moderately active CD. Participants were randomly assigned to the CD-TDI (n=16) or conventional management (CM) (n=12) for 13 weeks. The CD-TDI met weekly with a dietitian for coaching on the CD-TDI protocol. The CM followed their habitual diet. Stool and blood samples were collected at baseline and week 13 (W13). The fecal bacteriome was assessed using 16S rRNA gene sequencing. There were no differences between groups in fecal calprotectin, CRP or the Harvey-Bradshaw Index at baseline. At W13, in the CD-TDI, fecal calprotectin decreased from 130 ug/g (IQR:38-411) to 72 ug/g (IQR:3-153; (p=0.01) and was significantly different between groups (p=0.04). CRP was significantly decreased in the CD-TDI (1.0 mg/L, IQR:0.6-2.4) compared to CM (4.5 mg/L, IQR:2.6-13.0, p=0.002). The Harvey-Bradshaw Index decreased in the CD-TDI (W0:3.0, IQR:1.0-5.3; W13:2.0, IQR:0-3.0; p=0.05), but not in the CM. Differential abundance analysis revealed Lachnospiraceae, Selenomonadaceae, Sutterella spp. and Akkermansia muciniphila were more abundant in the CD-TDI at W13 (p<0.05), while Collinsella intestinalis was more abundant in the CM group. Alpha and beta diversity did not differ between groups. The CD-TDI decreased clinical and biochemical markers of inflammation in mild-to-moderately active CD, along with changes in the relative abundance of taxa. This preliminary analysis suggests CD-TDI could reduce the inflammatory burden in patients with CD and has the potential to be used as an adjunct therapy.
Courtney Hoskinson (Poster 6)
PhD Student, University of British Columbia (Canada)
Delayed gut microbiota maturation in the first year of life as a unifying feature in pediatric allergic disease
Courtney Hoskinson, Darlene L.Y. Dai, Kate L. Del Bel, Allan B. Becker, Theo J. Moraes, Piushkumar J. Mandhane, B. Brett Finlay, Elinor Simons, Anita L. Kozyrskyj, Meghan B. Azad, Padmaja Subbarao, Charisse Petersen, Stuart E. Turvey
BACKGROUND: Allergic diseases affect hundreds of millions of children worldwide and continue to increase in prevalence. These rising rates have coincided with social and environmental changes that have had an intergenerational impact on the stably colonizing microbes and their collective genes that make up our microbiota and microbiome, respectively. In this study, we analyzed highly characterized participants in CHILD (n=1,115) to test the hypothesis that early-life influences and microbiome features are uniformly associated with four distinct allergic diagnoses at 5 years: atopic dermatitis (AD, n=367), asthma (As, n=165), food allergy (FA, n=136), and allergic rhinitis (AR, n=187). RESULTS: We identified early-life clinical factors that are uniformly associated with all four of AD, As, FA, and AR. In a subset with shotgun metagenomic and metabolomic profiling (n=589), we discovered that impaired 1-year microbiota maturation may be universal to pediatric allergies (AD p<0.001; As p=0.0073; FA p<0.001; and AR p=0.0021). Extending this, we found a core set of functional and metabolic imbalances characterized by compromised mucous integrity, elevated oxidative activity, decreased secondary fermentation, and elevated trace amines, to be a significant mediator between microbiota maturation at age 1 year and allergic diagnoses at age 5 years (βindirect=-1.532; p=0.001). Microbiota maturation thus provides a focal point to identify deviations from normative development for allergic disease prediction and prevention. SIGNIFICANCE: In conclusion, we described detailed underpinnings driving decreased gut microbiome maturation, encompassed within the alteration of a core group of species, functional pathways, and metabolic imbalance associated with reduced microbiota-maturation age and elevated risk of allergy. This study provides new insight into underappreciated and nuanced aspects of the infant microbiome that will enable improved prevention and prediction of allergic disease.
Eszter Pal (Poster 45)
Graduate Student, McGill University (Canada)
Cardiac surgery induces sex-specific alterations in the gut microbiome that may hamper post-surgical recovery through a proinflammatory immune response
Eszter Pal, Brandon Shokoples, Yao Lu, Ken Dewar, Jeff Xia, Lorraine Chalifour
Introduction: Cardiac surgeries are among Canada’s most common surgical procedures, with serious complications arising in up to 20% of cases, requiring rehospitalization. Besides the physical stress of surgery, antibiotic use, malnutrition, and older age can further predispose patients to disruptions in gut microbiome composition and function, which have been linked to cardiovascular disease and systemic inflammation. We propose that cardiac surgery induces alterations to gut microbial composition, which may contribute to negative post-surgical outcomes through the promotion of a proinflammatory immune response. Methods: Gut microbiome profiles of retired breeder mice recovering from permanent left anterior descending coronary artery (LAD) ligation were temporally characterized through 16S rRNA sequencing of fecal DNA. Immune cell profiles were characterized using flow cytometry analysis and echocardiography was performed prior to euthanasia to assess cardiac remodeling and wound healing. Results: Community profiling and differential abundance analyses revealed significant sex-specific microbiome remodeling by day 3 post-surgery. We observed decreases in Firmicutes and Bacteroidetes ratios as well as time- and sex-specific expansions of Proteobacteria and Verrucomicrobia post-surgery, mirroring aspects of microbial profiles associated with inflammation. Flow cytometry analyses show an acute expansion of inflammatory innate cells and immunopathological T cell subsets (such as TCRγδ+ T cells) in the heart and small intestine that is more aggressive in males and delayed in females. Echocardiography shows substantial damage to cardiac function and structure that is worse in males and similarly delayed in females. Conclusion: Ultimately, we characterize pro-inflammatory immune responses associated with surgery-induced alterations in the gut microbiome which may delay wound healing and hamper recovery. Importantly, we also reveal major sex-specific differences in microbiome remodeling and post-surgical response, such as a more aggressive inflammatory response in males resulting in worse surgical outcomes.
Kenny Shin (Poster 46)
MSc Student, University of Ottawa (Canada)
The Utility of long-read 16S rRNA sequencing for species-level bacterial identification of metagenomic samples
Kenny Shin, Peter Dobranowski, Jennifer Li, Alain Stintzi
Background: 16S rRNA (16S) targeted amplicon sequencing is a widely-used method for bacterial taxonomic assignment which is much more economical than whole-genome sequencing (WGS) methods. Current methods using short-read (SR) DNA sequencers from Illumina and IonTorrent for 16S-analysis can effectively resolve samples to the genus level. However, species within the same genus can have opposing associations with disease, underlining the need for finer taxonomic resolution. Recently developed long-read (LR) and synthetic-LR (SLR) technologies like Nanopore and LoopSeq enable the sequencing of entire 16S genes and promise higher taxonomic resolution. Knowledge gap: Currently, there is no comprehensive benchmark comparison testing the utility of 16S sequencing for species-level taxonomic classification across all 4 of the aforementioned sequencing platforms. Methods: Here, the Illumina, IonTorrent, Nanopore, and LoopSeq technologies were used for 16S sequencing of two different mock community (Zymo) types of known quantity and composition (ground truth). Multiple 16S analysis pipelines (dada2, Emu, NanoCLUST) and reference databases (Silva 138.1, Emu) were employed to determine the best combinations for species-level taxonomic resolution. Detection and abundance estimation of different combinations were evaluated through area under the precision-recall curves (AUPRC) and beta-diversity analysis. Results: Using the Loopseq and Nanopore platforms along with the Emu program and the Emu reference database yielded the highest AUPRC and smallest Bray-Curtis dissimilarity to the ground truth composition at both the genus- and species-levels. In contrast, SR 16S sequences using dada2 were unable to fully resolve taxa at the genus level. Conclusion: We highlight the superior fidelity of LR and SLR technologies and corresponding optimal bioinformatic pipelines for species-level resolution among other 16S sequencing approaches. Outcomes: Being able to conduct species-level analysis using LR- and SLR-16S approaches together with the optimized pipelines enables microbiome researchers to tease apart microbiome-disease more efficiently.
Dr. Matthew Sorbara (Poster 47)
Assistant Professor, University of Guelph (Canada)
Urease activity enhances survival and regulates the colonization resistance-linked parameters of short-chain fatty acid production and acidification in Lachnospiraceae isolates
Firth I. Fitzgerald B. Sweeney A. Sim M. Sorbara M.T.
The gut microbiome provides important benefits to the host, including colonization resistance against the expansion of enteric pathogens. One mechanism of colonization resistance is the release of high concentrations of short-chain fatty acids (SCFA) and the generation of a slightly acidified environment in the colon by commensal anaerobes in the microbiota. The combination of high SCFA and an acidic pH triggers intracellular acidification in enteric pathogens including members of the Enterobacteriaceae family. Importantly, isolates of Enterobacteriaceae are increasingly antibiotic resistant in clinical settings. However, the mechanisms by which commensals themselves resist this stress remains unclear, which hinders the development of live biotherapeutics designed to restore colonization resistance. Here, we investigate the ability of Lachnospiraceae to tolerate pH and SCFA stress. The Lachnospiraceae are a family of anaerobes that are commonly associated with SCFA production, show high levels of genomic diversity, and vary in their capacity to acidify culture media. Unexpectedly, training a random forest model to predict Lachnospiraceae pH based on gene presence/absence identified multiple subunits of urease as important predictors of acidification. Urease cleaves urea into ammonia and carbon dioxide, providing assimilable nitrogen and acid tolerance. Urease is a well characterized virulence factor for many pathogens, but its role in commensals is largely unknown. Here we demonstrate that urease-encoding Lachnospiraceae show urea-dependent changes in SCFA production, acidification, growth, and urease activity. Encoding urease increases the tolerance of some Lachnospiraceae to acid stress and increases SCFA production under acidic conditions. As the parameters of SCFA production and acidification are central to the microbiota’s colonization resistance, elucidating the role of commensal-encoded urease will aid in ongoing development of live-biotherapeutics restoring microbiota function.
Gillian Tanabe (Poster 48)
PhD Student, University of Ottawa (Canada)
Characterization of the Gut Microbiota in Pediatric Inflammatory Bowel Disease: A Culturomics Approach
Tanabe, G; Mack, D; Stintzi, A
Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. The prevalence of pediatric-onset IBD is increasing globally, especially within Canada. Previous studies have linked changes in gut microbiota composition to the development of IBD. However, these studies have primarily used 16S rRNA gene sequencing, which has limited sensitivity for detecting low-abundance bacteria. Recent studies have highlighted the importance of culturing the gut microbiota for the detection of both low abundant and previously unidentified taxa. Despite this, few studies have employed culturomics to study the gut microbiota in IBD. Therefore, our work aims to address this gap using a culturomics-based 16S gene sequencing approach to identify novel and low abundant taxa in pediatric IBD. Briefly, mucosal-luminal interface (MLI) aspirates were collected from three sites of the gastrointestinal tract of pediatric IBD patients and non-IBD controls during colonoscopy. MLI aspirates were 16S rRNA sequenced directly and cultured on agar plates under six conditions (fastidious anaerobe, brain heart infusion, colistin nalidixic acid; both aerobically and anaerobically) prior to sequencing, to identify the culturable microbiota composition. Culturomics methods identified over 260 novel amplicon sequence variants (ASV), which were undetectable by standard 16S rRNA sequencing. Additionally, >150 low abundance ASVs were detected at higher resolution compared to the uncultured MLI. Under our six conditions, we cultured approximately 40% of the ASVs and 70% of the bacterial relative abundances detected by uncultured methods. Continuing data analyses will investigate taxonomic alterations in the culturable microbiota of IBD patients compared to controls. Our work uses a novel method, culturing bacteria from MLI aspirates of IBD patients combined with 16S rRNA gene sequencing, to identify unique and low abundant bacteria not detected by traditional approaches. Continued analyses are aimed at uncovering novel taxonomic distinctions in IBD and future elucidation of their role(s) in IBD pathogenesis.
Dr. Carolyn Thomson (Poster 49)
Research Associate, University of Calgary (Canada)
The shape of things to come: how the maternal microbiota shapes susceptibility to Type 1 Diabetes
Thomson, CA, Brown, K, Davoli-Ferreira, M, Burkhard, R, Pett, NJ, Ohland, CL, Ignacio, AS, Geuking, MB, McCoy, KD
Our microbiota and immune systems have co-evolved in a manner that has profound implications for health and disease susceptibility. Although microbial education of the immune system was thought to begin with colonization during birth, it is becoming increasingly clear that signals derived from the maternal microbiota can begin this process in utero. The long-term significance of this perinatal immune education remains unclear, particularly when it comes to disease development later in life. To explore this further, we have transiently colonized germ-free C57BL/6 and non-obese diabetic (NOD) dams with an auxotrophic strain of E. coli. By restricting colonization to pregnancy alone, this model allows us to focus specifically on the impact of the maternal microbiota on the germ-free offspring. Strikingly, the vertical transfer of microbial signals from mother to offspring in utero, and via the milk, is sufficient to reduce the incidence of type 1 diabetes development (T1D) in male NOD mice later in life. This was associated with profound transcriptional changes in the offspring’s gastrointestinal tract and pancreas early in life, with several immunological and metabolic pathways being differentially regulated in response to the maternal microbiota. Moreover, we observed long-lasting metabolic changes in the serum, urine, and pancreas. This metabolic reprogramming was characterised by a marked reduction in circulating dicarboxylic acids, which are elevated in the serum of diabetic patients in response to a shift from b- to w-oxidation of fatty acids. Similar metabolic phenotypes were observed in C57BL/6 mice, which have no genetic predisposition to diabetes, suggesting that altered metabolism is a long-term consequence of bacterial exposure in utero rather than a confounding effect of insulitis. Thus, exposure to bacterial products in utero has a long-lasting impact on the immune system and metabolome, invoking biological changes that may influence susceptibility to immunological disorders such as T1D.
Dr. Weilan Wang (Poster 50)
Postdoctoral Fellow, Baylor College of Medicine (USA)
Anticancer mechanism of dietary restriction of sulfur amino acids
Weilan Wang, Holly Quang, Katie Lu, Cayla Xue, Maryam Tabatabaei, Xia Gao
Dietary restriction of sulfur amino acids, methionine and cysteine, inhibits tumor growth alone and synergizes with chemotherapy or radiation in mouse cancer models. However, the antitumor mechanisms of sulfur restriction remain unclear. Dietary methionine restriction interferes with redox balance and nucleotide metabolism in tumors. While addition of nucleotides and N-acetyl-cysteine each rescued in part the methionine restriction-inhibited cancer cell growth in vitro, whether sulfur supplementation will restore the inhibited tumor growth in vivo remains unexplored. Here, we show that dietary methionine restriction and cysteine deprivation (MRCD) inhibits tumor growth in a syngeneic GEMM6 melanoma mouse model, and such inhibition were completely reversed by cysteine supplementation. Mechanistically, MRCD reduced glutathione peroxidase GPX4 in the tumor, which was reversed by cysteine supplementation (MRCS). Metabolomics analysis showed that MRCD elevated systemic levels of serine, glucose, and nicotinamide, which were inversely correlated with endpoint tumor volume. MRCS increased circulating sulfur-containing taurine, cysteine-S-sulfate, creatine and allantoin which positively correlated with increased tumor size. Furthermore, MRCS restored mucin secretion. Correspondingly, 16S rRNA gene sequencing analysis revealed a significant reduction of sulfite reducing Romboutsia spp. in the ileum of mice fed with MRCD, which was reversed by MRCS. Romboutsia spp. positively correlated with circulating accumulation of sulfur-containing metabolites and endpoint tumor volume. Collectively, our findings suggest that dietary sulfur restriction suppresses tumor growth by altering systemic metabolism, with important mediation from gut bacteria.
Poster Presenters
Spencer Abbott (Poster 7)
MSc Student, University of Calgary
Investigating how maternal antibodies modulate T1D outcomes in offspring
Spencer Abbott, Carolyn Thomson, Marcela Davoli-Ferreira, Baweleta Isho, Philippe Poussier, Kathy D. McCoy
The microbiome is involved in the education of the immune system, especially in early life. This can then impact susceptibility to immune-related diseases, including type-1 diabetes (T1D). The transfer of maternal antibodies to offspring in the non-obese diabetic (NOD) mouse model has been shown to increase the incidence of T1D under SPF conditions. This has been interpreted as evidence that maternal IgG antibodies from NOD dams are diabetogenic. In contrast, we hypothesize that maternal IgG acts to limit microbial translocation in early life and that bacterial translocation in the absence of maternal antibodies is protective. If true, the pathogenic effect of maternal antibodies would be microbiome dependent. Thus, our aim is to establish whether the transfer of maternal antibodies accelerates T1D development by limiting bacterial translocation. NOD dams were crossed with NOD-scid males and NOD males with NOD-scid dams, under germ-free (GF) or gnotobiotic conditions, giving rise to antibody-sufficient offspring born to antibody-sufficient or -deficient dams. T1D incidence was then monitored in the immunocompetent offspring. To assess how maternal antibodies influence microbe-mediated immune responses, we then compared germinal center responses in offspring at 3 and 5 weeks of age. Under GF conditions, there was no difference in T1D incidence, suggesting that the phenotype is microbiome dependent. In contrast, OMM12-colonized mice that did not receive maternal antibodies exhibited increased germinal center responses in the mesenteric lymph nodes and spleen at 3-weeks of age, compared to controls. These changes had returned to baseline by 5 weeks of age, suggesting that maternal immunoglobulins can transiently influence adaptive immune responses in a microbe-mediated manner. Our preliminary data suggests that the vertical transfer of maternal antibodies may influence T1D progression in the offspring by restricting bacterial translocation.
Dr. Sima Afsharnezhad (Poster 8)
Postdoctoral Fellow, Queen’s University
Combination of deep learning and blocking primers provide new insights into the tick microbiome
Sima. Afsharnezhad, Damian Bourne, Zhengxin Sun and Robert I. Colautti
In North America, Ixodes scapularis carries bacteria in the Borrelia burgdorferi, which causes Lyme disease. There are three major challenges regarding the disease. First, due to a 72- hour window, a fast and reliable method for species identification of the tick is important for determining whether prophylactic treatment with antibiotics is warranted. Second, all female Ixodes scapularis contain bacteria in the Rickettsia.buchneri which has been reported to be the most common bacterial with a mean relative abundance of 97.9, potentially concealing the identification of rare bacteria. Third, while most studies focus on Borrelia burgdorferi, ticks are known to transmit a wide variety of other pathogens that are less studied. This is especially important for epidemiological risk mapping and public health interventions. To assist with tick identification, we trained a convolutional neural network (CNN) on 500 dorsal and ventral tick photos to predict tick species and sex. We used Inception-Resnet with ImageNet to generate 53 million trainable parameters. Furthermore, we employed the whole 16S rRNA sequencing to improve detection of tick microbial communities. We also developed annealing inhibition blocking oligos to limit amplification of Rickettsia.buchneri sequences and thereby increase representation of other bacteria. We then collected 10 female adult I. scapularis ticks from Kingston, Ontario, and confirmed species and sex using our CNN model before extracting and pooling DNA to test our blocking primers. Our CNN classifier achieved a best accuracy of 100% for identifying a single tick species and 98.98% for its sex. Our best blocking primer (Blk1) reduced Rickettsia sequences by more than 98% and enhanced detection of relatively rare Borrelia pathogens and other bacteria. The use of deep learning for tick species identification in combination with blocking primers for microbiome analysis can accelerate field-based approaches to detect, track and diagnose Lyme and other tick-borne diseases.
Dr. Shokouh Ahmadi (Poster 9)
Postdoctoral Fellow, University of Calgary
Novel auxotrophic Bacteroides models for transient colonization of germ-free mice
Shokouh Ahmadi, Sharon Dong, Carolyn A. Thomson, Isla Skalosky, Philip Ahern, Mohammed E. Dwidar, Kathy D. McCoy, Markus B. Geuking
Aims: Auxotrophic bacteria lack functional biosynthetic pathways to synthesize compounds essential for growth. This auxotrophy can be used to transiently colonize germ-free mice with live in vitro-grown bacteria that can not persistently colonize in vivo. This approach was successfully employed for a commensal Escherichia coli strain (HA107). Using HA107 strain to colonize pregnant mice, Gomez et al. have demonstrated that maternal microbiota shapes the offspring’s immune system. To expand this toolbox and study the effects of other intestinal bacterial species on the immune system, we generated and characterized three auxotrophic Bacteroides strains including B. thetaiotaomicron, B. ovatus and B. caccae. Methods: An allelic exchange recombination system was used to delete the gene required for synthesis of D-alanine (D-Ala), a uniquely-bacterial peptidoglycan amino acid. The B. theta, B. ovatus and B. caccae strains were characterized in an in vitro culture using media with and without D-Ala. In addition, in vivo characterization was performed by gavaging germ-free C57BL/6 mice with either the wild-type or mutant strains. Fecal pellets were plated to quantify bacterial load and the immunological responses were investigated. Results: The auxotrophic Bacteriodes strains mutants only grow in vitro when media is supplemented with D-Ala. While initial colonization levels of auxotrophic strains are identical to the WT, the numbers of fecal live auxotrophic mutants decreased overtime and could no longer be detected 72 hours post-gavage. We also performed initial immunological characterization of the effects mediated by transient colonization with the different strains. Conclusions: These transiently-colonizing bacterial strains allow for tempral control of colonization and provide insights into how bacteria communicate with the immune system, like during gestation, where maternal intestinal microbes influence fetal immune system development and health outcomes.
Nazanin Arjomand-Fard (Poster 10)
PhD Student, University of Alberta
Biofilms in the appendix and other non-inflamed sections of the colon in pediatric patients with inflammatory bowel diseases
Nazanin Arjomand Fard, Michael Bording-Jorgensen, Jesse Webb, Simona Veniamin, Christopher Cheng, Troy Perry, Eytan Wine
Background and Aims: Biofilms are clusters of bacteria that are associated with the mucosa of inflammatory bowel diseases (IBD) patients with higher densities observed in IBD patients compared to healthy individuals. The appendix, which is a highly immune organ, seems to be involved in IBD pathogenesis. In this study, we aimed to evaluate biofilms in the appendix and other non-inflamed regions of the colon in pediatric IBD patients. We hypothesized that biofilms composed of pathobionts in these sections could drive inflammation in IBD patients. Methods: Combined fluorescence in situ hybridization (FISH) for biofilms (probe: EUB338) and immunofluorescence (IF) mucin staining (MUC2) identified biofilms in the appendix, peri-appendicular region (PA), cecum, and ascending colon (ASC) tissues, collected from the resected colons of 9 pediatric IBD patients and one pediatric non-IBD patient. Biofilm formation capacity of culturable bacteria (identified through 16S DNA Sanger sequencing) from these sections was measured. Interleukin (IL)-8 (proinflammatory chemokine) and IL-10 (anti-inflammatory cytokine) expressions were assessed by tissue qPCR. Results: FISH demonstrated biofilms in these sections, in close proximity to epithelial cells. Enterococcus avium showed a significantly higher ability to form biofilms than the negative control. qPCR data indicates a directional pattern with the appendix showing the highest levels of IL-10, and IL-8 expression is the highest in the PA, indicating an attempt to regulate immune responses. Mechanistic experiments are in progress to investigate the type of bacteria involved in the biofilms and their effects on the gut barrier integrity. Discussion: The presence of biofilms and immune activity in these sections, especially in the appendix and peri-appendix suggests an altered microenvironment in IBD patients, which may be involved in IBD pathogenesis. Identifying the bacteria involved in the biofilms and clarifying their characteristics will enable us in developing novel microbe-altering treatment strategies or personalized medicine.
TBD (Poster 11)
Jérémie Auger (Poster 12)
Bioinformatician, Lallemand
Group comparisons for multivariate datasets. The case of the microbiome.
Jérémie Auger
The worlds of molecular biology, genomics, metabolomics, chemistry screenings, (and all the related omics) all now have one thing in common: Almost all of the data generated is multivariate/ highly dimensional. In other words, for a simple outcome variable, there are hundreds if not millions of datapoints associated. Statistical principles must be adapted. And methods like dimensionality reduction and machine learning can be used to answer the question all of us want answered: did my treatment have an effect. Or how can we assess group differences for a specific outcome variable using 16S metabarcoding of microbiome samples. This bioinformatics methods poster demonstrates qiime2 (from MiSeq reads to Machine Learning classifiers) and associated packages. And how to compare groups reliably and robustly for microbiome research.
Abimbola Badejo (Poster 13)
Undergraduate Student, University of Calgary
To Supplement or Not? A Systematic Review and Meta-Analysis on the Effects of Biotic Supplementation on Infant Behaviors.
Matin, M., Brockway, M., Badejo, A., Kouroupis, A., Janke, R., and Keys, E.
Objective: Infant gut microbiota profiles are modulated by their dietary patterns and may influence state regulation (i.e., sleep and crying behaviors). This systematic review and meta-analysis aimed to determine the effect of biotic supplementation on healthy full-term infant sleep and crying behaviors. Methods: Medline, Embase, CINAHL, PsycINFO, DARE, CENTRAL databases and one trial registry were searched using the terms “infant” AND “sleep” AND “biotic”. Two independent reviewers screened records. Data on sleep and crying-related behavior outcomes, reported adverse/side effects, co-morbid conditions, and (probiotic, prebiotic, synbiotic and postbiotic) were extracted for analysis. Using the modified Cochrane Collaboration tool, two independent reviewers judged the risk of bias as low, high, or unclear. Meta-analyses were conducted using RevMan5. Results: The search yielded 424 unique studies and 25 randomized control trials were included in the analysis. Probiotic supplementation was provided in 52% of studies, while 28% and 20% of studies offered prebiotic and synbiotic supplementation, respectively. Sleep duration was the most common (95%) outcome measured for probiotics. Infants who received probiotics demonstrated significantly longer sleep duration compared to infants who received placebo at two, three, and four weeks of intervention (MD = 29.43 minutes [95% CI: 3.33 – 55.52], 27.92 minutes [95% CI: 6.10 – 49.74], and 27.22 minutes [95% CI: 7.06 – 47.38], respectively). However, in the 5th week of intervention, infants who received placebo slept longer (MD = -35.17 minutes [95% CI: -69.72 – 0.62]). No significant differences were found for crying-related outcomes. Conclusions: Based on this study, probiotics may improve sleep duration in healthy full-term infants for up to 4 weeks of administration. Future research on biotic supplementation should assess the full range of sleep behaviors, infant feeding type, and mechanistic evidence via microbiome analysis. In addition, researchers should also consider adverse events investigation, longer-term follow-up, and assessing associated immune markers.
Sahar Bagheri (Poster 14)
Bioinformatics Specialist, University of Calgary
Differences in human fecal sample processing impact microbiome studies
Sahar Bagheri, Paloma Cavalcante, Hena Ramay, Olabisi Coker, Christina Ohland, Kathy McCoy and Laura K. Sycuro
The fecal microbiome is widely researched as a surrogate for communities residing in the human gut. However, there is no ‘gold standard’ procedure for collecting, processing, and storing fecal samples, the contents of which may be sensitive to fluctuations in temperature and exposure to oxygen. The goal of our study is to examine how fecal sample handling procedures affect downstream sequencing applications, metabolomics, and bacterial culture. We analyzed 10 fecal samples from healthy volunteers. Samples were received within one hour of collection and immediately aliquoted in an anaerobic chamber to allow for comparisons of processing procedure (scooping vs. homogenization), short-term storage time/temperature (4 vs. 24 hrs at -20, 4, 25, or 37°C) and storage solution (preservative vs. none). Following these treatments samples were stored at -80°C for 4–6 months and then analyzed using 16S rRNA taxonomic profiling and liquid chromatography for short chain fatty acids (SCFA) metabolites. Analyses of alpha and beta diversity revealed that procedural variability was insignificant compared to participant variability, suggesting that these measures robustly capture an individual’s fecal microbiome signature regardless of short-term processing/storage conditions. Surprisingly, homogenization, rather than temperature, had the greatest impact on community composition. Regardless of homogenization method/solution (PBS, BHI/glycerol, preservative) this procedure was associated with an increased abundance of Prevotella and Phocaeicola in most samples. SCFA also appeared to be impacted by homogenization, showing reduced levels in PBS and preservative (OMNImet). In conclusion, this study highlights the importance of standardizing fecal processing procedures to obtain reliable and reproducible results. We are continuing to investigate why homogenization shifts community composition (possibly by releasing host cell- or biofilm-associated species) and evaluate linkages between handling procedure and the breadth of bacterial species cultured from the human samples.
Delaney Barth (Poster 15)
Graduate Student, University of Calgary
Sex-specific differences in the gut microbiome and metabolome at 3 months of age
Delaney Barth, Marcel van de Wouw, Marie-Claire Arrieta, Lianne Tomfohr-Madsen, Catherine Lebel, Gerald Giesbrecht
The composition and function of the gut microbiome influence brain and behavioral development. Animal studies have shown clear sex-specific differences in the way the microbiome affects behavior and brain function, but human data, particularly during early life, are still relatively unexplored. This study aims to characterize sex-specific differences in the early life gut microbiome and its metabolites. Data are from the Canadian Pregnancy During the COVID-19 pandemic cohort. Stool samples were collected at 3M of age for metagenomic shotgun sequencing (n=311) and metabolomics (n=878). Differences in the infant microbiome and metabolome between infant sex were investigated using Mann-Whitney U tests with a correction for multiple testing using the Benjamini-Hochberg procedure. Male infants had increased stool ophthalmate and reduced hippurate levels (Adj. P<0.001, Adj. P=0.003, respectively). Interestingly, ophthalmate is produced by enzyme EC6.3.2.3, which is expressed by 19 different bacterial taxa in the infant gut microbiome. Of these taxa, there was a trend towards increased Escherichia coli relative abundances in males (p=0.071). The microbiota’s richness was assessed using Shannon and Simpson alpha diversity measures, which indicated females having higher values (p < 0.05). Behavioral outcomes, evaluated using the Ages and Stages Questionnaires (ASQ) and the Infant Behavior Questionnaire-Revised (IBQR), showed several correlations. Females exhibited negative correlations between Shannon and Simpson alpha diversity and problem-solving skills at 12 months (p < 0.05), as well as crying episodes per week at 3 months (p < 0.05). Additionally, a positive association was observed between problem-solving skills in females and hippurate levels (p < 0.05), whereas male positive affectivity/surgency at 6 months exhibited a significant inverse relationship with ophthalmate levels (p < 0.05). These findings unveil novel associations in the early life microbiome and behavioral outcomes based on sex. Further research is necessary to elucidate the underlying mechanisms and long-term implications of these results.
Dr. Evandro Beraldi (Poster 16)
Postdoctoral Fellow, University of Calgary
Depletion of gut microbiota accelerates disease progression in the TDP43 mouse model of amyotrophic lateral sclerosis
Evandro J. Beraldi, Sukyoung Lee, Oluwamolakun Bankole, Yulan Jiang, Catherine M. Keenan, Kathy D. McCoy, Gerald Pfeffer, Keith A. Sharkey, Minh Dang Nguyen
Background: Amyotrophic lateral sclerosis (ALS) is a progressive, fatal motoneuron disease. Biological sex is an important factor in the presentation and risk of ALS, but the mechanisms underlying these sex differences are poorly understood. Emerging studies suggest that the gut microbiota plays a role in ALS initiation and progression, and the different microbiota composition between males and females suggests that such variation may account for the male susceptibility to disease. Hypothesis: We investigated if the gut microbiota is critical for disease progression and sexual dimorphism in a mouse model of ALS. Methods: SPF-housed, male and female mice expressing a mutant human form of TDP43 (A315T) under the prion promoter that specifically targets expression to neurons (TDP43 mice) were used. Mice were maintained on autoclaved water or an antibiotic (Abx) cocktail diluted in autoclaved water. Treatment started at 5 weeks of age and continued until the mice showed signs of severe motoneuron disease. In parallel, germ-free TDP43 mice are also being generated. Results: TDP43 mice show marked sexual dimorphism in ALS progression, with males having reduced average lifespan (106±28 days [mean±SD], n=42) compared to females (238±84 days, n=16). Strikingly, the depletion of gut microbiota with Abx treatment reduces the survival rates of TDP43 males (48±4 days, n=10) and females (62±10 days, n=10). TDP43 mice, both on water or Abx-treatment, had impaired gastrointestinal function, with enlarged terminal ileum and dry feces in the cecum and colon at the end stage of disease. Similarly, germ-free TDP43 of both sexes also develop precocious motoneuron disease onset and die as early as Abx-treated mice. Conclusions: Gut microbiota are protective against TDP43 toxicity and contributes to sexual dimorphism in this mouse model. These data suggest that specific bacteria and their metabolites can influence the disease progression and could potentially be modulated to increase lifespan in ALS.
Dr. Dominique Bihan (Poster 17)
Senior Scientist, University of Calgary
Calgary Metabolomic Research Facility: Canada’s national hub for microbiome metabolomics.
Dominique G. Bihan, Marija Drikic, Ryan Groves, Mohammad M. Banoei, Sharanya Menon, Kirsty Brown, Joseph Wang, Kathy D. McCoy, and Ian A. Lewis
The Calgary Metabolomics Research Facility (CMRF) features a state-of-the-art suite of analytical liquid chromatography-mass spectrometry (LC-MS) instrumentation. We support a wide range of collaborative and service research projects that seek to understand the metabolic complexities inherent to host-microbe interactions. Serving as the metabolomics lead of the International Microbiome Centre and the metabolomics hub of the CIHR IMPACTT pan Canadian Microbiome Core, CMRF provides extensive metabolomics support to Canadian and international researchers. Herein, we showcase selected data, from two collaborative research projects, highlighting the relative influence of commensal bacteria and sampling sites on host metabolic profiles. The first study depicts the targeted analysis and quantification of short chain fatty acids (SCFAs) and bile acids (BAs) performed on contents collected from different sites of the human gastrointestinal tract (GIT) alongside feces. The study demonstrates that the concentration of SCFAs and BAs correlates with the presence or absence of species able to either produce or modify the aforementioned metabolites. Specifically, SCFAs concentration increases with the increase in density and diversity of microbiome communities, while the concentration of glycine-and taurine-conjugated BAs decreases under the same conditions, displaying the physiological roles of the gut microbiome. The second study, performed on gnotobiotic mice, illustrates the use of a semi-targeted LC-MS method to elucidate the contribution of various biological factors (including sampling sites, microbiome, age and sex) on host metabolism. The study reveals a different metabolic profile between the upper and lower GIT mostly attributed to the difference in the density and composition of the bacterial community, whereas age was the prevalent factor influencing the metabolic profile in systemic sites. In conclusion, CMRF’s metabolic analytical platforms provide a comprehensive and robust tool to unravel the complexity of the host-microbe interactions, and decipher the contribution of the microbiome in host health and disease.
Dr. Kirsty Brown (Poster 18)
Postdoctoral Fellow, University of Calgary
Exploring the Impact of Bacterial Colonization on Intestinal Mesenchymal Stromal Cells
Kirsty Brown, Marco Gallo, Kathy McCoy
Intestinal mesenchymal stromal cells (iMSC) are non-endothelial, non-epithelial and non-haematopoietic cells which constitutes a mixed population of cells. iMSC have pleiotropic roles in maintaining homeostasis, including providing structural support and maintaining epithelial cell turnover. Immunological roles of MSC have long been appreciated in lymphoid tissues, and their immunological role within mucosal tissues is increasingly being recognized. Despite established connections between the microbiota and epithelial and immune cells in the intestine, how the microbiota impacts the development and regulation of iMSC is not well defined. To better understand microbe-iMSC connections in the small intestine, we performed single cell RNA sequencing of iMSC in germ-free (GF), gnotobiotic, and specific pathogen free (SPF) mice. We found that while the subclusters of iMSC develop independently of the microbiota, Il33 expression was increased in myofibroblasts with increasing exposure to the microbiota. Expression of IL33 on submucosal myofibroblasts was validated using 3D imaging of the small intestine and qPCR on sorted myofibroblasts confirmed the increase in SPF compared to GF mice. Finally, depletion of Il33 in iMSC using a Cxcl13cre x Il33fl/fl system, revealed a decrease in Il33 and an increase in Il6 transcripts in small intestinal tissue. Together, our results uncover microbe-iMSC interactions in the small intestine and suggest that iMSC-derived Il33 plays a role in maintaining homeostasis. Understanding how microbes regulate may provide insights that can be leveraged to promote intestinal health.
Dr. Larissa Celiberto (Poster 19)
Postdoctoral Fellow, University of British Columbia
Intestinal mucus: regulating resistance to gut bacterial infection and dysbiosis
Larissa S. Celiberto, Kirk Bergstrom, Wesley Zandberg, Lijun Xia, Hongbing Yu, Bruce A. Vallance
The intestinal mucus layer is a carbohydrate-rich barrier that protects the intestinal epithelium from harmful substances and inflammation. It is produced by goblet cells and mainly consists of a heavily O-glycosylated mucin called Muc2. In addition to its physical and biochemical barrier function, mucus plays a key role in nourishing and shaping the gut microbiota profile, thereby contributing to gut homeostasis and protecting the host against bacterial pathogens. We infected mice deficient in Muc2 or Muc2’s core 1 O-glycosylation with the enteric pathogen C. rodentium. Both mouse strains suffered rapid and severe infections as compared to their littermate controls, including increased pathogen burdens (100 fold), greater intestinal macroscopic and histopathology scores, and heightened barrier disruption. Although no microbiota signature was identified in mucin-deficient mice using 16S microbiome analysis, the functionality of microbes assessed by gas chromatography was affected in the absence of mucus, with mice showing lower levels of the bacterial metabolite butyrate. Colonization of germfree Muc2 deficient mice with butyrate-producing bacteria and fecal microbiota transplant with littermate control samples were unable to normalize butyrate levels and protect mice against C. rodentium infection. However, the same stool-derived microbes showed normalized butyrate production when cultured anaerobically in vitro with mucins or purified O-glycans. Moreover, mucin-deficient mice were protected from severe infection when supplemented with the pro-drug tributyrin every other day after infection, displaying reduced weight loss and clinical signs of colitis, while pathogen burdens were greatly reduced as was tissue pathology. These findings demonstrate the essential interplay between mucus and microbes to promote gut homeostasis and host protection via the bacterial metabolite butyrate, and highlight the need to explore the mechanisms by which endogenous host factors, such as the mucus layer, modulate the resident microbiota and its metabolites.
Catherine Chan (Poster 20)
Research Technician, BC Children’s Hospital Research Institute
A customizable stool ex vivo assay for detecting microbiome and metabolic changes in the gut
Catherine S. Chan, Bernice Wong, Christine McCulloch, Larissa Celiberto, Bruce A. Vallance, Ho Pan Sham
The gut microbiota is critical for maintaining health by aiding in digestion and defending against pathogenic microbes. Specific commensal bacteria in the gut produce short-chain fatty acids (SCFAs) when they metabolize indigestible carbohydrates within the intestinal tract. SCFAs serve as functional markers for microbiome changes and are important in studying inflammatory diseases such as inflammatory bowel disease (IBD). The most abundant and well-studied SCFAs are butyrate, acetate, and propionate, and their levels reflect the state of the microbiome. An in vitro assay to examine SCFA production by the resident microbiota, in response to different nutrients, environmental changes or disease states is needed to overcome the complexity challenges an in vivo system. Here we describe an ex vivo assay developed using donor stool collected from humans and mice (C57BL/6Cr) to mimic the interactions between the microbiota and external stimuli (such as fibre). We explored different insoluble fibres found in fibre-rich diets that drive the production of beneficial SCFAs. Microbiome composition and SCFA concentrations before and after anaerobic incubation with nutrient stimuli was analyzed using 16S rRNA sequencing and gas chromatography-tandem mass spectrometry respectively. We found a positive correlation between microbes known to produce beneficial SCFAs and increased SCFA levels. When this assay was applied to IBD-prone mouse models, we found a correlation between in vivo and ex vivo data, supporting the notion that IBD clinical progression can be regulated through a fibre-rich diet. Our assay provides researchers with a quick and high-throughput option for pre-testing diets in patients with gastrointestinal conditions such as IBD. Clinicians can track disease progression in patients by monitoring changes in microbiome composition and their functional implication, then apply a personalised diet recommendation to increase SCFA production. Further development of the assay will provide a unique opportunity for screening microbiota targeting drugs in patients with gastrointestinal disease.
Diana Changirwa (Poster 21)
PhD Student, University of Calgary
Gastrointestinal colonization by Candida albicans modulates systemic host defence in sepsis
Diana Changirwa, Amanda Zucoloto, Katrina Yu, and Braedon McDonald
Pathological dysbiosis of the intestinal microbiome can lead to defects in immune homeostasis and a breakdown in host defense that increases susceptibility to infection. This is particularly relevant in patients with sepsis, a disorder of infection-induced systemic immune dysregulation and multi-organ dysfunction, where dysbiosis of the bacterial microbiome in these patients has been associated with adverse clinical outcomes. However, the impact of the fungal microbiome on sepsis pathogenesis has not been characterized. Analysis of the gut fungal microbiome in critically ill patients with sepsis identified severe fungal dysbiosis driven by marked overgrowth of Candida. Based on these clinical observation, we next sought to investigate the functional impact of intestinal Candida overgrowth on systemic inflammation and host defense using mouse models. We colonized the gastrointestinal tracts of antibiotic-conditioned specific pathogen free (SPF) C57BL/6 mice with different strains of Candida albicans (including both hyphal and yeast morphotypes) for 10 days prior to infection/sepsis with a well-characterized model of S. aureus bloodstream infection. We observed that mice colonized with a yeast-locked strain of C. albicans displayed reduced illness severity, reduced systemic inflammation (lower concentrations of proinflammatory cytokines IL-12p70, IL-1β and IL-6), and enhanced protection against systemic pathogen dissemination compared to uncolonized controls. In contrast, mice colonized with hyphal/filamentous C. albicans displayed similar illness severity, systemic inflammation, and pathogen burden as uncolonized controls. Together, these data demonstrate that fungal dysbiosis with C. albicans overgrowth can modulate the systemic host response to sepsis, and that selective gut colonization by yeast-locked C. albicans may confer beneficial systemic immune modulation that is protective in sepsis.
Leah D’Aloisio (Poster 22)
MSc Student, University of British Columbia Okanagan
Underrepresented populations in gut microbiome research: exploring changes in the gut microbiome of Indian immigrants in Canada
Leah D. D’Aloisio, Natasha Haskey, Nijitai Abulizi, Carson McComb, Mekenna Smith, Jacqueline Barnett, Vignesh Shetty, Ushasi Bhaumik, Mamatha Ballal, Hemanta Koley, Sanjoy Ghosh & Deanna L. Gibson
Background: Young Indian immigrants and Indo-Canadians are at a significantly higher risk for developing IBD once living in a westernized country. While the etiology of IBD remains unclear, a gut microbiome that is no longer symbiotic with its host is a key player. However, Indians are one of the least represented in microbiome research, therefore we cannot accurately assess the role of their gut microbiome in IBD. To understand why Indians are at a greater risk for IBD in Canada, we must first characterize their gut microbiome. Purpose: Our study investigates how immigration to Canada impacts the gut microbiome of Indian populations and explores how it differs from those with European descent. Method: Stool samples from healthy volunteers (ages 18-55) were collected from Indians in India, Indian immigrants, Indo-Canadians, Euro-Canadians, and westernized immigrants. DNA was extracted for 16S sequencing on the Illumina MiSeq platform and shotgun sequencing at 10M reads/sample on the Illumina Novaseq platform. QIIME2 was used for microbiome analysis, and linear discriminate analysis (LDA) effect size (LEfSe) was used to identify differentially abundant taxa (a = 0.05, LDA threshold greater than 3.0). Results: When comparing Euro-Canadians to Indians residing in India, we found that alpha diversity was significantly lower in Indians (Pielou’s evenness, p= 4.51e-14), whereas beta diversity was significantly higher in Indians (Weighted Unifrac, p= 0.001). Furthermore, LDA revealed Prevotella was 5 times more abundant in the gut microbiome of Indians, while Bacteroides was ~4 times more abundant in the Euro-Canadian gut. Conclusion: So far, we found substantial differences in the gut microbiomes between those living in India versus Euro-Canadians. Our next steps include 16S analysis of the additional cohorts, and metagenomic analysis for all groups. We predict that Indo-Canadians will harbour a more industrialized microbiome, sharing more taxa with Euro-Canadians than with those living in India.
Darlene Dai (Poster 23)
PhD Student, BC Children’s Hospital Research Institute
Breastfeeding enrichment of B. longum subsp. infantis mitigates the effect of antibiotics on the microbiota and childhood asthma risk
Darlene L.Y. Dai, Charisse Petersen, Courtney Hoskinson, Kate L. Del Bel, Allan B. Becker, Theo J. Moraes, Piushkumar J. Mandhane, B. Brett Finlay, Elinor Simons, Anita L. Kozyrskyj, David M. Patrick, Padmaja Subbarao, Lars Bode, Meghan B. Azad, Stuart E. Turvey
BACKGROUND: Early antibiotic exposure is linked to persistent disruption of the infant gut microbiome and subsequent elevated pediatric asthma risk. Breastfeeding acts as a primary modulator of the gut microbiome during early life, but its effect on asthma development has remained unclear. OBJECTIVES: This study aims to explore the effect of breastfeeding on antibiotic-associated asthma risk and infant gut microbiome. METHODS: We harnessed the CHILD cohort to interrogate the influence of breastfeeding on antibiotic-associated asthma risk in a subset of children (n = 2,521). We then profiled the infant microbiomes in a subset of these children (n = 1,338) using shotgun metagenomic sequencing and compared human milk oligosaccharide and fatty acid composition from paired maternal human milk samples for 561 of these infants. FINDINGS: Children who took antibiotics without breastfeeding had 3-fold higher asthma odds, whereas there was no such association in children who received antibiotics while breastfeeding. Meanwhile, functional diversity and 94% of the top 50 most significant KEGG orthologs (KOs) found to be altered in children given antibiotics were responsive to breastfeeding, supporting the argument that breastfeeding and antibiotics act as opposing forces on the microbiome. Functional changes associated with asthma protection were linked to enriched Bifidobacterium longum subsp. infantis colonization. Network analysis identified a selection of fucosylated human milk oligosaccharides in paired maternal samples that were positively associated with B. infantis and these broader functional changes. CONCLUSIONS: In conclusion, we identified a clear protective association for asthma when children are breastfed at the time of antibiotics usage. This protection may be in part due to breastfeeding’s sweeping impacts on “rebalancing” microbiome diversity and relative abundances to pre-antibiotic levels, and enriching B. infantis. Our study provides key findings that will inform clinical recommendations regarding breastfeeding to prevent pediatric asthma following antibiotics exposure.
Dr. Marcela Davoli-Ferreira (Poster 24)
Postdoctoral Fellow, University of Calgary
Investigating the role of human commensal bacteria in promoting autism spectrum disorder
Marcela Davoli-Ferreira, Lukas Mager, Aline Ignacio, Kirsty Brown, Fernanda Castanheira, Carolyn Thomson, Jenine Yee, Linda Ward, Thomas Louie, Markus B. Geuking, Kathy D. McCoy
Clinical trials using fecal microbiota transplantation (FMT) in children with autism spectrum disorder (ASD) induce beneficial long-term effect, improving ASD behavioral and neurological symptoms, suggesting that the gut microbiota contributes to ASD etiology. In a pilot study using FMT to treat ASD individuals, our group identified several changes in bacterial composition following FMT, with the most predominant effect being the loss of Clostridium innocuum correlating with the improvement of ASD symptoms. Here, we aimed to investigate whether these patient-derived C. innocuum strains contribute to ASD development and/or severity. We used a gnotobiotic ASD genetic model (BTBR T+Itpr3tf/J mice) colonized with C. innocuum strains isolated from ASD or neurotypical individuals and we assessed ASD-like gastrointestinal symptoms and microglial phenotype using flow cytometry and qPCR, as well as the ASD-like behaviours using three-chamber sociability and social novelty tests. The colonization with C. innocuum isolated from ASD donors induced an altered immune phenotype, including increased frequencies of monocytes and CD11b+CD103- and CD11b+CD103+ dendritic cells, as well as exacerbated Th1 cells in the gut of BTBR mice when compared to mice colonized with the neurotypical-derived bacteria. Mice colonized with ASD-derived strains also showed reduced expression of tight junctions in the ileum and increased intestinal permeability, resembling gastrointestinal symptoms often observed in ASD individuals. Notably, ASD-like behaviours and some alterations in the central nervous system were also observed under ASD- C. innocuum colonization, with microglial cells showing increased levels of CSF1R and reduced CX3CR1expression, suggesting an ‘immature’ microglial phenotype. In conclusion, our preliminary results show that C. innocuum derived from ASD individuals can induce gastrointestinal and behavioural alterations and microglial phenotype traditionally associated with ASD, suggesting that this bacterial strain could contribute to the etiology of this neurological disorder.
Dr. Vanessa DeClercq (Poster 25)
Research Associate, Dalhousie University
Oral microbial signatures associated with age and frailty in Canadian adults.
Vanessa DeClercq, Jacob T Nearing, Morgan GI Langille
Background: Frailty is an ageing-related syndrome of physiological deterioration that is associated with the risk of adverse health outcomes. Research suggests a role of the gut microbiome in frailty, but little is known about the association between frailty and the oral microbiome. Thus, this study aimed to assess the association between frailty and microbial diversity and composition of the oral cavity. Methodology: Data and saliva samples were obtained from male and female participants aged 35-70 years from a Canadian population cohort. The Frailty Index (FI) was assessed in 0.1 increments, with a FI score of ≥ 0.3 representing 5% of the participants and those with the highest level of frailty. Samples (n=1,374) were analyzed by 16S rRNA gene sequencing and differences in microbial diversity and community compositions were examined among frailty and age categories. Results: Most alpha diversity measures were similar with age, except for Evenness which showed a significant increase with age. In contrast, all alpha diversity measures (Evenness, Observed Taxa, Shannon Diversity, and Faith’s Phylogenetic Diversity) show a significant decline with high frailty. Beta diversity as measured by Bray-Curtis (R2=0.008 and R2=0.004) and weighted UniFrac (R2=0.012 and R2=0.006) show significant association between age group and frailty, respectively. Using three differential abundance tools, Veillonella was increased with higher frailty, whereas a reduction was observed with increased age. In addition, significant differences in a taxon belonging to the family Lachnospiraceae were significantly different with increasing frailty and Alloprevotella, Abiotrophia, Neisseria, and Porphyromonas were altered with age. Conclusion: The results from this study show age and frailty are differentially associated with measures of microbial diversity and composition of the salivary microbiome. Findings suggest that the oral microbiome may be a useful indicator of increased risk of frailty or a potential target for improving health in ageing adults.
Dr. Dhwani Desai (Poster 26)
Bioinformatics Specialist, Dalhousie University
Visualising Taxa-Function relationships from meta-omic data
Dhwani K. Desai, André Comeau, Morgan G.I. Langille
High-throughput sequencing has necessitated and facilitated the development of various tools for dissecting the taxonomic structure and molecular function of microbial communities residing in diverse ecological niches. A wide variety of tools and protocols have been developed to process amplicon sequencing as well as meta-omic (metagenomic and metatranscriptomic) data to generate relative abundances of taxonomic groups or functional categories for samples. A key output from many of these protocols is a stratified relative abundance table that specifies the taxonomic contribution to each function. However, there are very few tools which can effectively visualize the different taxa-function relationships from these stratified outputs. Here we introduce an R Shiny application called JarrVis (Just Another stRatified Rpkm VISualizer) which can visualize the taxa-function relationships resulting from different types of 16S amplicon sequencing or meta-omic data. JarrVis can visualize relationships between samples (which can be combined based on metadata categories), the taxa that are detected in these samples (at any given taxonomic level) and the functions encoded by these taxa. We have utilized JarrVis to study taxa-function relationships in 1) a 16S amplicon time-series spanning 4 years with samples collected weekly, 2) a list of function-significant nodes identified using a balance-tree approach in a dataset of metagenome-assembled genomes from marine metagenomes and 3) a set of metatrascriptomes from gut samples of patients with Crohn’s Disease, Ulcerative Colitis and non-IBD controls. JarrVis and related scripts and data are available at https://github.com/dhwanidesai/JarrVis.
Pedro Dimitriu (Poster 27)
Head of Research and Senior Director, Bioinformatics, Microbiome Insights
Exploring multi-omics Integration for characterizing microbial-based products used in biotechnology applications
Pedro Dimitriu, Erick Cardenas, Ben Tantika, Gord Stewart, Bill Mohn Malcolm Kendall
Microbial-based products (MBPs) are a type of “green” product that contains microbes as active ingredients. In 2019, Health Canada and Innovative Solutions Canada put forward a challenge seeking robust and cost-effective solutions to identify, characterize and predict possible interactions among the micro-organisms in MBPs that may mask or enhance adverse effects (e.g. pathogenicity, toxicity) in humans. In response, Microbiome Insights, is developing a prototype to (1) accurately identify and annotate microorganisms in mixtures, (2) develop a strain-based relational database and (3) characterize the microbial population dynamics and interactions that may attenuate or augment adverse effects. We used Illumina- and Nanopore-based genome-resolved metagenomics to characterize the taxonomic composition and functional potential of MBPs and mock communities designed to contain representative bacterial species. We constructed a relational database containing functional annotations from metagenome-assembled genomes, which enabled the integration of essential genomic features for each species, relevant literature and patents sourced from public repositories. We developed a multi-well assay for tracking gene expression changes of two mock communities and an MBP in response to short-term environmental stressors. We used metatranscriptomics to examine population dynamics and interactions that may affect pathogenicity and inferred co-expression dynamics of genes of interest (KEGG functional hierarchies and modules, antimicrobial resistance, and virulence). We were able to recover 116 high-quality bacterial genomes present at ~30X coverage using long reads. Comprehensive functional annotation revealed several genes involved in a variety of antimicrobial resistance and virulence mechanisms. Exposure to heavy metals, increase or decrease in pH levels, and antibiotics were associated with the strongest transcriptional responses. While gene-level expression was mock community-specific, certain responses, such as metal transport upon cadmium exposure and serine proteolysis at low pH, were common to all mock communities. These observations have potential implications for predicting the potential for pathogenicity and effectiveness of microbial-based products.
Liz-Audrey Djomnang (Poster 28)
Graduate Student, Cornell University
Urinary cell-free DNA is an informative bio-analyte to measure the dynamics of the urinary microbiome after kidney transplantation.
Liz-Audrey Djomnang, Omary Mzava, Lars Westblade, John R. Lee, Manikkam Suthanthiran, Darshana Dadhania, Iwijn De Vlaminck
Urine is frequently used to detect and monitor Urinary Tract Infections (UTIs) and solid organs post-transplantation. Within a year of receiving a kidney transplant, 25% of recipients risk losing their kidney to complications arising from UTIs. Advances in microbiome studies found correlations between microbiome changes and disease. Documenting the regular changes in the urinary microbiome could help us prepare for or predict the onset of disease. Urinary cell-free DNA (cfDNA) is a reliable biomarker with utility in allograft monitoring and infection detection. While urinary cfDNA can be used to study the urine microbiome, urine typically has a low microbial cfDNA biomass, making it prone to DNA contamination during handling.To reduce DNA contamination, we developed an assay (SIFT-seq) to tag and recover sample intrinsic cfDNA molecules. We tagged DNA molecules via bisulfite conversion of unmethylated cytosines to thymine. Non converted DNA molecules were considered contaminants and filtered out. We applied SIFT-seq to a cohort of 10 kidney transplant recipients, each provided 7-13 urine samples between 4 – 360 days post-kidney transplant. We found that the urinary microbiome is mostly composed of bacteria, with Proteobacteria, Bacteroides, and Actinobacteria as the most abundant phyla. We also detected viruses usually associated with opportunistic urinary tract infections. Through alpha diversity measurements, we find that over time, the urinary microbiome changes in complexity and evenness. Beta diversity measurements showed that samples originating from the same patient were more similar than those from a different patient. In sum, the urinary microbiome is complex, with some volatility, and is unique to each patient. Microbiome dysbiosis is often associated with disease onset. Recording regular temporal changes in the urinary microbiome of kidney transplant patients can bring us closer to predicting disease and rethinking antibiotic therapies following the kidney transplant.
Peter Dobranowski (Poster 29)
PhD Student, University of Ottawa
Individualized ex vivo resistant starch fermentation by pediatric inflammatory bowel disease patient microbiomes
Peter Dobranowski, Haonan Duan, Mais Jubouri, Alex Dewar, Adrian Beltran, Krystal Walker, Leyuan Li, James Butcher, Janice Mayne, Ruth Singleton, David Mack, Daniel Figeys, Alain Stintzi
Interindividual variability in responses to prebiotic fibers is an obstacle to improving health via microbiome modulation. Rather than a one-size-fits-all approach to fiber-based microbiome modulation, recent advances in ex vitro screening have enabled selecting prebiotics on an individual basis. Such approaches coupled to multi-omic analyses may also reveal microbial factors underlying interindividual variability. Furthermore, most prebiotic-based therapies target butyrate production, which is dysregulated in conditions like inflammatory bowel disease (IBD). Other metabolites and functional pathways are rarely considered as targets of microbiome modulation. We performed high-throughput microbiome culturing using stool samples obtained from 15 pediatric IBD patients with a panel of 9 different resistant starches (RS). After an 18 h culture, we performed multi-omic analyses (16S metagenomics, semi-targeted metabolomics, and shotgun metaproteomics) to examine changes to the microbiomes. Parallel group-level and individual-level analyses revealed RS- and individual-specific changes in fermentation, taxonomic diversity, and functional output. We also highlight RS-driven increases in reportedly anti-inflammatory metabolites, such as alpha-ketoglutaric acid, and preferential production of acetate over butyrate. Our ex vivo findings reinforce recent calls to individualize prebiotic therapies, particularly in clinically relevant populations. We present preliminary evidence to reconceptualize the beneficial properties of prebiotics to include fermentation by-products beyond butyrate.
Breenna Dobson (Poster 30)
MSc Student, University of Calgary
Sexual Dimorphism of Disease Severity in Sepsis is Independent of the Gut Microbiota
Breenna Dobson, Katrina Yu, Braedon McDonald
Sepsis is a life-threatening disease characterized by an exaggerated host response to infection leading to collateral damage to tissues and organ dysfunction. Despite many promising pre-clinical discoveries of sepsis in animal models, translation to humans has universally failed, and there remains no disease-modifying treatments. A key reason for the failed animal-to-human pipeline in sepsis research is that pre-clinical sepsis models have largely failed to consider the impact of heterogeneity (genetic, environmental, phenotypic, and other) that is paramount in human disease, including the fundamental contributions of biological sex on disease pathogenesis and treatment response. Clinical studies in humans have established that the epidemiology and outcomes of sepsis differ between males and females, highlighted by a significantly higher incidence of disease and mortality in males than females. Despite this, the mechanisms underlying this sex bias have not yet been defined. The gut microbiome and its influence on immune development and function has emerged as an important diver of sexual dimorphism in several immune-mediated disease. Therefore, we tested the hypothesis that sexual dimorphism of systemic inflammation, organ damage, and disease severity in sepsis is mediated through sex-based differences in the gut microbiota. Using a well-established model of abdominal sepsis (fecal-induced peritonitis, in which a defined amount of donor SPF feces is injected into the peritoneal cavity), we determined the role of the microbiota in sepsis pathogenesis and outcomes by comparing systemic inflammatory response, pathogen dissemination, organ dysfunction, and disease severity in SPF (specific pathogen-free, ie. conventional laboratory mice) versus germ-free (GF) mice. In SPF mice, we observed that males demonstrated a significantly higher illness severity than females. In germ free (GF) mice, the same sex-bias in sepsis pathogenesis was observed, with increased illness severity in GF males compared to GF females, indicating that sexual dimorphism of sepsis severity is independent of the gut microbiota. Interestingly, increased disease severity in males was not linked to an exaggerated systemic inflammatory response, nor differences in host defense or dissemination of the underlying infection. Together, these findings demonstrate that sexual dimorphism of illness severity in sepsis is independent of the gut microbiota.
Toluwatise Ehindero (Poster 31)
Undergraduate Student, University of Calgary
Re-defining Candida albicans colonization of the lungs in critical illness: friend or foe?
Toluwatise Ehindero, Diana Changirwa, Katrina Yu, Christina Thornton, and Braedon McDonald
Critically ill patients who require mechanical ventilation in Intensive Care Units (ICU) are at extremely high risk of lung infections (ventilator-associated pneumonia, VAP). A common organism isolated from the lungs of ICU patients is Candida albicans, yet this microbe is not considered a lung pathogen in immunocompetent hosts, leaving physicians with a conundrum – is this colonization or infection? Commensal or pathogen? This dichotomy is too simplistic in contemporary microbiome science where microbe-host interactions can blur the lines between commensal and pathogen. Furthermore, the dimorphic nature of C. albicans also contributes to its impact on the host, with hyphal morphologies typically associated with invasiveness and pathogenesis compared to yeast forms. Therefore, we hypothesized that in ICU patients, airway colonization by C. albicans has differential impact on the lung microbiome depending on whether it is a yeast or hyphal morphotype. We performed ITS2 amplicon sequencing to characterize the airway fungal microbiome in 96 tracheal aspirate samples collected from 49 adult patients receiving mechanical ventilation over the course of their ICU admissions, and found that 62/96 demonstrated Candida dominance (>30% relative abundance), with 28/96 demonstrating marked Candida overgrowth (>75% relative abundance). Next, we performed culture-based analysis of samples with Candida dominant mycobiomes, and found that 100% of culturable fungi were Candida, of which 54% were C. albicans, and the remainder were non-albicans species. Of cultured C. albicans isolates, 72% were hyphal morphotypes, while only 28% were yeast-locked morphotypes. These results indicate that the vast majority of C. albicans colonization in the lungs of ICU patients is mediated by potentially pathogenic hyphal morphotypes. Further investigations will define the impact hyphal vs yeast morphotypes on microbiome ecology, microbiome-host interactions in the lungs, and clinical outcomes in critically ill patients.
Dr. Han Fang (Poster 32)
Postdoctoral Fellow, McMaster University
Microbial D-lactate contributes to host blood glucose metabolism
Han Fang, Fernando F. Anhê, Breanne McAlpin, Jonathan D. Schertzer
L-lactate and D-lactate are the two forms of lactate. During anaerobic exercise, skeletal muscle produces L-lactate through glycolysis. L-lactate transits out of muscle, into the blood and is taken up by the liver to produce blood glucose via gluconeogenesis. This glucose then travels back to the muscle to complete the Cori cycle. Unlike L-lactate produced by the host, most D-lactate originates from gut bacteria. However, little is known about the role of gut microbiota derived D-lactate in host metabolism. D-lactate and L-lactate are absorbed at the same rate by the intestine, but D-lactate delivery in rodents leads to more glycogen deposition in the liver compared to L-lactate. People with diabetes and rodent models of diabetes both have higher levels of blood and urine D-lactate. Therefore, we hypothesize that gut microbiota derived D-lactate engages an alternate branch of the Cori cycle and provides a key substrate for gluconeogenesis and lipogenesis in the host liver. By injecting mice with L-lactate or D-lactate, we showed that D-lactate fuels liver glycogen and triglyceride storage in mice. Colonization of germ free mice with bacteria that produce significant amount of D-lactate increased blood glucose. We also tested a new way to trap microbial D-lactate in the gut in order to lower blood glucose and liver fat during obesity. Oral delivery of biocompatible polymer that trapped D-lactate in the gut lumen and forced its excretion in the feces, lowered blood glucose, fasting insulin, and liver fat in obese mice. We continue to optimize biopolymers that trap D-lactate and lower blood glucose and we have found that these effects are polymer length dependent. Collectively, these findings demonstrate that microbial-derived D-lactate contributes to host glucose production and can be used as a potential therapeutic target to combat hyperglycemia.
Zhi Yi Fang (Poster 33)
PhD Student, Queen’s University
Networks of co-occurring human milk microbiota are associated with host genomics and the development of childhood atopy and asthma in the CHILD Cohort Study
Zhi Yi Fang, Sara A. Stickley, Amirthagowri Ambalavanan, Yang Zhang, Kelsey Fehr, Shirin Moossavi, Charisse Petersen, Stuart E. Turvey, Piushkumar J. Mandhane, Elinor Simons, Theo J. Moraes, Malcolm R. Sears, Padmaja Subbarao, Meghan B. Azad, Qingling Duan
The diverse microbial community found in human milk has been proposed to seed the infant gut microbiota, which could impact children’s long-term health outcomes such as atopy and asthma. Earlier studies have identified non-genetic determinants (e.g., antibiotic use) of the human milk microbiota (HMM) composition as well as candidate gut microbes (e.g., Veillonella) associated with risk of allergic diseases in children. However, the influence of host genomics on HMM remains poorly understood. In addition, the connections among co-occurring microbes in human milk and their modulating effects on risk of childhood allergic diseases have yet to be investigated. This is the first study to employ a network analysis approach to identify connections among co-occurring microbes in human milk and to determine their associations with maternal genomics and allergic outcomes in human milk-fed infants. HMM composition was assessed by 16S rRNA sequencing of breastmilk samples collected 3-4 months postpartum from 885 mothers of the CHILD Cohort Study. Genomic profiles of the mothers were obtained from the Illumina HumanCoreExome BeadChip. Using an unsupervised machine-learning method, we identified clusters of co-occurring microbes and determined their associations with childhood atopy and asthma at age 5 years using a linear regression model. For example, we identified a microbial cluster containing Pseudomonas–Stenotrophomonas in mothers’ milk associated with their children’s risk of asthma. In addition, increased alpha-diversity and the Veillonella–Prevotella containing cluster are associated with reduced risk of childhood atopy. Genome-wide association analyses of HMM revealed that genomic variants on chromosomes 1 (e.g., rs4970535, P=4.7×10-10) and 2 (e.g., rs756018, P=1.8×10-11) are significantly associated with Lachnoanaerobaculum and Pseudomonas, which are members of microbial clusters associated with atopy and asthma, respectively. Thus, our findings suggest that maternal genomics contribute to the microbial community in human milk, which may modulate risk of atopy and asthma during childhood.
Bradey Fitzgerald (Poster 34)
MSc Student, University of Guelph
Variable Resistance of Lachnospiraceae Isolates to Intestinal Stressors
Bradley Fitzgerald, Autumn Sweeney and Matthew Sorbara
Beneficial microbes of the gut microbiota provide colonization resistance (CR) that prevents the establishment of pathogenic bacteria and provides protection from increasingly prevalent multi-drug resistant pathogens. Competition for nutrients and other mechanisms of CR impose stress on beneficial microbes and pathogens alike. These beneficial microbes are a pivotal part of CR, however, their resistance to these stressors in the gut environment is understudied. In particular, the Lachnospiraceae family produces high levels of short-chain fatty acids (SCFAs), which directly inhibit the expansion of numerous multi-drug resistant pathogens. The present study categorizes the inter- and intra-species resistance patterns of isolates from the Lachnospiraceae family to pH, bile acid, osmotic, SCFAs and antibiotic stress. Each stress condition was independently evaluated for isolates using in vitro analysis. The genetic basis for the variable resistance patterns was investigated by correlating resistance with differences in their whole genome sequences. Further, alterations in metabolic profiles of isolates under stress were elucidated through gas-chromatography mass spectrometry to determine their stress-triggered contributions to CR. A high degree of inter- and intra-species variability was observed in isolates under multiple stress conditions. Specifically, the species Anaerostipes hadrus displayed a variable phenotype of SCFAs stress resistance in acidic conditions. A. hadrus are prominent producers of the SCFA, butyrate. Under SCFA stress, however, these isolates shift their metabolic profiles to limit the presence of butyrate. Overall, this project provides insight into the impact of CR stressors on isolates from the Lachnospiraceae family, which play a pivotal role in maintaining CR. The increased knowledge of how beneficial isolates behave under CR has the potential to be an important part of our arsenal against multidrug resistant pathogens and complements ongoing efforts of antibiotic discovery.
Dr. Fabien Franco (Poster 35)
Postdoctoral Fellow, University of Calgary
Regulatory circuits of mitophagy restrict cell death for mounting memory CD8+ T cell formation
Fabien Franco, Alessio Bevilacqua, Ruey-Mei Wu, Chun-Pu Lin, Lorène Rousseau, Fu-Ti Peng, Yu-Ming Chuang, Jhan-Jie Peng, Jaeoh Park, Yingxi Xu, Kung-Chi Kao, Antonino Cassotta, Yi-Ru Yu, Daniel E. Speiser, Federica Sallusto and Ping-Chih H
CD8+ T cell metabolism is dynamically regulated during infection. Notably, oxidative metabolism supports differentiation and survival of memory CD8+ T cells. However, it remains unclear whether the maintenance of mitochondrial quality is needed for memory T cells. Interestingly, mitochondrial quality maintenance by mitophagy is critical to prevent the development of Parkinson’s disease in humans. Here, we found that Parkinson’s patients had decreased levels of CD8+ memory T cells at steady state and failed to form memory T cells upon COVID-19 vaccination. In mice, we unveiled that mitophagy proteins were upregulated during memory development in response to IL-15. Deletion of Parkin or NIX led to severe impairment in CD8+ memory T cell formation resulting from declined survival. Mechanistically, NIX expression prevents ferroptosis by avoiding metabolic deregulation. In contrast, Parkin prevents apoptosis induction. Together, our findings indicate that the mitophagy machinery plays a pivotal role during memory T cell formation.
Dr. Thaís Glatthardt (Poster 36)
Postdoctoral Fellow, University of Calgary
Malassezia restricta Gut Colonization Impacts Lung Immune Responses to Respiratory Syncytial Virus
Thaís Glatthardt, Erik van Tilburg Bernardes, Mackenzie Gutierrez, Leanne Bilawchuk, David Marchant, Eugenia Corrales Aguilar, Marie-Claire Arrieta
Respiratory syncytial virus (RSV) is a major cause of infant bronchiolitis and pneumonia worldwide. Besides the risk of hospitalization and severe disease, early life RSV infection is associated with increased asthma risk. While the mechanisms involved in this connection are incompletely understood, the gut-lung axis is hypothesized to be involved given the roles of the gut microbiome in immune development, asthma risk, and lung immune responses to other respiratory viruses. While most microbiome studies are focused on gut bacteria, we and others have demonstrated that colonization with gut fungi potentiates mucosal and systemic immunity. Further, we recently showed that infant antibiotic use leads to an overgrowth of the fungi Malassezia restricta, and that colonization with this fungus increased susceptibility to allergic airway inflammation in mice. Thus, we hypothesized that colonization of M. restricta may also exacerbate inflammation after RSV infection and increase susceptibility to allergic airway inflammation post-infection. Eighteen-day-old gnotobiotic BALB/C mice colonized with bacterial consortium Oligo-MM12 alone or in combination with M. restricta were infected intranasally with RSV (A2 strain). Lungs were collected for immune cell profiling via FACS on the fifth day post-infection. Despite poor viral particle recovery from infected lungs, we detected an increase in monocytes (CD45+Ly6C+IRF5+) and macrophages (CD45+CD64+CX3CR1+IRF5+) and Th1 T cells (CD45+TCRb+CD4+Tbet+) in RSV infected mice colonized with M. restricta compared to mice colonized only with Oligo-MM12. This suggests that colonization with M. restricta exacerbates inflammation to control RSV infection. Further experiments are necessary to evaluate the impact of M. restricta on susceptibility to airway inflammation post RSV infection. Results from this work will help elucidate the mechanistic underpinnings of the gut-lung axis during early life, how it contributes to the risk of asthma and potential therapeutic strategies.
Dr. Simon Hirota (Poster 37)
Associate Professor, University of Calgary
Gut-residing bacteria can shape host drug metabolism in the small intestine through an innate lymphoid cell-IL-22 driven axis
Kyle L Flannigan, Michael Johnston, Sarah L Erickson, Kristoff M Nieves, Humberto Jijon, Marco Gallo, Kathy D McCoy, Simon A Hirota
The microbiota’s influence on drug metabolism has been recognized, however the mechanism(s) are unclear. Colonization of germ-free mice with an SPF microbiota induces the expression of cytochrome P450 (CYP) enzymes in the liver and small intestine (SI), two important sites of drug metabolism. In this study, we sought to investigate the role of a specific gut-residing microbe (segmented filamentous bacteria; SFB) in shaping the expression and activity of host drug metabolism enzymes. SFB-negative (SFB-) mice were obtained from Jackson (Jax) and colonized with feces from SFB-mono-associated mice. 14 days later, the expression of drug metabolism genes was assessed, and SI lamina propria cells isolated for flow cytometry. A monoclonal antibody for Thy1.2 was used to deplete innate lymphoid cells (ILCs) in RAG1-/- mice. SI organoids were generated from mice and humans and treated with IL-22 to further assess the dynamics of CYP3A11/CYP3A4 expression and activity. Colonization of Jax mice with SFB suppressed various CYP genes in the SI (but not liver), with Cyp3a11 being the most downregulated gene. SFB-induced IL-22 production by ILC3s correlated with reduced SI Cyp3a11 expression. Additionally, SFB colonization had no effect on the expression of Cyp3a11 in the SI of mice in which ILCs were depleted. Both SFB colonization and administration of IL-23, to induce IL-22 from ILC3, suppressed the CYP3A11-mediated metabolism of orally administered drugs. In mouse SI enteroid cultures, IL-22 reduced the expression of Cyp3a11 and decreased the ability of SI enteroids to metabolize CYP3A11-specific substrates. Finally, in human ileal enteroids, IL-22 suppressed genes in a drug metabolism pathway centred around CYP3A4. Our data suggest that a single gut-resident microbe can influence drug metabolism in the SI through an ILC3-IL-22-dependent mechanism. These findings provide an understanding of how the microbiota and its induction of IL-22 could impact therapeutic outcomes.
Dr. Aline Ignacio (Poster 38)
Postdoctoral Fellow, University of Calgary
Investigating the role of intestinal commensal bacteria in reprogramming intestinal macrophage
Aline Ignacio, Sonia Czyz, Marcela Davoli Ferreira, Kathy McCoy
AIM: The profound importance of microbiota-host interactions in regulating immunity has been demonstrated in multiple illnesses. However, whether the gut microbiota can induce innate memory (also referred as ‘trained immunity’) in innate cells has not yet been studied. Our group demonstrated that transient colonization of pregnant dams-only results in germ-free (GF) offspring with altered immune system landscape later in life, providing strong evidence of memory-like phenotype induced by intestinal microbiota. Therefore, I hypothesized that microbiota-derived products and metabolites induce reprogramming of intestinal macrophages, which will then show increased responsiveness upon inflammation. This study aims to investigate whether commensal bacteria can reprogram intestinal macrophage immune responses, resulting in increased effector function upon secondary stimulation. METHODOLOGY: GF pups were gavaged with the auxotrophic strain Escherichia coli HA107. Because this bacterium cannot survive more than 48h in vivo, the pups return to GF status, which allows the induction of innate memory to be disassociated from continuous microbial exposure. Control pups were gavaged with PBS. At 8-weeks of age, GF mice were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS) or PBS, and small intestine and colon lamina propria cells were evaluated 6h and 24h post-injection by flow cytometry. GF mice were gavaged with E. coli JM83 and bacterial translocation was evaluated 18h post gavage. RESULTS: Preliminary results have revealed that in response to LPS injection, transient colonization in early life increased macrophage cytokine production and numbers in the intestines. Transient colonization in early life reduced bacterial translocation in adulthood. CONCLUSION: Transient exposure to commensal bacterium modulates innate immune response in adulthood. Epigenetic modifications will be assessed and the functional consequences of trained immunity will be determined by analysis of host immune defense against enteric infection. Funding: Canadian Institutes of Health Research (CIHR)
Maira Jiménez-Sanchez (Poster 39)
PhD Student, University of British Columbia
Exposing the skin to UVB light modulates the intestinal microbiota
Maira Jiménez, Hyungjun Yang, Jan Dutz, Bruce A. Vallance
Immune and inflammatory illnesses, such as inflammatory bowel disease (IBD) and multiple sclerosis (MS) are increasing in Western society due to changes in modern lifestyle and environment, such as the overuse of antibiotics and reduced exposure to sunlight, including ultraviolet B (UVB) light. Notably, low sunlight exposure and overuse of antibiotics drive changes in gut microbiome composition, which are also associated with the pathogenesis of these chronic inflammatory diseases. In this study, we hypothesized that UVB light exposure on the skin of mice, and its induction of vitamin D (25(OH)D3) can subsequently modulate the intestinal microbiome’s composition and function. After 5 weeks of feeding mice either vitamin D deficient (Vit D -) and sufficient diets (Vit D +), we shaved the backs of mice and we exposed them to UVB light (492 seconds, 99.23 m J/ cm 2). Exposing mice fed a Vit D (-) diet to UVB light significantly increased their serum concentrations of 25(OH)D3 after 21 days of treatment (9 exposures). In contrast, UVB exposure only caused a modest increase in vitamin D levels in mice fed a Vit D (+) diet. Microbiome analysis showed a significant difference in alpha diversity after UVB treatment only in mice fed the Vit D (-) diet, suggesting that vitamin D levels impact the gut microbiota’s diversity. Moreover, in the Vit D (-) group, we saw significant differences in the phyla Actinobacteriota and Proteobacteria after treatment, whereas the relative abundances of Firmicutes and Actinobacteriota were altered after UVB exposure in the Vit D (+) group. In addition, UVB treatment led to a significant increase in the levels of the short chain fatty acid propionic acid in mice fed the Vit D (-) deficient diet. These results thus show that (1) UVB treatment can alter the composition and function of the intestinal microbiome, and (2), the microbiome shift of mice treated with UVB depends on vitamin D.
Wasif Khan (Poster 41)
PhD Student, University of Manitoba
A cross-cohort analysis of dental plaque microbiome to identify common biomarkers in early childhood caries
Mohd Wasif Khan, Daryl Fung Lerh Xing, Robert J. Schroth, Prashen Chelikani, Pingzhao Hu
Background: Early Childhood Caries (ECC) is a common dental disease affecting half of children worldwide. Several published studies have identified differences in the dental plaque microbiome between ECC and healthy teeth. However, most of these studies suffer from small sample sizes and low reproducibility. Objectives: In this meta-analysis, we aimed to evaluate the collective evidence from previous studies that investigated the microbiome data in ECC and caries-free (CF) teeth using differential abundance and machine learning based methods. Methods: We searched previously published studies for dental plaque microbiome where raw sequencing data available for 16S rRNA amplicon sequencing. The raw sequences were downloaded and processed in Qiime2 program to obtain the operational taxonomic units (OTU) table for each study. The study-associated batch effects in the OTU tables were corrected using the sPLSDA method. A meta-analysis was conducted using these OTU tables to identify differentially abundant OTUs. We also compared the classification performance of Lasso, random forest, decision trees, support vector machine, and XGBoost methods for CF and ECC samples on these datasets using a leave-one-dataset-out cross-validation (LODO-CV) approach. Results: Five published studies matched the selection criteria with a total of 117 CF and 106 ECC samples. Beta diversity analysis revealed that the sPLSDA batch correction method increased homogeneity among studies. The meta-analysis showed a higher abundance of Prevotella salivae, Selenomonas sputigena, and Prevotella oulorum in ECC samples. Among all the tested methods, the random forest classifier had the highest disease classification performance in terms of AUROC for both, the pooled dataset (0.91) and LODO-CV analysis (0.62-0.86). Conclusions: Our meta-analysis pipeline provides more robust results for ECC classification and identification of taxonomic biomarkers. The machine learning analysis is effective for ECC classification in both single and combined studies. The LODO-CV shows that the model has generalizability for ECC classification across different studies.
Dr. Yeganeh Khaniani (Poster 42)
Research Associate, University of Calgary
Revealing Metabolite Consumption and Production Pattern in Bacterial Species using LC-MS Profiling
Yeganeh Khaniani, Soren Wacker, Gopal Ramamourthy, Ryan Groves, Thomas Rydzak, Ian A. Lewis
The metabolite profile of a bacterial species can provide valuable information for diagnosis, monitoring, and treatment of bacterial infections. For instance, metabolic preferences of bacteria may influence competition and pathogenesis. Detecting such metabolites can provide early warning signs of a potential infection and help to initiate appropriate intervention measures. The metabolite profile of a bacterium species refers to the specific set of metabolic products or by-products present within the bacterial cell or secreted into the surrounding environment. These metabolites vary depending on the bacterial species, its growth conditions, and its metabolic pathways. In this study, we employed liquid chromatography-mass spectrometry (LC-MS) to profile the consumption and production patterns of metabolites in both Gram-positive and Gram-negative species: Enterococcus faecalis, Enterococcus faecium, Klebsiella oxytoca and Klebsiella pneumoniae. The strains were cultured up to a late exponential phase. Extracts were then diluted with 50/50 methanol/water (v/v) and the resulting samples were subsequently analyzed using a Thermo Fisher Scientific Vanquish UHPLC platform and a Thermo Scientific Q ExactiveTM mass spectrometer. Our results revealed distinct metabolic profiles for each species, with differences in both the types and quantities of metabolites consumed and produced. K. oxytoca displayed a unique metabolic profile characterized by the consumption of complex carbohydrates and the production of various organic acids and polyols. E. faecium, on the other hand, exhibited a broader metabolic repertoire, including utilization of carbohydrates, amino acids, and organic acids. Furthermore, we identified species-specific metabolites that were exclusively consumed or produced by a particular species, providing valuable clues about their unique metabolic capabilities and potential ecological roles.
Reihane Khorasaniha (Poster 43)
PhD Student, University of Manitoba
Consumption of β-fructan fibers changes significantly during disease flare in inflammatory bowel disease patients, associated with altered gut microbiota
Reihane Khorasaniha, Ismaila Ba, Juan Jovel, Kathy Vagianos, Cassandra Dolovich, Michael Sargent, Kevin McGregor, Charles N Bernstein, Heather K. Armstrong
Dietary fibers are not digested, they are fermented by gut microbes, producing beneficial short-chain fatty acids (SCFAs). Gut microbiota composition is altered and SCFAs are reduced in inflammatory bowel disease (IBD) patients, suggesting fiber fermentation is less successful. Our team previously demonstrated specific unfermented dietary fibers (e.g., β-fructans) can induce inflammation in IBD patients. IBD patients who displayed inflammatory responses to β-fructans consumed less β-fructan suggesting avoidance patterns. Here, we aimed to understand how the avoidance of specific dietary fiber subtypes relates to disease progression in IBD patients. 155 IBD patients were enrolled in the Living With IBD Study. Flare was defined by fecal calprotectin (≥250μg/g stool; collected at baseline, week 26, and week 52). Food frequency questionnaires (FFQ) were collected at baseline and 52 weeks. Average daily fiber subtype intakes were calculated, and Kcal-adjusted fiber intakes were estimated by applying the residual method, and the correlation between fiber avoidance patterns and disease severity (flare/remission) was assessed by logistic regression using SPSS26. Gut microbiota abundance and functions were determined (shotgun metagenomics; Kraken2 and HUMAnN3) and correlated with fiber consumption and disease activity. Only patients who progressed from remission to flare had a high probability of greater fold change in consumption of β-fructans (baseline to week 52), fructooligosaccharides (odds ratio, 1.23; 95% confidence interval [CI], 1.01–1.49; P = 0.04), and inulin (odds ratio, 1.4; 95% confidence interval [CI], 1.06–1.85; P = 0.02). Changes in the consumption of β-fructans correlated with changes in microbiota composition and functions. We previously showed β-fructans can induce gut damage in IBD if not fermented by gut microbes. Therefore, our findings may suggest that IBD patients’ tolerance of specific dietary fibers may change during disease progression from remission to flare, dependent on changes in the ability of gut microbiota to ferment fibers.
Pardis Kiani (Poster 44)
MSc Student, University of Calgary
Brain-first versus Body-first: Exploring aspects of gut microbiota in Parkinson’s disease
Pardis Kiani, Marcela Davoli Ferreira, Kathy McCoy
Parkinson’s disease (PD) is characterized by the presence of intraneuronal a-synuclein inclusions, known as Lewy Pathology. Despite extensive research, the initiation of the a-synuclein aggregation remains unclear. Recently, researchers have proposed that PD can be classified into two subtypes, ‘brain-first’ and ‘body-first’, and that the microbiome plays a role in PD pathology. This study aims to investigate whether microbiota from Parkinson’s disease subtypes will induce divergent gastrointestinal functions and motor deficits in microbiome-associated mice. To accomplish this, human PD microbiome-associated mouse models were generated from ‘brain-first’ and ‘body-first’ PD donors. We assessed gut transit time and intestinal permeability through carmine red dye and FITC-Dextran, respectively, to examine gastrointestinal dysfunction in the two subtypes in microbiota-associated mouse models. We assessed gut transit time and intestinal permeability through carmine red dye and FITC-Dextran, respectively, to examine gastrointestinal dysfunction in the two subtypes in microbiota-associated mouse models. Additionally, we evaluated motor deficits through various behavioral experiments, including pole descend, nasal adhesive removal, and hindlimb score. We also assessed changes in olfaction function through olfactory preference and olfactory avoidance tests on a subset of human PD microbiome-associated mice. We observed more than 80 percent engraftment of bacterial phyla in the recipient mice, indicating successful bacterial engraftment. Analyzing gastrointestinal function assays showed increased intestinal transit time in males in the ‘brody-first’ subtype. Moreover, ‘body-first’ male mice showed increased motor deficits and hyposmia compared to the “brain-first” subtype. Our data suggest the effect of gut microbiota on presenting Parkinson’s disease motor and nonmotor symptoms. These findings indicate the need for further investigations to confirm the differential gastrointestinal function and motor deficits between the two PD subtypes.
Dr. Larisa Kovtonyuk (Poster 51)
Postdoctoral Fellow, University of Calgary
The role of intestinal microbiome in CD19 directed CAR T cell therapy
Larisa V. Kovtonyuk, Sacha Benaudia, Douglas Mahoney, Kathy D. McCoy
Adoptive T cell therapies have been under intense development for the past decades. One of the most promising therapies is chimeric antigen receptor (CAR) T cells, which has been approved by FDA. Nearly 50% of CAR T cell patients either do not respond to the therapy or relapse after treatment. In addition, almost half of patients experience severe adverse effects due to cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). Recently the role of microbiome and their secreted metabolites in modulating the anti-cancer function of T cells in immune checkpoint inhibitor therapy was established. Although there are likely many reasons for non-responsiveness, relapse or toxicity, differences in the patient’s microbiome composition may be one underlying factor. We hypothesize that specific intestinal microbial species can influence CD19-CAR T cell therapy via secreted metabolites. Recent studies have demonstrated differences in the composition of the microbiome between responder and nonresponder patients and have identified some bacterial species that are associated with responsiveness or absence of toxicity. We have established germ-free NSG (KbDb)null mice to investigate whether the presence of a microbiota alters the efficacy of CAR T cell therapy and to elucidate the underlying mechanisms involved. Our preliminary data show that CD19-CAR T cells transferred into germ free NSG (KbDb)null mice that received luciferase expressing Nalm6 cells can eliminate tumor cells in vivo. However, CD19-CAR T cells perform better and eliminate tumor cells more efficiently in NSG (KbDb)null mice harboring a diverse specific pathogen-free (SPF) microbiota. In addition, CD19-CAR T cells in germ free NSG (KbDb)null mice acquired higher expression of PD-1 and LAG3 exhaustion markers. This preliminary data indicates that the microbiome can modulate CAR T cell performance in vivo. Further studies will elucidate the specific bacteria and metabolites involved.
Samuel Lawal (Poster 52)
MSc Student, University of Manitoba
Changes in microbiota composition and functions (enzymes) correlate with changes in dietary β-fructan fibre consumption patterns during IBD disease progression from remission to flare
Samuel A. Lawal, Reihane Khorasaniha, Ismaila Ba, Juan Jovel, Ramsha Mahmood, Richard Miller, Athalia Voisin, Kevin McGregor, Charles N. Bernstein, Heather K. Armstrong
Dietary fibers can be beneficial in inflammatory bowel disease (IBD) patients, yet many IBD patients describe intolerance of high-fiber foods, particularly during disease flare. Dietary fibers are not digested; they are fermented by microbiota producing beneficial short-chain fatty acids (SCFA). Gut microbiota are altered in IBD and SCFA are often found to be decreased, suggesting fiber fermentation is reduced. We previously showed that unfermented β-fructan fibers can drive inflammation and symptomatic flare in certain IBD settings, associated with changes in gut microbiota functions. Furthermore, patients who displayed a pro-inflammatory response to β-fructans, also consumed less β-fructans, suggesting patients may avoid consuming foods high in β-fructans to avoid worsened symptoms. Here we aimed to examine how changes in β-fructan consumption and gut microbiota were associated with changes in IBD disease activity. We enrolled 155 adult IBD patients; 27 patients did not complete the study, food frequency questionnaires (FFQ) were collected at weeks 0 and 52 (n=128), and stool was collected at weeks 0, 26, and 52 (n=52). Active disease was defined by stool fecal calprotectin (>250 μg/g). Fiber subtype consumption patterns were calculated. Shotgun metagenomics sequencing was performed on the stool and the microbiota taxonomic (Kraken2) and metabolic (HuManN3) profiles were analyzed. Stool metabolomics (metabolites) and GC-VFA (short chain fatty acids; SCFA) were performed. Fiber consumption, microbiota, metabolomics, SCFA, and demographics were correlated. Changes in microbiota functions, metabolomics, and SCFA from baseline to week 26 to week 52 correlated with changes in β-fructan consumption from baseline to endpoint. These changes were most significant in patients progressing from remission to flare (n=23), not remission-remission (n=49), flare-remission (n=28), or flare-flare (n=28). Our results suggest IBD patient sensitivity to β-fructans may occur primarily during IBD flare, related to changes in gut microbiota, however, the directionality of these interactions have yet to be fully elucidated.
Dr. Karen Lithgow (Poster 53)
Postdoctoral Fellow, University of Calgary
Secreted Proteases from Bacterial Vaginosis-Associated Bacteria Activate Human MMPs and Modulate Endocervical Barrier Function
Karen V. Lithgow, Antoine Dufour, Laura K. Sycuro
Bacterial vaginosis (BV) is a prevalent dysbiotic vaginal condition that increases the risk of sexually transmitted infection (STI). The presence of select BV-associated Prevotella species increases HIV risk by up to 13-fold. However, we lack mechanistic understanding of how Prevotella species augment STI risk. Elevated cervicovaginal proteolysis occurs during BV and increased human matrix metalloproteinase (MMP) expression is linked to HIV acquisition, yet the role of bacterial proteases in initiating and exacerbating this proteolysis is poorly defined. We hypothesize that BV-associated Prevotella species secrete proteases that dysregulate proteolysis, disrupt the endocervical barrier, and promote STI susceptibility in the female genital tract. Using fluorophore-quenched substrates and protease inhibitors, vaginal Prevotella species were screened for different classes of secreted proteases targeting structural components of the human cervix (collagens, elastin). Five out of eight vaginal Prevotella species were found to produce diverse and redundant enzymes that degrade key cervical barrier proteins. An MMP-8 activation assay using collagen zymography revealed that proteolytic vaginal Prevotella species target MMP-8 for activation and degradation, producing unique proteolyzed breakdown products over time. Furthermore, Prevotella species metalloproteases degrade a fluorogenic peptide substrate specifically designed to assess human MMP activity. These results suggest that Prevotella proteases not only activate MMPs, but also mimic their activity by degrading the same protein/peptide substrates. Intriguingly, we found that Prevotella bivia, Prevotella corporis and Prevotella melaninogenica traversed a polarized endocervical transwell barrier model, while beneficial vaginal bacteria and protease-deficient BV associated bacteria did not. Importantly, epidemiological studies reveal that two of these barrier traversing species, P. bivia and P. melaninogenica, are linked to HIV risk. Our findings suggest a new mechanism whereby proteolytic Prevotella species directly degrade structural proteins involved in cervical barrier maintenance, while amplifying their effects via MMP activation and MMP mimicry to increase susceptibility to STIs in the female genital tract.
Dana Lowry (Poster 54)
PhD Student, University of Calgary
Maternal gut microbiota transplantation in germ-free mice partially replicates offspring phenotype
Dana E. Lowry, Erin W. Noye Tuplin, Kate M. Sales, Melissa Biddle, Kathy D. McCoy, Raylene A. Reimer
Early life gut microbiota is integral in the development of host immune, nervous, and metabolic systems. Disrupting this microbial colonization leads to increased risk of chronic diseases such as obesity and diabetes. We showed in rats that antibiotic-induced disruptions in maternal gut microbiota increase offspring obesity risk, while maternal co-consumption of prebiotics protect offspring from this risk. We used fecal microbiota transplants to determine whether the obesity-prone phenotype is directly transferred via maternal gut microbiota. Cecal matter from Sprague-Dawley rat dams was pooled to create slurries from: 1) Control (CT), 2) Antibiotic [AB] (low-dose penicillin (LDP)), 3) Oligofructose [OF] (10%) or 4) Antibiotic+Oligofructose [AO]. Slurries were transferred to germ-free C57BL/6 male mice fed standard chow. Weight, food intake and fecal samples were collected over two weeks, at which point body composition, insulin and oral glucose tolerance was measured. By Day 14, AB mice gained more weight than OF and AO, whereas body fat % was higher in OF and AO vs CT mice. Cecum size, fasting insulin and leptin, insulin and oral glucose tolerance did not differ. Fecal microbiota remained clustered according to group over time. Alpha diversity (Observed, Shannon and Chao1) increased over time in all groups except for the AO group. By Day 13, alpha diversity was decreased in the OF and AO vs AB and CT mice. From days 2-13, the AB mice had enriched Enterobacteriaceae, Prevotellaceae and Mucispirillum, and reduced Akkermansiaceae. Bifidobacteriaceae decreased and Akkermansiaceae and Coriobacteriaceae increased in OF and AO mice from days 2-13. Two ASVs, Lachnospiraceae AC2044 group and Alistipes finegoldii, were positively and negatively correlated with change in body weight and increased and decreased in AB vs OF and AO groups, respectively. The resultant phenotype of mice receiving maternal microbiota replicates the phenotype of rat weanlings from the respective maternal treatment groups.
Yao Lu (Poster 55)
PhD Student, McGill University
MicrobiomeAnalyst 2.0: comprehensive statistical, functional and integrative analysis of microbiome data
Yao Lu, Guangyan Zhou, Jessica Ewald, Zhiqiang Pang, Tanisha Shiri, Jianguo Xia
Microbiome studies have become routine in biomedical, agricultural, and environmental sciences with diverse aims, including diversity profiling, functional characterization, and translational applications. The resulting complex, often multi-omics datasets demand powerful, yet user-friendly bioinformatics tools to reveal key patterns, biomarkers, and functional insights. Here we introduce MicrobiomeAnalyst 2.0 to support comprehensive statistics, visualization, functional interpretation, and integrative analysis of common data formats generated from microbiome studies. Compared to the previous version, MicrobiomeAnalyst 2.0 features three new modules: 1) a Raw Data Processing module for amplicon data processing and taxonomy annotation that connects directly with the Marker Data Profiling module for downstream statistical analysis; 2) a Microbiome Metabolomics Profiling module to help dissect associations between community compositions and metabolic functions through joint analysis of paired microbiome and metabolomics datasets; and 3) a Statistical Meta-Analysis module to help identify consistent signatures by integrating datasets across multiple studies. Other important improvements include added support for multi-factor differential analysis by considering complex metadata, interactive visualizations for popular graphical outputs, updated methods for functional prediction and correlation analysis, and significantly expanded taxon set libraries based on the latest literature. These new features are demonstrated using a multi-omics dataset from a recent type 1 diabetes study. MicrobiomeAnalyst 2.0 is freely available at microbiomeanalyst.ca.
Madeline Mellet (Poster 56)
MSc Student, University of Calgary
Development of a primary intestinal mesenchymal stromal cell culture to assess microbially induced inflammatory mediators
Madeline Mellett, Kirsty Brown, Kathy D. McCoy
The gastrointestinal tract harbours a complex community of microorganisms that constantly interact with the intestinal immune system. While many studies have begun to elucidate microbial induced changes of intestinal immune cells, the impact of microbes on non-immune cells such as intestinal mesenchymal stromal cells (iMSCs) has been largely overlooked. iMSCs are a heterogeneous cell population that are primarily described as maintaining tissue structure, but they also play important roles in immune modulation. For instance, iMSCs can produce pro-inflammatory cytokines and chemokines that could influence innate immune responses. We aimed to explore how the microbiota may shape the production of inflammatory mediators using a primary cell culture model. Single cell suspensions were generated from the small intestines of C57BL/6 mice and primary iMSC were isolated by differential adherence to culture flasks. Flow cytometry analysis revealed that cultured cells expressed phenotypic markers of iMSCs including GP38, CD81, NCAM, CD140α and αSMA. To understand the influence of bacteria on cytokine production from these cells, they were incubated with the bacterial component lipopolysaccharide (LPS) and compared to incubation with TNFα and IL1β, which would mimic an inflammatory environment. Exposure to LPS led to transcriptional upregulation of immunomodulatory cytokines compared to treatment with TNFα and IL-1β. Furthermore, these cytokines could be identified in LPS treated cells through immunofluorescence. These results suggest that microorganisms may induce cytokine expression from iMSCs, rather than iMSCs responding to an inflammatory environment. Future experiments will assess the ability of additional bacterial features to induce cytokine expression and which pathways are important for induction from iMSCs. These findings contribute to an increasing understanding of iMSC communication with both intestinal microbes and the immune system, which may translate to improved understanding of diseases like inflammatory bowel disease where iMSCs have been implicated.
Emily Mercer (Poster 57)
PhD Student, University of Calgary
Divergent maturational patterns of the infant bacterial and fungal gut microbiome are associated with interkingdom dynamics and nutrition
Emily Mercer, Hena Ramay, Allan Becker, Elinor Simons, Piushkumar Mandhane, Stuart Turvey, Theo Moraes6 Malcolm Sears, Padmaja Subbarao, Meghan Azad & Marie-Claire Arrieta
Background: In early-life, the gut microbiome undergoes ecological shifts characterized by increasing bacterial alpha-diversity and variable changes in fungal alpha-diversity. However, not all infants follow these patterns, but our understanding of the factors linked to atypical microbiome maturation is limited. We assessed bacterial and fungal microbiome maturation in early-life to determine if atypical maturational patterns were observed in otherwise healthy infants and identify factors underlying these patterns. Methods: 16S and ITS2 Illumina sequencing was performed on stool samples from 100 CHILD Cohort Study infants at 3 and 12 months of age to characterize the bacterial and fungal gut microbiome, respectively. Untargeted metabolomics was performed on urine samples collected at the same timepoints using LC-MS/MS. Ecological and multivariate analyses were performed using RStudio. Results: Atypical alpha-diversity trajectories were observed in 24% and 20% of infants in the first year of life for bacteria and fungi, respectively. Atypical trends were linked to reduced abundance of Bacteroides and increased Candida at 3 months. Functional analysis revealed an atypical bacterial trend was associated with elevated urinary trimethylamine N-oxide, creatine, and indole acetic acid, and an atypical fungal trend with elevated lactate. Using decision trees, the strongest predictors of atypical alpha diversity trends were interkingdom dynamics, breastfeeding duration, and maternal diet during pregnancy. Interkingdom network analyses revealed the microbiome of infants with an atypical bacterial or fungal alpha-diversity trend displayed substantially more co-occurrence dynamics than typical trends, reflective of less stable communities and delayed microbiome maturation. Conclusion: Our analysis revealed a substantial proportion of infants display atypical patterns of gut microbiome maturation in the first year of life. These patterns are associated known microbiome-modifying factors and interkingdom influences and are characterized by less mature ecological dynamics. This work highlights the importance of interkingdom analyses at the individual level to generate improved understandings of microbiome maturation in early-life.
Dr. Selene Meza-Perez (Poster 58)
Instructor, University of Alabama at Birmingham
Gut microbiota impairs anti-tumor immunity by catabolizing arginine
Meza-Perez S, Liu M, Silva-Sanchez A, Morrow CD, Eipers PG, Lefkowitz EJ, Ptacek T, Scharer CD, Arend RC, Gray MJ, Randall TD
The composition of gut microbial communities influences anti-tumor immunity. Compiled evidence shows that microbiota modulates antitumor responses along with the use of immune checkpoint inhibitors across several cancer types. However, the mechanisms by which microbiota positively or negatively regulate anti-tumor immunity are still poorly understood. Here we show that microbiota-replete mice accumulate immune suppressive Tregs following tumor implantation in the omentum (visceral adipose tissue used as metastatic site for peritoneal tumors), process that is reversed in neomycin treated mice which mostly lack Proteobacteria. Mechanistically we found that the absence of Proteobacteria depletes microbial genes for arginine catabolism increasing serum arginine, phenomenon that is associated with high expression of mTOR and impaired suppressive capacity on Tregs. Suggesting that the absence of Proteobacteria, an arginine catabolic bacteria group, increases arginine availability affecting Treg metabolism and function leading to tumor rejection. Thus, in addition to generating immune modulating metabolites, the microbiota alters anti-tumor immunity by consuming metabolites such as arginine. Our data also suggest reasonably simple strategies to alter arginine availability and increase the efficacy of immunotherapy.
Dr. Chunlong Mu (Poster 59)
Postdoctoral Fellow, University of Calgary
Microbiome features associated with single and multi-medication use in cardiometabolic disease
Jane Shearer, Shrushti Shah, Grace Shen-Tu, Kristina Schlicht, Nathalie Rohmann, Matthias Laudes, Andre Franke, Chunlong Mu
Human gut microbiota is recognized as a critical player in both metabolic disease and drug metabolism. However, drug-microbiota interactions in cardiometabolic disease are not well understood. To obtain a comprehensive view of how drug intake affects the gut microbiota, we investigated the association of microbial structure with single and multi-medication use in 134 middle-aged adults with diagnosed cardiometabolic disease from the Alberta’s Tomorrow Project, a longitudinal cohort tracking the health of adults in Alberta. Multivariate Association with Linear Model in MaAsLin2was used to evaluate the relationship between microbial features and single or multi-medication use. Highly individualized microbiota profiles were observed across participants. Increasing drug use was negatively correlated with alpha diversity as assessed by the Shannon index. A total of 50 associations were identified between microbial composition and single drugs, exemplified by the depletion of Akkermansia muciniphila by beta blockers and statins, and the enrichment of Escherichia/Shigella and depletion of Bacteroides xylanisolvens by metformin. Associations with microbial composition were also found for levothyroxine, selective serotonin reuptake inhibitors antidepressants, anticonvulsants, and 5-aminosalicylic acid. Metagenomics function prediction further indicated alterations in microbial functions associated with drugs, such as the depletion of enzymes involved in energy metabolism encoded by Eggerthella lenta due to beta blocker use. Interestingly, statin plus non-steroidal anti-inflammatory drugs, a combination that has been used for lowering cardiovascular risk, led to an enrichment of Akkermansia muciniphila. Several other associations were identified for dual drug combinations, particularly the depletion of Ruminococcus and Bacteroidetes by angiotensin receptor blocker plus nonsteroidal anti-inflammatory drugs. Together, these results show profound reductions in bacterial diversity as well as species specific and microbiota functional potential impacts associated with both single and multi-medication use in cardiometabolic disease.
Kevin Muirhead (Poster 60)
PhD Student, University of Calgary
Mechanisms of Lactobacillus dominance and pathogen exclusion in the cervicovaginal niche
Kevin Muirhead, Karen V. Lithgow, Shaelen Konschuh, Nelly R. Mugo, Alison C. Roxby and Laura K. Sycuro
Prior to sexual debut, the adolescent vaginal microbiome frequently exhibits Lactobacillus crispatus (Lc) dominance, whereas communities dominated by Lactobacillus iners (Li) are more common in sexually active adults. For unknown reasons, Li is less proficient at niche acidification, an important factor for stable dominance and exclusion of sexually-transmitted pathogens. Glycogen metabolism is thought to drive acid production and in this study we profiled glycogen-targeting enzyme activities to elucidate factors controlling Lc/Li dominance. Of the six dominant Lactobacillus species, only Lc and Li encode cell surface/secreted type I pullulanses (pulA) predicted to debranch glycogen at α1-6 cross-links. pulA exhibits allelic variation with gene absence or functional inactivation in 24% of Lc and 19% of Li genomes/metagenomes. Although both species grow on glycogen, only Lc grows on pullulan (requiring α1-6 debranching) and exhibits pullulanase (debranching) activity. To profile these activities in vivo, we analyzed vaginal samples from Kenyan Girls Health Study participants (N=86). Supporting our in vitro findings, pullulanase activity was 3 fold higher in Lc-dominated (N=32) versus Li-dominated samples (N=32). Furthermore, total lactic acid levels were 2.5-fold higher in Lc-dominated compared to Li-dominated samples. Among Lactobacillus-dominated samples (N=69), the more protective D-lactic acid isoform correlated with pullulanase activity (Spearman rho=0.6214, p<0.0001) and amylase activity (Spearman rho=0.3235, p=0.0067), while L-lactic acid did not. Multivariate modelling revealed that the strongest predictor of total lactic acid was Lc presence, but pullulanase activity contributed significantly to models predicting D-lactic acid, even when Lc was sub-dominant. This work is the first to demonstrate that although Li can directly catabolize glycogen, it lacks pullulanase activity; this could explain why it produces less lactic acid and is more permissive in vivo. In contrast, Lc pullulanase activity is associated with the production of protective D-lactic, highlighting the therapeutic potential of pullulanase-active probiotics to promote sexual health.
Hana Olof (Poster 61)
MSc Student, University of Manitoba
Changes in cooking temperature and gut microenvironment pH can impact dietary fiber composition, in turn impacting interactions with gut microbiota and gut immune cells.
Hana Olof, Samuel A. Lawal, Jun Gao, Xinyun Liu, Ramsha Mahmood, Thava Vasanthan, Heather K. Armstrong
Dietary fibers can promote gut health in inflammatory bowel diseases (IBD), yet many IBD patients express intolerance of fiber consumption. Our recent study showed that the ability of gut microbes to ferment specific fibers is significantly altered in IBD. Unfermented β-fructan fibers induced gut inflammation via TLR2 and NLRP3 inflammasome activation. The physiochemical properties of dietary fibres are impacted by heat (cooking) and pH (gut environment). Our research aims to investigate how food preparation methods (heat) and gut microenvironment (pH; altered in IBD) can impact fiber physiochemical properties and fiber fermentation by colonic microbiota, in turn impacting gut immune response to fibers. We exposed β-fructan, β-D-glucan, pectin, and arabinoxylan to varying temperatures and pHs. We determined fiber subtype concentrations and physiochemical properties by AOC2009.01. Fibers were then cultured with whole microbiota gut washes from non-IBD (n=5) and IBD (n=5) patients in the anaerobic chamber to examine if altering fiber composition through cooking or different pHs can impact the ability of microbiota to ferment the fibers. Lastly, immune responses to fiber isolates were examined in cell lines, primary blood mononuclear cells (PBMCs), and gut biopsies cultured ex vivo from non-IBD (n=5) and IBD (n=5) patients (qPCR, ELISA, microscopy). Heat and pH impacted fiber composition and physiochemical properties, along with their fermentability by microbiota. The isolates that contained greater amounts of β-fructan or AX induced greater pro-inflammatory cytokine secretion. Exposure of dietary fibers to heat (cooking) or pH (gut environment) can impact the ability of gut microbiota to ferment fibers and in turn, can alter the gut immune cell response to fibers. These results demonstrate how cooking may improve fiber tolerability in IBD patients, and how changes in the IBD patient gut microenvironment, such as pH, may reduce fiber fermentation, increasing risk of negative interactions with specific dietary fibers in the gut.
Dr. Mona Parizadeh (Poster 62)
Postdoctoral Fellow, University of Calgary
Early-life Temporal Dynamics of the Human Gut Microbial Diversity, Composition and Co-occurrence Networks in a Rural District Setting
M. Parizadeh, I. Laforest-Lapointe, A. Valdez-Salazar, P. Morán-Silva, L. Rojas-Velázquez, J. Torres, C. Ximénez-García, M.C. Arrieta
Gut microbial colonization begins during birth and is highly heterogeneous until the age of 2-3 years. Many aspects of human development that rely on microbe-host and microbe-microbe interactions are affected by several factors, including social geography. Studies have shown that the microbial species richness in rural infants is greater than in urban infants. Yet, little is known about the early-life gut microbial composition in rural areas from less industrialized world regions. Moreover, our overall knowledge of the non-bacterial community composition is limited although microbial eukaryotes are increasingly recognized to have critical roles in ecosystem ecology and host crosstalk. To address these gaps, we characterized the bacterial and eukaryotic microbiomes in fecal samples from a longitudinal human cohort of nine children established in Morelos, Mexico, sampled monthly from birth until 18 months of age (average 14 samples/child). Using shotgun metagenomic and 18S rRNA sequencing, we studied the bacterial and eukaryotic microbiomes and their co-occurrence networks in these samples in response to the mode of birth (vaginal vs c-section). Our results showed that both bacterial and eukaryotic compositions were affected by birth mode and proximity to animals. Gut bacterial Shannon diversity increased over time in all infants but was higher in infants delivered vaginally. Vaginal birth favoured certain families from the Actinobacteria and Firmicutes phyla, while some families from the Proteobacteria phylum increased in infants delivered via c-section. Most microbial communities that co-occurred in both modes of birth belong to these bacterial phyla, as well as the Opisthokonta fungal phylum. This study highlights the importance of considering both bacterial and eukaryotic components of the gut microbiome in early-life development and demonstrates that infant gut microbial composition and co-occurrence patterns vary with birth mode. Further research is needed to understand the dynamics of early-life gut microbiomes and the ecological processes that influence them.
Dr. Sarah Piché-Choquette (Poster 64)
Postdoctoral Fellow, Université du Québec à Montréal
Joint Species Distribution Modeling of the contribution of the microbiome to asthma risk in premature infants
Sarah Piché-Choquette, Marie-Claire Arrieta, Steven Kembel
Asthma is a chronic respiratory disease affecting 1 in 8 Canadian children and accounting for approximately 30% of pediatric healthcare expenditure. Premature infants are at the highest risk of developing asthma and display widely different gut microbiomes from their term counterparts. Considering that recent studies have brought forward convincing evidence supporting causal links between early life dysbiosis and the development of asthma, our aim is to pinpoint microbiome features as well as environmental factors that could lead to the development of asthma. More specifically, in collaboration with the Alberta BLOOM research initiative, we applied a Joint Species Distribution Modeling approach to detect biomarkers of microbiome development and environmental factors associated with atopic wheeze at age 1. Our study focussed on the ongoing longitudinal cohort study BLOOM, including microbiome data from preterm and term infants and related familial, stress and immune factors. Our preliminary analysis of a subset of 60 infants led to the identification of 4 distinct microbial community types among the gut microbiomes of preterm infants between birth and equivalent term age. One of these community types is much closer to the microbiome of healthy term infants, both in terms of composition and function, and is therefore considered more mature and expected to be less prone to the development of asthma. We discuss whether microbiome maturation in premature infants could be promoted by probiotics.
Erika Prando Munhoz (Poster 65)
Laboratory Technician, University of Calgary
Evaluating the liver immune environment in a preclinical model of pathobiont accelerated non-alcoholic fatty liver disease (NAFLD)
Erika Prando Munhoz, Carolyn A. Thomson, Henry H. Nguyen
Non-alcoholic fatty liver disease (NAFLD) encompasses a range of liver pathology including simple fat accumulation in the liver (steatosis) to progressive liver inflammation and scarring (NASH). Treatment options for NAFLD/NASH are currently very limited. Understanding the drivers that promote the progression of liver injury will be important for the diagnosis and treatment of NAFLD patients. We have consistently observed that a particular gut pathobiont can accelerate histological liver steatosis in a high-fat diet (HFD) animal model of NAFLD. We aim to evaluate the impact of this pathobiont on local immune responses within the liver in the setting of accelerated steatosis. 6-week-old C57BL/6 mice were gavaged with the pathobiont and fed a high-fat diet (HFD) for a total of 8 weeks, an early time point that does not typically lead to NAFLD development in SPF mice not colonized with this pathobiont. The livers were subsequently collected and analyzed for immune cell profiling using flow cytometry and histology. At 8 weeks, mice that were colonized with the pathobiont had a significantly higher degree of liver steatosis noted on H&E histology. Blinded quantification of steatosis with Oil Red staining confirmed this. Immune cells within the liver of pathobiont colonized mice vs pathobiont absent mice fed HFD showed decreased frequencies of Treg cells (1.50% vs 2.47%), CD8+ T cells (10.94% vs 14.96%), B cells (32.13% vs 36.61%) and NK cells (9.79% vs 13.97%) and increased frequencies of Mo/MF cells (7.63% vs 5.19%). Although segmented filamentous bacteria (SFB) has been implicated in metabolic disease, we did not find any differences in SFB levels in stool of both groups, using quantitative PCR. An altered pathobiont associated liver immune environment may drive progression of liver injury in the setting of NAFLD. Additional investigation is necessary to better elucidate the contributions of these immune cells to NAFLD progression.
Dr. Charlène Roussel (Poster 66)
Postdoctoral Fellow, Université Laval
Exploring the ex vivo effects of stearidonic acid (SDA)-rich Ahiflower oil on the gut microbiota of individual humans and their capacity to produce SDA-derived N-stearidonoyl ethanolamine (SDEA)
Charlène Roussel, Mathilde Sola, Nayudu Nallabelli, Frédéric Raymond, Yves Desjardins, Vincenzo Di Marzo, Cristoforo Silvestri
The seed oil from Buglossoides arvensis (AhiflowerTM) is the richest source of the ω-3 PUFA stearidonic acid. Within the palette of mechanisms potentially explaining the beneficial effects of dietary PUFAs, the bidirectional interaction between dietary PUFAs and the gut microbiome is of great interest. Dietary PUFAs may in fact exhibit prebiotic properties, by positively modulating resident microorganism populations and their metabolic activities, while the gut microbiota can impact the metabolism, biotransformation, and absorption of dietary PUFAs. However, such studies on the effects of Ahiflower oil on the gut microbiota and its metabolic functions are lacking, warranting further investigations. In this context, we utilized the TWIN-M-SHIME®, which mimics both the ileum and proximal colon lumen and mucosal microbiome ecosystems, as an ecological framework to characterize the compositional and metabolic (in terms of SCFA, targeted lipidomic) responses of the human gut microbiota to 14-days of Ahiflower oil supplementation. This exploratory investigation utilized a mini cohort of four unique fecal donors to elucidate the personalized spatial-temporal fermentation effects using 16S rRNA sequencing and LC-MS/MS approaches. Cluster analysis revealed two distinct groups (cluster 1 and 2) in response to Ahiflower oil supplementation at the microbial community composition level. Cluster 2 exhibited an increased abundance of potential second-generation probiotics like Akkermansia muciniphila with an increased metabolic response with higher propionate production in both the ileum and proximal colon. Spearman correlation analysis identified the potential role of bacteria utilizing the succinate pathway in propionate production. Additionally, our findings indicated the ability of gut bacteria, particularly in the ileum of both clusters, to produce N-stearidonoyl ethanolamine, SDEA. Relevance network analysis to predict SDEA biosynthesis by bacterial taxa is currently ongoing. Further research in this field will help deepen our understanding of the intricate interactions between dietary PUFAs, the gut microbiota, and human health.
Mahana Sabachvili (Poster 67)
PhD Student, University of Calgary
Pathobionts and Precision Medicine in Inflammatory Bowel Disease
Mahana Sabachvili, Carolyn Thomson, Kathy McCoy, Lukas Mager
Inflammatory bowel diseases (IBD), such as Crohn’s disease (CD), are chronic inflammatory disorders of the gastrointestinal tract affecting around 10 million people worldwide. The pathogenesis of IBD is unclear; however, studies have shown the importance of gut microbiota. Indeed, a subgroup of IBD patients responds to microbial manipulation therapy such as fecal microbiota transplantation (FMT). We hypothesized that specific bacterial taxa drive inflammation in a subset of IBD patients. We aim at identifying these bacteria and to understand the underlying mechanisms how they do so. In the future this may lead to microbiome-based precision therapy approaches in IBD. First, we have recruited CD patients that show reduced gut inflammation after FMT treatment. Germ-free (GF) Muc2-deficient mice were colonized with stool samples collected from patients before and after FMT. Six weeks after colonization, gut inflammation was assessed by histology. Moreover, immune responses were analyzed in the spleen, the mesenteric lymph nodes and the large intestine by flow cytometry, and the intestinal content was collected to culture bacteria from the Pre- and Post-FMT microbiomes with the objective of identifying specific bacteria with a high inflammatory potential. Our data indicates that the pre-FMT microbiome induces gut inflammation, characterized by a shorter colon length and an enhanced immune response. Moreover, we cultured several patients derived intestinal bacteria taxa enriched in the pre-FMT samples and identified 4 candidats. To determine there pathogenic potential, GF Muc2-deficient mice were monocolonized with each bug and the gut inflammation was assessed six weeks later as described before. Our data indicates that 3 of these bacteria induce an immune response in the large intestine. Together, our datas suggest that patient derived microbiomes have different inflammatory potentials and may be a good tool to identify specific inflammation inducing bacteria.
Maricarmen Salas-López (Poster 68)
Graduate Student, CINVESTAV Mexico
Characterization of the archaeal community of colostrum and fecal samples of Mexican mothers and newborns.
Maricarmen Salas-López, Carmen Josefina Juárez-Castelán, Karina Corona-Cervantes, Alberto PiñaEscobedo, José Manuel Hernández-Hernández, Martín Noé Rangel-Calvillo, Claudia Pérez-Cruz and Jaime García-Mena
Human milk is the perfect food for a newborn. It is mainly composed of water, proteins, lipids, carbohydrates, and biological components, such as bacteria, viruses, and archaea. It appears that the central bacteriome of human milk is composed of taxa like Staphylococcus, Streptococcus, Serratia, Pseudomonas, and Corynebacterium. These bacteria are considered to reach human milk via the entero-mammary route, to colonize the intestine of the neonate by vertical transference during lactation. In recent reports, archaea have been cultivated from human milk; however, their taxa composition is not well characterized, and it is unclear if as occurs with bacteria, they are vertically transmitted. Also, their role in health remains to be elucidated. In this study, we sought to characterize the diversity of the archaeal community by next-generation sequencing of V5-V6 regions of the 16S-rRNA gene from colostrum and fecal samples of 42 mothers and neonate pairs. It was hypothesized that the archaeal community diversity present in the colostrum samples would be similar to the diversity in the neonate feces, supporting the hypothesis that vertical transfer occurs during lactation. Our results indicate that alpha diversity differences between groups are significant, possibly due to the difference in the milieu which is the source of the samples. The beta diversity analysis disclosed that the microbial diversities in the two different samples are similar. Relative abundance results show a dominance of the archaeal genera Methanoculleus and Methanosarcina in both fecal and colostrum samples. Additional analyzes revealed the absence of differential taxa between both groups, which is consistent with our hypothesis. This result is confirmed by the prediction of metabolic pathways, which indicates that there are no differential metabolic pathways between colostrum and feces, giving the idea that these archaea may be specifically selected to fulfill an important role in the newborn intestine.
Jared Schlechte (Poster 69)
PhD Student, University of Calgary
Engraftment of microbiome-immune dysfunction in a humanized microbiota model of critical illness.
Jared Schlechte, Amanda Z Zucoloto, Ian-ling Yu, Braedon McDonald
Critically ill patients in the ICU experience high rates of secondary infections (up to 50%), largely attributed to immune dysfunction and impaired host defense. We recently reported that the enrichment of Enterobacteriaceae in critically ill patients is associated with expansion of hypofunctional immature neutrophils, resulting in a significant reduction in nosocomial-infection-free survival (Schlechte et al., 2023). To further establish the causal impact of Enterobacteriaceae enrichment on neutrophil dysfunction, we established a humanized microbiota mouse model of critical illness. We colonized the gut of germ-free mice with fecal samples from six healthy volunteers and six critically ill patients with Enterobacteriaceae dysbiosis. Intestinal contents as well as whole blood, liver, and spleen were collected for meta-omics analysis in which we compared microbiome composition (16S amplicon sequencing), immune landscape (single-cell mass cytometry), and plasma biomarkers after three weeks of colonization. Using this humanized microbiota model of ICU dysbiosis, we found that mice colonized with the microbiome from critically ill patients displayed a similar microbiome composition to their donors, with significant enrichment of Enterobacteriaceae compared to mice colonized with a healthy volunteer microbiome. In support of our previous findings, ICU-microbiota colonized mice had a significantly altered immune landscape in both the systemic circulation as well as in tissues (liver, spleen). These alterations in immune landscape were independent of systemic inflammation or intestinal barrier dysfunction as we observed no difference between mice colonized by a healthy microbiome or a critically ill microbiome. Lastly, we observed significant alterations to the neutrophil compartment mirroring those observed in our human cohort, with expansion of immature neutrophils which display an immunosuppressive phenotype. Taken together, these findings further establish intestinal dysbiosis in critical illness as a driver of neutrophil dysfunction. Therefore, precision-based microbiota modifications targeting Enterobacteriaceae enrichment may be a useful therapeutic adjunct in critical illness.
Shrushti Shah (Poster 70)
PhD Student, University of Calgary
Interplay between Gut Microbiota, Tryptophan Metabolites and Hypertension in Perimenopausal Women
Shrushti Shah, Chunlong Mu, Grace Shen-Tu, Kristina Schlicht, Nathalie Rohmann, Matthias Laudes, Andre Franke, Harald Köfeler, Jane Shearer
Hypertension (HT) is a multifactorial health condition affecting 1.28 billion people globally. Although the gut microbiome is implicated in the onset and progression of HT, there is a paucity of studies investigating the link between the gut microbiota, its metabolites, and HT during the perimenopausal period, which poses an increased risk of HT for many women. This study examines the relationship of gut microbiota and its metabolites in age (56.6±6.6 years) and BMI-matched (24.2±2.0 kg/m2) normotensive vs. hypertensive (≥130 mmHg systolic, ≥80 mmHg diastolic blood pressure) women (n=108). Blood samples were collected for metabolomics and cytokine analysis while fecal samples were used for 16S rRNA sequencing. Serum metabolomics showed participants with HT to have elevated tryptophan and kynurenine and that the kynurenine:tryptophan ratio (IDO1 activity) had a strong positive relationship with blood pressure (r=0.7, p≤0.01). HT was also associated with lower levels of health-promoting indoles produced by the hydrolyzation of tryptophan (via tryptophanase) by the gut microbiota. These findings were confirmed in fecal microbiome analysis where individuals with HT had a lower abundance of Proteobacteria, Alistipes shahii, and Bacteroides (B. faecichinchillae and B. stercoris) suggesting a lower predicted activity of indole-producing enzymes, tryptophan synthase (TrpA) and indole-3-glycerol phosphate synthase (TrpC) in HT compared to healthy controls. Additionally, HT induced alterations in tryptophan metabolism were tightly correlated with inflammation. Indole was found to negatively correlate with IFN-γ (r=-0.5, p<0.05) whereas kynurenine showed a positive association with C-reactive protein (r=0.4, p<0.05) demonstrating the protective role of indoles in mitigating inflammation. In conclusion, our data indicate a dysbiosis in microbiota-regulated tryptophan metabolism in HT which likely contributes to systemic inflammation and further HT progression in perimenopausal women.
Shrushti Shah (Poster 71)
PhD Student, University of Calgary
Physical Activity-Induced Alterations of the Gut Microbiome are BMI-Dependent
Shrushti Shah, Chunlong Mu, Shirin Moossavi, Grace Shen-Tu, Kristina Schlicht, Nathalie Rohmann, Corinna Geisler, Matthias Laudes, Andre Franke, Thomas Züllig, Harald Köfeler, Jane Shearer
Physical inactivity is one of the leading causes of chronic metabolic disease including obesity. Increasing physical activity (PA) has been shown to improve cardiometabolic and musculoskeletal health and to be associated with a distinct gut microbiota composition in trained athletes. However, the impact of PA on the gut microbiota is inconclusive for individuals performing PA in their day-to-day life. This study examined the role of PA and hand-grip strength on gut microbiome composition in middle aged adults (40-65y, n=350) with normal (18.5–24.9kg/m2) and overweight (25–29.9kg/m2) body mass index (BMI). PA was recorded using the International Physical Activity Questionnaire and hand-grip strength was measured using a dynamometer. Serum samples were assessed for lipidomics while DNA was extracted from fecal samples for microbiome analysis. Overweight participants showed a higher concentration of triacylglycerols, and lower concentrations of cholesteryl esters, sphingomyelin and lyso-phosphotidylcholine lipids (p<0.05) compared to those with normal BMI. Additionally, overweight participants had a lower abundance of the Oscillibacter genus (p<0.05). The impact of PA duration on the gut microbiome was BMI dependent. In normal but not overweight participants, high PA duration showed greater relative abundance of commensal taxa such as Actinobacteria and Proteobacteria phyla, as well as Collinsella and Prevotella genera (p<0.05). Furthermore, in males with normal BMI, a stronger grip strength was associated with a higher relative abundance of Faecalibacterium and F. prausnitzii (p<0.05) compared to lower grip strength. Taken together, data suggest that BMI plays a significant role in modelling PA induced changes in gut microbiota.
Sara Shama (Poster 72)
PhD Student, University of Toronto
Characterizing the Microbiota Shared Between Mother’s Milk and the Very-Low-Birth-Weight Infant Gut
Sara Shama, Michelle R Asbury, Alex Kiss, Nicole Bando, Amel Taibi, James Butcher, Elena M Comelli, Julia K Copeland, Adrianna Greco, Akash Kothari, Philip M Sherman, Alain Stintzi, Christopher Tomlinson, Sharon Unger, Pauline W Wang, Deborah L O’Connor, for the OptiMoM Feeding Group
Background: Preterm mother’s milk contains microbial communities, although their colonization potential in very-low-birth-weight (VLBW, <1500g) infants remains unknown. Objective: To determine relationships between the microbiota present in preterm mother’s milk and the VLBW infant gut during initial hospitalization. Methods: Infants born <1250g (n=94) from the OptiMoM Fortifier Study (NCT02137473) were included. Weekly paired milk (n=334) and stool samples (n=422) from each mother-infant dyad were prospectively collected during postnatal weeks 1-8. Samples were analyzed by V4-16S rRNA gene sequencing. Results: A positive association was observed with microbial richness (number of operational taxonomic units [OTUs]) in paired milk-stool samples, but only when milk samples contained >200 OTUs (P<0.001). Weighted UniFrac distances (beta-diversity) increased longitudinally (P<0.001), while unweighted UniFrac distance decreased (P=0.04), suggesting that low-abundance taxa are increasingly shared over time. On average, 7 OTUs (standard deviation, 3) were shared between paired milk-stool samples. This amount did not change over time but was higher for infants fed predominantly mother’s milk (P=0.03). Shared OTUs represented 43.1% (95%CI: 41.0, 45.2) of the OTUs found in the infant microbiota during postnatal week 1, which decreased to 28.7% (95%CI: 27.5, 29.9) by week 8 (P=0.001). OTUs shared frequently mapped to Enterobacteriaceae, Staphylococcaceae, Veillonellaceae, Clostridiaceae-1, and Erysipelotrichaceae; although, sharing Staphylococcaceae was 3% (incidence rate ratio: 0.97, 95%CI: 0.95, 0.99) less likely with each additional postnatal week (PFDR=0.007). Predominant mother’s milk and nutrient-fortifiers were not associated with milk-stool sharing of individual taxa; however, direct breastfeeding increased the likelihood of sharing Veillonellaceae (PFDR=0.04), Erysipelotrichaceae (PFDR=0.003), Streptococcaceae (PFDR=0.004), and Pasteurellaceae (PFDR=0.003). Conclusions: Longitudinal relationships exist between the microbiota in preterm mother’s milk and the VLBW infant gut. Future research is needed to delineate strain-level and functional relationships of the shared microbiota and to define the potential for mother’s milk to rehabilitate perturbed (dysbiotic) gut microbiomes in VLBW infants. (CIHR-funded #FHG129919;#FDN143233;#FBD181488).
Dr. Saif Sikdar (Poster 73)
Postdoctoral Fellow, University of Calgary
Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity
Saif U. Sikdar, Daesun Kim, Franz Zemp, Douglas J. Mahoney
The microbiome has a significant impact on immune health and response to immune-stimulating treatments, including cancer immunotherapy. However, the molecular mechanisms by which commensal microbes influence cancer immunology remain poorly understood. Here, we report on the discovery of a class of microbiome-derived metabolites called hydroxyphenyl propanoates (HPP) that enhance tumour immune surveillance in mice and synergize with immune checkpoint blockade (ICB) therapy. HPP molecules act as broad spectrum potentiators of innate immune signalling pathways in tumour-associated myeloid cells by promoting cleavage of the pore-forming protein gasdermin D (GSDMD), a critical effector of canonical and non-canonical inflammasome signalling. Heightened secretion of proinflammatory cytokines from HPP-treated myeloid cells, including IL-1β, promotes NF-κB activity within tumour-infiltrating leukocytes. This leads to improved anticancer CD8 T cell function, significant tumour regression, and better long-term cancer control by immune checkpoint therapy in mice. Human peripheral blood mononuclear cells respond to HPP treatment in a similar way. GSDMD cleavage also associates with a favourable response to ICB therapy in advanced stage melanoma patients. Taken together, our study uncovers a novel mechanism of microbiome-mediated modulation of host antitumour immunity that is modifiable and can be harnessed to enhance the efficacy of cancer immunotherapy.
Isla Skalosky (Poster 74)
PhD Student, University of Calgary
Impact of Intestinal Microbiota Complexity and Molecualr Mimicry of a Vial Epitope on Anti-viral T Cell Responses
Isla Skalosky, Shokouh Ahmadi, Marcela Davoli Ferreira, Regula Burkhard, and Markus Geuking
Aims: It is well known that the intestinal microbiota and microbial metabolites impact the development and regulation of the immune system. This also affects immune-mediated diseases later in life. How the intestinal microbiota affects anti-viral immune responses or vaccine efficacy is less clear. It has been demonstrated that microbial mimicry of self-antigens can be either beneficial or detrimental for autoimmunity, depending on the context. The consequences of microbial antigen mimicry of a viral epitope on anti-viral T cell responses has not been studied in detail. Methods: Germ-free or gnotobiotic mice colonized with precisely defined sDMDMm2 microbiota were challenged with an acute systemic lymphocytic choriomeningitis virus (LCMV) infection. In addition, germ-free and gnotobiotic mice were monocolonized with either a wild-type or genetically modified intestinal commensal Escherichia coli strain expressing an LCMV-derived epitope. The impact of colonization status and the presence or absence of the LCMV epitope mimic on anti-viral T cell responses was characterized by tetramer staining and intracellular cytokine staining for flow cytometry. Results: Preliminary results show no improvement of anti-LCMV T cell responses in sDMDMm2 colonized mice compared to germ-free mice. Intestinal colonization with LCMV mimic E. coli significantly suppressed systemic epitope-specific T cell activity in terms of interferon cytokine production. Conclusions: The LCMV infection model has been established in germ-free, E. coli monocolonized, and gnotobiotic sDMDMm2 colonized C57BL/6 mice with corresponding readouts. Presence of a viral epitope mimic within the intestinal microbiota appears to inhibit mimic-specific anti-viral T cell activity, possibly through the induction of microbiota-induced epitope-specific regulatory T cells. Research funded by the Canada Institutes for Health Research (CIHR) Canada, Crohn’s and Colitis Canada (CCC) Grant-In-Aid of Research, and the Alberta Graduate Excellence Scholarship (AGES).
Sara Stickley (Poster 75)
PhD Student, Queen’s University
Network clusters of co-occurring gut microbes are associated with childhood atopy and asthma, environmental exposures, and host genomics in the CHILD Cohort Study
Sara A. Stickley, Zhi Y. Fang, Amirthagowri Ambalavanan, Yang Zhang, Charisse Petersen, Darlene Dai, Piushkumar J. Mandhane, Elinor Simons, Jeffrey R. Brook, Theo J. Moraes, Malcolm R. Sears, Meghan B. Azad, Stuart E. Turvey, Padmaja Subbarao, Qingling Duan
Taxa-specific abundance shifts in the gut microbiota during early-life have been previously associated with development of childhood allergic diseases. These earlier studies, however, did not investigate the connections among individual microbes and their combined effects on health and disease. In this investigation, we employ an unsupervised machine learning approach to determine network clusters of co-occurring microbes and examine their associations with atopy and asthma susceptibility in children. Moreover, we integrate host genomics and environmental exposures to explore their main and interaction effects on these microbial groups. We leverage microbiota data from the CHILD Cohort Study generated from 16S rRNA sequencing of stool samples collected at two ages: 3 months (N=747) and 1 year (N=766). We also utilize genomic profiles obtained from the Illumina HumanCoreExome Bead Chip and environmental data (e.g. breastfeeding practices, human milk composition, antibiotic use, and pet ownership). We identified 10 groups of co-occurring microbes at 3 months, such as a cluster correlated with decreased risk of inhalant allergy (ages 3-5 years; P=0.035). Genome-wide association analyses identified that microbes within this cluster, Anerostipes sp. and Blautia obeum, were associated with variants on chromosome 20 (e.g. rs6072373, P=4.8×10-8) and 2 (e.g. rs13031049, P=9.4×10-11) respectively. Additionally, we observed 13 microbial clusters at 1 year, including one containing Streptococcus salivarius that is correlated with increased food allergy (ages 3-5 years; P=4.8×10-3 ). This cluster was also associated with shorter duration of breastfeeding (P=6.3×10-3 ) and exposure to a less diverse human milk microbiota (P=0.021). Lastly, analyses stratified by sex determined that a cluster at 1 year, including multiple microbes from the Blautia genus, was associated with increased asthma among males (age 5 years; P=0.045). Our findings suggest that the gut microbiota during early-life are modulated by both environmental and genetic factors, which in turn may impact atopy and asthma susceptibility.
Dr. Lena Takayasu (Poster 76)
Research Associate, Cornell University
Profiling the dynamics of the gut microbiome in mice at high temporal resolution.
Lena Takayasu
The gut microbiome plays a crucial role in maintaining host homeostasis and has been linked to various life events, including aging, childbirth, and disease. Despite significant progress in understanding the gut microbiome, much remains unknown about its dynamics and evolution throughout the host’s lifetime. To address this, we longitudinally collected a total of 1,815 fecal samples from 10 mice (eight siblings and parents) with an average sampling interval of 4.3 days and obtained a total of 18.0 million high-quality reads of the 16S rRNA gene V1-2 region from fecal DNA samples using the Illumina MiSeq platform. We discovered that changes in gut microbes are associated with various life events. Our findings revealed a bimodal distribution of gut species, with persistent and transient species in all mice. The persistent species were primarily composed of Bacteroidetes, which showed a strong correlation with the mice’s lifespan. To further explore these dynamics, we constructed Metagenomic Assembled Genomes from the data using the Illumina Novaseq 6000 system and estimated horizontal gene transfer events that occurred throughout their lives. Our data suggest that the gut microbiome undergoes dynamic changes and interactions throughout the host’s lifetime.
Dr. Marcel van de Wouw (Poster 77)
Postdoctoral Fellow, University of Calgary
The Impact of Prenatal Stress on the Infant Gut Microbiome During the COVID-19 Pandemic
Marcel van de Wouw, Marie-Claire Arrieta, Lianne Tomfohr-Madsen, Catherine Lebel, Gerald Giesbrech
Introduction: Substantial stress exposure during pregnancy results in changes in child physiology (e.g., gut microbiome), which may contribute to the biological embedding of prenatal stress in children’s development. Even though we know that prenatal stress impacts the composition of the infant gut microbiome, it is currently unclear how different types of stress (objective vs subjective) affect the gut microbiome and how gut microbiome function is impacted. Methods: Data are from the Pregnancy During the COVID-19 Pandemic Cohort. Prenatal measures of Pandemic Objective Hardship (POHI) and subjective mental health outcomes, such as anxiety (PROMIS) and depression (EPDS), were quantified. Stool samples were collected from infants at 3 months of age to analyze gut microbiome composition using metagenomic shotgun sequencing (n=311), and the stool metabolome as a measure of gut microbiome function (n=878). Results: POHI correlated positively with the diversity of the infant gut microbiome and 15 bacterial taxa. POHI also correlated with 51 of the 154 detected stool metabolites, which were implicated in metabolic pathways related to lipid, carbohydrate and bile acid metabolism. These effects on the infant gut microbiome were not observed for measures of prenatal mental health. Conclusion: Prenatal objective stress has a greater impact on the infant gut microbiome and metabolome compared to subjective stress. These data provide novel insights into the biological embedding of prenatal stress.
Erik van Tilburg Bernardes (Poster 78)
PhD Student, University of Calgary
Antibiotic-induced Malassezia spp. expansion in infants elicits intestinal immune dysregulation and increased airway inflammation in mice
Erik van Tilburg Bernardes, Mackenzie W. Gutierrez, Carolyn A. Thomson, Kathy D. McCoy, Stephen B. Freedman, Marie-Claire Arrieta
The neonatal immune system undergoes important developmental changes that are dependent on microbial colonization post-birth. Early-life antibiotic use impacts the gut microbiome, leading to immune dysregulation and increased risk of childhood asthma. However, it is currently unknown if the fungal microbiome (mycobiome) contributes to antibiotic-induced immune dysregulation conducive to allergic asthma. We aimed to evaluate the effect of antibiotic-induced mycobiome changes on neonatal immune development and experimental airway inflammation model. We ran an observational, prospective clinical study of 47 young infants (<6 months of age) receiving antibiotics. We compared the bacterial and fungal microbiome in fecal samples collected before and after antibiotics via shallow shotgun (bacteria) and ITS2 (fungi) sequencing, as well as qPCR for fungal and bacterial DNA quantification. Antibiotic use decreased bacterial and increased fecal fungal DNA and induced expansion of Malassezia spp. in infants. To evaluate the effect of Malassezia spp. colonization on host immunity, we compared immune development and susceptibility to airway inflammation in gnotobiotic mice colonized with consortia of 12 mouse-derived bacteria (Oligo-MM12), or bacteria and Malassezia restricta, as representative taxon expanded following antibiotic treatment. M. restricta colonization increased Th2 cells, eosinophils, macrophage infiltration, and neutrophils counts in the colonic lamina propria. M. restricta also increased migratory dendritic cells, eosinophils, Th2, and Th17 in mesenteric lymph nodes, suggesting elevated immune responses deemed critical in atopy development. M. restricta also increased house dust mite-induced airway inflammation, with elevated cellularity and marked eosinophilia in the bronchoalveolar lavage fluid obtained from challenged mice. This translational work shows that fungal overgrowth and expansion of Malassezia spp. are previously overlooked collateral effects of infant antibiotic use, which causally contribute to immune dysregulation and increased susceptibility to allergic airway inflammation in mice.
Dr. Jennifer Vena (Poster 79)
Scientific Director, Alberta Health Services
Microbiome research opportunities in Alberta’s Tomorrow Project, a large prospective cohort study
Jennifer Vena, Shandra Harman, Valez Lumi, Kathleen Murdoch, Wendy Powell, Will Rosner, Grace Shen-Tu & Bud Skiffington
Initiated in 2000, Alberta’s Tomorrow Project (ATP; www.myATP.ca) is a longitudinal cohort study following 55,000 Albertans (age 35-69 at enrollment with no previous history of cancer) recruited from across the province, and a data/biosamples platform that supports high-quality research to understand why some people develop cancer and chronic diseases while others do not. ATP collects health and lifestyle information from participants, and makes this data available to researchers through a structured access process. ATP has also collected biosamples from participants at multiple timepoints, including samples to support microbiome research. First, during the rollout of large-scale biosample collection during recruitment (2009-2015), ~9400 participants provided saliva samples using Oragene DNA kits (and almost 2300 of these participants also provided a blood sample). In 2018/19, ~400 participants (those with metabolic syndrome or colitis, and controls) participated in a gut microbiome sub-study and provided blood and stool samples (DNA has been extracted). Finally, in 2020-2022 ATP conducted a COVID-19 antibody testing sub-study wherein participants attended a study center in one of four locations (Calgary, Edmonton, Red Deer and Lethbridge, representing urban (88%) and rural (12%) communities) to provide blood samples. Saliva samples were also collected from ~3700 participants via ‘swish and spit’ mouthwash. At every collection, participants also completed detailed health and lifestyle questionnaires, often including diet and physical activity questionnaires. The research opportunities afforded by the samples collected to date combined with the health and lifestyle information captured longitudinally can be expanded by linkage to administrative health databases (e.g. health service utilization, medications, cancer registry) and environmental data (via the Canadian Urban Environmental Health Research Consortium). In summary, ATP’s data and biosamples can support comprehensive research investigations into the microbiome and cancer and chronic disease outcomes. ATP can also provide support for grant applications, data/sample requests and coordination with other databases.
Sasanka Weerasingha (Poster 80)
Graduate Student, University of Ottawa
Identifying resistant starch primary degraders in human gut microbiomes
Sasanka Weerasingha, David Mack, and Alain Stintzi
The presence of dietary fibers or resistant starches (RS) significantly impacts the composition and metabolism of the human gut microbiota with significant effects on host physiology. Despite notable strides in comprehending how human gut bacteria metabolize RS, there remains a need for techniques to identify bacteria that consume them. Ruminococcus bromii and Bifidobacterium spp are the most studied primary degraders of RS within the human gut. However, it is possible that there are other prevalent species yet to be discovered, particularly in altered gut microbiota such as those found in individuals with inflammatory bowel disease (IBD). Here, we optimized and used an in-vitro assay to identify RS primary degraders from the microbiota of IBD patients. Our approach relies on the identification of bacteria that closely attach to RS. Briefly, stool samples from 12 IBD patients were cultured in a medium containing RS as the sole carbon source. Different types of RS were tested in our assay with the objective to identify RS-specific primary degraders. To isolate closely attached bacteria, the cultures underwent repeated pelleting and washing. Baseline microbiome and closely attached bacteria were analyzed separately by conducting 16S rRNA sequencing on raw stool and RS-treated stool samples, respectively. Primary analysis confirmed previous findings with the identification of Ruminococcus and Bifidobacterium spp as primary degraders validating our in-vitro assay. In some patients, an exclusive co-occurrence of Ruminococcus and Bifidobacterium spp was observed. Moreover, certain individuals exhibited elevated levels of the Lachnospiraceae and Clostridiaceae families, which were particularly abundant in those with a low abundance of the known primary degraders. This suggests that microbial members from these families may potentially function as novel primary degraders in the context of IBD. Further investigations will be conducted to explore whether the novel taxa identified in this study exhibit functional capabilities as primary degraders of RS.
Logan Wisteard (Poster 81)
MSc Student, Queen’s University
Effect of Feeding Time on Microbiome Community in Engorged Ticks using Nanopore Sequencing
Logan Wisteard, Sima Afsharnezhad, Damian Bourne, Robert I Colautti
With the increasing northern range expansion of Ixodes scapularis (deer tick, blacklegged tick), there has been an increase in reported cases of tick-borne diseases in Canada, including Lyme disease. Understanding the interactions between ticks, their pathogens, and their shared environment requires studying microbial changes in the tick microbiome sampled from ticks in their natural habitats. To better understand how tick microbiome communities are affected by feeding time, measurements of morphological features were done on 42 engorged ticks collected from 8 veterinary clinics in a Lyme disease hotspot (Kingston, ON). Each tick’s DNA was sequenced using full-length 16S rRNA gene amplicons on the Oxford Nanopore MinION platform, and the emu pipeline to analyze. To characterize bacterial microbiome communities at the species level, idisosmal length, scutal width, and coxal gap were measured to estimate feeding time. Principal component analysis (PCA) of morphological features with K-means clustering revealed three morphological clusters. Borrelia (the causative agent of Lyme disease) was found in the cluster associated with the shortest amount of time. A total of 263,264 reads were classified, with read counts ranged from 44 to 26,708 per tick. The highest bacterial abundance found was Rickettsia buchneri at 59%. Borreliella burgdorferi was found in approximately 2% of the samples. Nanopore sequencing holds promise for real-time, field-based detection of rare pathogens to advance our understanding of tick microbiomes and disease transmission.
Anthony Wong (Poster 82)
Lab Project Coordinator, The Hospital for Sick Children
Characterization of defined fecal-derived microbial consortia from DIABIMMUNE infants: colonization dynamics in a germ free NOD mouse model
Anthony Wong, Zhuohui Lin, Alessandra de Paiva Granato, Christina Ohland, Christopher Yau, Steve Mortin-Toth, Simone Renwick, Emma Allen-Vercoe, Kathy McCoy and Jayne Danska
Type 1 Diabetes (T1D) is a T-cell mediated autoimmune disease that has seen increased incidence over the past 60 years. Genetic risk factors alone cannot explain this increase, suggesting dynamic environmental factors play a role. The human intestine is an environment colonized by hundreds of bacterial species, and alterations in the intestinal microbiome are associated with autoimmune disease, including T1D. Previous work has shown that the composition of the intestinal microbiome in patients with T1D differs from healthy controls. However, to date, relationships between the composition of the fecal microbiome and appearance of islet autoantibodies (IAB), a clinical definition of T1D, have only achieved modest significance. This lack of association with disease may reflect confoundment by inter-individual taxonomic variability and dynamic fluctuations during early development. To overcome this, we used axenic microbial culture followed by sequence analysis to design defined microbial communities from young children who were members of a birth cohort at risk for T1D. The selected fecal samples isolated at 12-18 months of age from individuals within this study represent two clinical outcomes at 36 months of age: 1) IAB negative (non-converters) or, 2) IAB positive (seroconverters). These fecal-derived defined consortia were introduced by oral gavage to germ-free NOD mice and their fecal samples collected longitudinally. Using high throughput 16S rRNA sequencing, we have evaluated the assembly, dynamics and composition of these consortia over time. We are currently examining evidence for islet-directed autoimmunity and diabetes to determine how the non-converter and seroconverter consortia impact immune and metabolic functions.
Dr. Robyn Wright (Poster 83)
Postdoctoral Fellow, Dalhousie University
Linking the oral microbiome with child and adolescent mental health
Robyn Wright, Christie Burton, Vanessa DeClercq, Russell Schachar, Morgan Langille and Jennifer Crosbie
Introduction: With the increasing child mental health crisis, there is a need to identify personalized medicine targets for common childhood-onset disorders, both for early detection and treatment. The oral microbiome is a potential target as it has been linked with mental health in adults and links between the gut microbiome and child mental health have been documented. However, the relationship between the oral microbiome and mental health traits in children and adolescents over a dynamic age range is largely unknown. Methods: We collected saliva samples, demographic information and reported mental health diagnoses, including Attention-Deficit/Hyperactivity Disorder (ADHD), Obsessive-Compulsive Disorder (OCD) and Autism Spectrum Disorder (ASD), from children and adolescents (n=1344; ages 4-19) attending the Ontario Science Centre. Saliva samples were analyzed by 16S rRNA gene sequencing and we have examined the effects of age, gender and diagnosis on microbial diversity and composition. Results: Alpha diversity is significantly impacted by age, gender, and mental health diagnosis. When we compare those with a diagnosis versus age and gender-matched controls with no diagnosis, we find that those diagnosed with ASD (n=52) have lower evenness, while those diagnosed with ADHD (n=151) or OCD (n=17) have both lower evenness and diversity than those with no diagnosis. Both age and gender have a significant impact on oral microbiome composition (beta diversity), although the effect sizes of both are small and a large amount of variation remains unexplained by the variables we have examined. We also find associations of oral microbiome composition with ADHD, OCD and ASD diagnoses. Current and future work: Current work is aimed at determining whether the differences between those with and without ADHD, OCD or ASD diagnoses change with age and gender, identifying which microbes are associated with these changes and investigating associations between the microbiome and the traits underlying these diagnoses.
Wendy Zhang (Poster 84)
MSc Student, University of Calgary
Discerning Alveolar Macrophage Ontogeny in Mice
Zhang W, Castanheira F, Neupane A, Kubes P.
The adaptive and innate immune systems are the two branches of the immune system our body uses to defend against invading microbes. After each encounter with a pathogen, the adaptive immune system develops a memory to prevent an infection with the same microbe. This ability to remember has recently also been shown in the innate immune system and was termed “trained immunity”, describing the ability to generate a stronger response towards a broad range of pathogens despite infection with just a single pathogen. The lung is an organ directly exposed to the microbes in the outside world, making it a likely site for immune cells to develop trained immunity. The predominant innate immune cells that reside in the lungs are the alveolar macrophages. These cells encounter countless pathogens every day and therefore are a likely cell in which to study trained immunity. My lab recently figured out how to watch the macrophages crawl inside the lungs of living animals and this study examined how these macrophages behaved under different conditions (Neupane et al, 2020). These macrophages cannot live forever, so cells from the bone marrow constantly replace the macrophages, meaning that there are always two types of macrophages present in the lung. However, without an effective labelling tool, it was previously impossible to distinguish between the two macrophage populations, making their differences in behavior and function unknown. This study utilized a unique model to look at the two different macrophages and visualize these cells inside a living mouse to look for behavioural similarities and differences. Our findings determined the behavioral and functional similarities of these macrophages at homeostasis, their responses against the influenza virus, and provided support for both populations to be trained against influenza.
Dr. Reda Albadawy (Poster 85)
Professor, Benha University (Egypt)
A study whether the status of the generated mRNA – ncRNA network (related to gut microbiota derived mediators is involved in the potential improvement due to the microbiota treatment
Reda Albadawy, Amany Helmy Hasanin, Maha Saad, Mohamed Othman, Marwa Matboli
Abstract: Microbiome is a novel issue in health and diseases. Non Alcoholic Fatty Liver Disease (NAFLD) is the most health problem world wide. Not yet there is no drugs for Non-Alcoholic Steatohepatitis (NASH).
Aim: of the study to explore gut microbiome mediators in NAFLD diagnostic, therapeutic.
Methodology: An experimental study was done on 60 mice after feeding high fat diet for 14 weeks. Blood and stool sample taken for microbiome studies Analysis done by 16 sRNA sequencing.
Results: At the phylum level, the NASH mice had significantly increased relative abundance of Verrucomicrobia and unclassified colistridiae and decreased relative abundance of Bacteroidetes compared to the Naive mice, which were partially restored by Rosavin treatment. At the genus level, the NASH mice had a significantly higher abundance of Ruminococcus, Eubacterium, and Clostridium, Bacteroides and lower abundance of Cronobacter, Streptococcus, and Alistipes, which were also partially restored by Rosavin treatment.
Discussion: Since a large dose of Rosavin(30mg/kg) treatment had better efficiency as demonstrated above, 16S rRNA gene sequencing on the mice gut microbiota among naive, NASH model, and HFHD-Roavin (30 mg/kg) groups was performed to analyze the gut microbial community structure, indicating gut microbiota composition among the groups was different from each other. The HFHD diet led to an increased abundance of Firmicutes and decreased abundance of Bacteroidetes, which were considered as “fat bacteria” and “lean bacteria” respectively, and such dysbiosis was reversed after Rosavin treatment, Rosavin treatment might, at least in part, explain the improved hepatic inflammation of NASH without diet alteration. As Alistipes is an acetate producer, which is a type of short-chain fatty acids that have anti-inflammatory actions), it can be suggested that improvement of hepatic inflammatory after Rosavin treatment in the present study might be due to the restoration of Alistipes and its products.
Conclusion: the generated mRNA-ncRNA network (related to gut microbiota derived mediators) has involved in the potential improvement due to the microbiota treatment.
Acknowledgement: Academy of Scientific Research and Technology, Cairo, Egypt, source abstract here
Correspondence: [email protected]; [email protected]
