Having IMPACTT 4:
Advancing Microbiome Research
June 3-5, 2024
Selected Abstracts
Selected Abstracts for Oral and Poster Presentation
See below for the abstracts selected for oral and poster presentations for our Having IMPACTT 4: Advancing Microbiome Research Symposium on June 3-5, 2024 at the Malcolm Hotel in Canmore, AB, Canada.
Presenters
Short Talk Presenters
Session 1: Microbiome in Early Life (Monday, June 3, 2024)
Meggie Kuypers (+ Poster 32)
Dr. Van Ortega
Session 2: Innovative Approaches for Elucidating Microbiome Function (Monday, June 3, 2024)
Dr. Lauren Davey
Dr. Vanessa Dumeaux
Session 3: Harnessing the Microbiome (Tuesday, June 4, 2024)
Dr. Erik Bakkeren (+ Poster 66)
Juan Camilo Burckhardt (+ Poster 67)
Special Topic: Microbiome Sequencing & Computational Development (Tuesday, June 4, 2024)
Dr. Robyn Wright
Geoff Winsor
Dr. Ali Mirza
Session 4: Microbiome, Metabolites & Disease (Tuesday, June 4, 2024)
Dr. Riadh Hammami
Dr. Dina Kao (+ Poster 68)
Session 5: Microbiome Regulation in Cancer & Cancer Therapy (Wednesday, June 5, 2024)
Dr. Larisa Kovtonyuk (+ Poster 69)
Dr. Ann Gregory
Flash Talk & Poster Presenters
Flash Talk & Poster Session #1 (Monday, June 3, 2024)
Shirin Afroj (Poster 1)
Dr. Jacqueline Barnett (Poster 2)
Dr. Francisco Daniel Davila Aleman (Poster 3)
Hans Ghezzi (Poster 4)
Connor Gianetto-Hill (Poster 5)
Dr. Rebecca Jeffery (Poster 6)
Flash Talk & Poster Session #2 (Tuesday, June 4, 2024)
Jessica Josephson (Poster 37)
Simarpreet Kaur (Poster 38)
Dr. Maryam Kebbe (Poster 39)
Kathryn Strayer (Poster 40)
Athalia Voisin (Poster 41)
Christopher Yau (Poster 42)
Poster Presenters
Poster Session #1 (Monday, June 3, 2024)
Spencer Abbott (Poster 7)
Dr. Shokouh Ahmadi (Poster 8)
Nazanin Arjomand Fard (Poster 9)
Panuya Athithan (Poster 10)
Dr. Preethi Balasundaram (Poster 11)
Sabrin Bashar (Poster 12)
Diya Chandrasekhar (Poster 13)
Dr. Rinku Chaudhari (Poster 14)
Christopher Cheng (Poster 15)
Sonia Czyz (Poster 16)
Dr. Vanessa DeClercq (Poster 17)
Dr. Marcel de Leeuw (Poster 18)
Dr. Zerihun Demissie (Poster 19)
Dr. Kirti Dubli (Poster 20)
Saly El Salti (Poster 21)
Rose-Marie Fortin (Poster 22)
Arash Ghasemi (Poster 23)
Dr. Thaís Glatthardt (Poster 24)
David Good (Poster 25)
Dr. Riadh Hammami (Poster 26)
Jerry He (Poster 27)
Chadabhorn Insuk (Poster 28)
Fatimoh Kasaba (Poster 29)
Dr. Wei Wei Thwe Khine (Poster 30)
Grace Kim (Poster 31)
Meggie Kuypers (Poster 32)
Dana Lowry (Poster 33)
Christine Macpherson (Poster 34)
Isabelle Mainville (Poster 35)
Sarah Mansour (Poster 36)
Poster Session #2 (Tuesday, June 4, 2024)
Giselle McCallum (Poster 43)
Ailidh McGonigle (Poster 44)
Emily Mercer (Poster 45)
Fynn Mitchell (Poster 46)
Hana Olof (Poster 47)
Dr. Mona Parizadeh (Poster 48)
Dr. Leila Rezaei (Poster 50)
Dr. Jennifer Ronholm (Poster 51)
Dr. Elsa Rousseau (Poster 52)
Mahana Sabachvili (Poster 53)
Amy Shupe (Poster 54)
Claire Sie (Poster 55)
Isla Skalosky (Poster 56)
Dr. Matthew Sorbara (Poster 57)
Dr. Yves St-Pierre (Poster 58)
Dr. Marcel van de Wouw (Poster 59)
Dr. Joongjae Kim (Poster 60)
Ray Wang (Poster 61)
Dr. Amber Fedynak (Poster 62)
Noor Alsmadi (Poster 63)
Dr. Marcela Davoli Ferreira (Poster 64)
Lucas de Figueiredo Soveral (Poster 65)
Dr. Erik Bakkeren (Poster 66)
Juan Camilo Burckhardt (Poster 67)
Dr. Dina Kao (Poster 68)
Dr. Larisa Kovtonyuk (Poster 69)
Mursal Nader (Poster 70)
Dr. Xiaofan Jin (Poster 71)
Abstracts
Short Talk Presenters
Meggie Kuypers (Session 1: Microbiome in Early Life)
PhD Student, University of Toronto | Canada
Exposome attrition leads to transgenerational alterations in adaptive immune landscape
Kuypers M, Li Z, Bayer G, Liu Z, de Amat Herbozo C, Sephton V, Copeland J, Guttman D, Philpott D, Mallevaey T
The microbiota encompasses all of the commensal microorganisms living on and within the host. It is greatly impacted by the exposome, which is the cumulative sum of all environmental exposures encountered throughout an individual’s lifetime. Beyond genetic makeup, each person’s unique and diverse history of microbial exposure shapes their immune landscape. Importantly, recent advances have shown that laboratory mice raised in environmentally restricted, specific pathogen-free (SPF) conditions possess an underdeveloped immune phenotype equivalent to human newborns, while “dirty” pet store (PS) mice that are colonized with a greater array of microbes exhibit a more mature immune system reminiscent of human adults.
SPF mice released into the natural environment are able to efficiently acquire a phenotype closely resembling free-living animals, suggesting that exogenous microbial exposure is sufficient to induce immune system maturation. Conversely, PS mice housed under environmental restriction experience a transgenerational loss of their CD44hiCD62Llo (activated/effector memory) T cell population despite possessing a diverse endogenous microbiome, with a corresponding increase in CD44loCD62Lhi (naïve) T cells. Under exposome attrition, the immune phenotype of PS mice reverts to SPF levels within four generations (PS F0 – PS F3). This is also reflected in their functional response, with diminished cytokine production from PS F3 T cells following in vitro PMA/ionomycin stimulation when compared to newly purchased PS (NPS) mice that have rich exposome exposure. This effect can be mitigated through short-term co-housing of PS F3 mice with NPS mice, providing further evidence of the importance of exogenous microbial stimulation to immune system maturation. 16S rRNA sequencing suggests that these differences may not be bacterially driven, which is further supported by introducing either SPF or wild mouse-derived bacterial communities into germ-free C57BL/6 mice. Overall, this work will enrich our understanding of microbe-immune interactions and ultimately improve the translatability of preclinical research using animal models.
Dr. Van Ortega (Session 1: Microbiome in Early Life)
Postdoctoral Fellow, University of Calgary | Canada
Effect of the early-life premature microbiome on growth and neurodevelopment in mice in response to stress
Van A. Ortega, Simrit Rai, Kaetlyn Phillips, William Nguyen, Michelle R. Asbury, Thais Glatthardt Da Silva Dos Santos, Emily Mercer, Marie-Claire Arrieta
The establishment of the postnatal gut microbiome is critical for the appropriate development of various physiological systems, including immunity and the nervous system. Premature infants have altered microbiome communities compared to heathy term infants and also show delayed growth and poorer neurodevelopmental outcomes. We recently showed that extremely premature infants supplemented with bifidobacterial-containing probiotics exhibited accelerated microbiome maturation to a composition similar to healthy term-born infants. Further analysis yielded a positive correlation between infant growth patterns and microbiome maturation.
To test for causality and associated mechanisms, we colonized newborn germ-free mice with stool microbiomes from extreme (low-bifido) or late (high-bifido) premature infants (25- and 33-week gestation, respectively) collected through the Alberta BLOOM study. To elicit growth and neurodevelopmental dysfunction, half of the mice pups were subjected to daily chronic stress through maternal separation. We also tested the restorative potential of a daily-supplemented multi-strain probiotic (Florababy) historically provided to premature infants in Canada. Pup body weights were measured daily from postnatal (P) day P7-P21, while plasma and hair corticosterone levels, neurodevelopmental markers in brain (serotonin transporter, tryptophan hydroxylase 1/2, AMPA Receptor-GluA1/2), and active microglial phenotypes were determined at P21. Finally, maternal stool pellets were collected every 2 days from P2 to P21, with pup stools collected at P21 for qPCR, gene sequencing and metabolomic analysis.
Preliminary results from this study show that pups colonized with extreme preterm microbiomes have significantly lower rates of weight gain than pups colonized with late preterm microbiomes. Probiotic supplementation also significantly reduced weight gain in all microbiome conditions, while effects from chronic stress were less pronounced. Probiotics also reduced active microglia at P21, and particularly when administered to mouse pups harbouring a low-bifido microbiome and subjected to early-life stress. Additional results from this study show the potential impact of extreme preterm microbiomes to affect growth and neurodevelopment.
Dr. Lauren Davey (Session 2: Innovative Approaches for Elucidating Microbiome Function)
Assistant Professor, University of Victoria | Canada
A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization
Lauren E. Davey and Raphael H. Valdivia
Background: Akkermansia muciniphila, a mucin-degrading gut bacterium, holds therapeutic promise due to its protective effects against obesity, metabolic diseases, and its role in enhancing cancer immunotherapies. However, because A. muciniphila was genetically intractable, little is known as to how this bacterium colonizes the gut and mediates its positive health effects.
Methods: We developed a system for transposon (Tn) mutagenesis in A. muciniphila and applied transposon insertion sequencing (TnSeq) to identify determinants of mucin utilization and colonization. Host transcriptional responses to colonization by A. muciniphila and mutants unable to metabolize mucin were assessed using germ-free mice.
Results: TnSeq revealed the necessity of branched-chain amino acid biosynthesis, as well as sulfur and carbon metabolism for mucin-based growth. In vivo, additional amino acid biosynthesis genes were crucial for colonization. Notably, Tn mutants of unidentified proteins, clustered into two chromosomal regions that we termed Mucin Utilization Loci (MUL), were indispensable for both mucin utilization and colonization. These mutants were outcompeted by gut microbes in conventional mice but could colonize germ-free mice. Furthermore, mucin metabolism by A. muciniphila repressed host lipid biosynthesis genes, linking mucin catabolism to potential probiotic effects.
Conclusion: Genetic analysis of A. muciniphila revealed multiple uncharacterized genes that are required for harvesting mucin and colonization. We propose that mucin utilization via the MUL locus contributes to stable colonization of the intestinal tract and that the active catabolism of mucin by A. muciniphila may provide health benefits to the host.
Dr. Vanessa Dumeaux (Session 2: Innovative Approaches for Elucidating Microbiome Function)
Assistant Professor, Western University | Canada
Unraveling the functionality of microbiomes with novel computational and single-cell profiling approaches
Ray Wang, Mohamed Meawad, Teddy Lee, Alice Deng, Binh Tran, Vanessa Dumeaux
Host-adapted microbial communities, commonly referred to as microbiomes, consist of diverse microorganisms including commensal, symbiotic, and pathogenic species occupying specific anatomical niches. Traditionally, community compositions are quantified as vectors of relative abundances at various taxonomic levels. Studies have found that host-adapted microbiomes are variable across individuals, yet certain configurations are observed more frequently across the global population than what we would expect by chance. Multiple factors, ranging from environmental pressures to intrinsic microbe-microbe interactions, could explain the existence of these preferred configurations.
In humans, significant efforts have been dedicated to defining dominant microbial configurations (MCs) within various microbiomes, including those of the vagina, skin, oral cavity, and gut. In the gut, MCs, also called enterotypes, have been associated with dietary, and various metabolic or immunological markers. Despite these advancements, the functional roles and the underlying mechanisms contributing to the stability and resilience of these MCs remain poorly understood.
This presentation will introduce our research in developing novel computational and genomics approaches to identify and characterize specific MCs. We apply deep learning to analyze a large compendium of healthy adult gut microbiomes, capturing non-linear trends between features, such as taxa or pathway abundances. These efforts aim to refine the definition and understanding of both compositional and functional MCs. Alongside these computational advancements, we are developing a single-cell RNA profiling assay to offer granular insights into the activities of individual microbes within a complex community.
This multifaceted research is aimed towards enhancing our functional knowledge of microbiomes. Our computational approach enables a comprehensive analysis of healthy adult gut microbiomes on a global scale, while our single-cell assay dissects the functional roles of individual cells within distinct communities. As we delve deeper into the intricacies of MCs, the opportunities to modulate them for varied applications in biomedicine, environment, and synthetic biology will expand.
Dr. Erik Bakkeren (Session 3: Harnessing the Microbiome)
Postdoctoral Fellow, University of Oxford | United Kingdom
Ecology of the human microbiome: how diversity in the microbiome protects against pathogens
Erik Bakkeren, Frances Spragge, Martin T. Jahn, Elizete B. N. Araujo, Claire F. Pearson, Xuedan Wang, Louise Pankhurst, Olivier Cunrath, Kevin R. Foster
A diverse human gut microbiome plays an important role in resisting colonization of the host by pathogens. While many mechanisms have been found to influence the ability of the microbiome to provide colonization resistance, these mecahnsisms are often context-specific and dependent on individual species. Therefore, we currently lack general principles to predict which microbiota communities will be protective versus those that will allow a pathogen to colonize and cause disease. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. We then applied our findings to successfully predict communities that resist a novel target strain. Our results support that more diverse microbiomes protect better against pathogens. Crucially, although increased microbiome diversity increases the probability of protection against pathogens, the overlap in nutrient-utilization profiles between the community and the pathogen is key. Our work suggests a route to optimize the composition of microbiomes for protection against pathogens.
Juan Camilo Burckhardt (Session 3: Harnessing the Microbiome)
PhD Candidate, University of British Columbia | Canada
With a little help from our [tiny] friends: modifying gut bacteria to detect malabsorption
Juan C. Burckhardt, Giselle McCallum, Jerry He, Charlotte Clayton, Alice Hong, Carolina Tropini
Gut osmolality, reflecting particle concentration in intestinal fluid, is vital in gut physiology, governing functions such as water absorption and cell structure. Gut osmolality imbalances frequently occur in conditions such as inflammatory bowel disease, food intolerances, and laxative use. These imbalances arise as impairments in nutrient absorption that lead to an accumulation of particles in the gut lumen. Despite its importance, human intestinal osmolality remains understudied. Existing stool-based methods cannot capture the osmolality of different gut regions, limiting their use as a diagnostic tool. Furthermore, gut disease diagnosis usually necessitates invasive endoscopic procedures. Previous research from our lab revealed that certain bacterial families persist during gut osmotic upshifts. These osmotically-tolerant bacteria sense the gut osmotic environment and respond to its changes by regulating the transcription of specific sets of genes. Thus, we proposed to detect malabsorption by leveraging the genetic mechanisms that these commensals employ when responding to changes in the gut’s physical environment. We repurposed regulatory sequences of osmolality-responsive genes from the genetically tractable, osmotically-tolerant, and prevalent commensal Bacteroides thetaiotaomicron to create a biosensor for localized and fine-scale osmolality measurements. Through transcriptomics analyses, we identified B. theta genes selectively expressed across increased osmolality both in vitro and in vivo. Subsequently, we cloned the promoters from the identified genes into a synthetic reporter fluorophore circuit, creating osmolality biosensors in B. theta. We selected biosensors with a graded response to osmolality, and through further optimization, we achieved a 15- to 25-fold fluorescence difference in high osmolality conditions compared to baseline. Currently, we are validating our optimized biosensor in vivo by measuring its signal in the intestines of mice experiencing disrupted gut osmolality. Through this work, we are creating a platform for commensal-based microbial diagnostics that respond to disease-induced gut changes, improving our understanding of gut physical conditions.
Dr. Robyn Wright (Special Topic: Microbiome Sequencing & Computational Development)
Postdoctoral Fellow, Dalhousie University | Canada
Reducing false-positive taxa in metagenomes by coverage validation of reference genomes
Robyn J. Wright, Benjamin Fisher, André M. Comeau and Morgan G.I. Langille
In metagenomic analyses of microbiomes, one of the first steps is often the taxonomic classification of reads by comparison to a database of reference genomes. For samples from environments not represented well in databases, many taxonomic classifiers incorrectly identify very few microbial taxa. Lots of taxonomic classifiers also underestimate the already low diversity in low microbial biomass samples with high host contamination. In contrast, Kraken2 – one of the most used metagenomic taxonomic classification tools – identifies hundreds to thousands of microbial taxa in these samples. However, many of these taxa are likely to be false positives, for example, Mycobacterium tuberculosis in blood samples from healthy humans and tumour tissue samples. Increasing the confidence threshold used by Kraken2 increases precision in simulated, mock, and human microbiome samples from well-characterised body sites. However, for environmental samples or those with low microbial biomass, e.g., tumour samples, this often results in most or all of the reads remaining unclassified. We have developed a method to verify the taxa identified by Kraken2 by mapping the classified reads to representative genomes of these taxa. By doing this, we can calculate several metrics that indicate the likelihood that the taxon is actually present. For example, a comparison of the actual versus expected fraction of the representative genome that is covered, the median identity of reads to the representative genome, and an indication of whether mapped reads all map to one area of the genome or not. These metrics can also be pooled across sample groupings. This ensures higher precision without a large decrease in recall or making the computational time untenable and, importantly, is effective even if a taxon is present at very low abundances. We will discuss our current assessments of the efficacy of this method on samples from different environments, including from tumour tissue samples.
Geoff Winsor (Special Topic: Microbiome Sequencing & Computational Development)
Graduate Student, Simon Fraser University | Canada
Challenges and opportunities with integrating microbiome data with other metadata
Dr. Ali Mirza (Special Topic: Microbiome Sequencing & Computational Development)
Postdoctoral Fellow, Simon Fraser University | Canada
IMPACTT Update: Defining the Effects of Sample Handling on Metagenomic Amplicon Sequencing and Culturomic Profiles of the Gut Microbiome
Dr. Riadh Hammami (Session 4: Microbiome, Metabolites & Disease)
Associate Professor, University of Ottawa | Canada
Are microbiome extracellular vesicles the missing connection to the gut-brain axis?
Walid Mottawea, Basit Yousuf, Salma Sultan, Tamer Ahmed, JuDong Yeo, Nico Hüttmann, Yingxi Li, Nour Elhouda Bouhlel, Hebatoallah Hassan, Xu Zhang, Zoran Minic, and Riadh Hammami
Microbiota-released extracellular vesicles (MEVs) have emerged as significant contributors to intercellular communication, yet their impact on the gut-brain axis remains underexplored. This study aimed to assess MEVs capacity to cargo bioactive metabolites and interact with the host. In this study, a range of neuro-related compounds was found enclosed within MEVs, including arachidonyl-dopamine, gabapentin, glutamate, and N-acylethanolamines. Metaproteomics revealed an abundance of enzymes involved in neuronal metabolism, particularly in the glutamine/glutamate/GABA pathway. These neuro-related proteins and metabolites were found to be associated with Bacteroides spp. Interestingly, a GABA-producing Bacteroides isolate, B. finegoldii, released EVs containing high GABA content (4 µM), unlike a low GABA-producing isolate, Phocaeicola massiliensis. MEVs displayed dose-dependent paracellular transport and were taken up by Caco-2 and hCMEC/D3 cells through endocytosis. Exposure of Caco-2 cells to MEVs did not show a differential expression of genes related to intestinal barrier integrity, while affected many genes included in several immune pathways and cell apoptosis process as revealed by RNA-Seq analyses. In vivo biodistribution confirmed MEVs’ presence in the brain, liver, stomach, and spleen. Overall, these findings underscore the capability of MEVs to cross both the intestinal and blood-brain barriers, delivering their cargo to remote organs, including the brain, potentially influencing organ functions. MEVs might play a critical role in microbiome-host communication, impacting the gut-brain axis.
Dr. Dina Kao (Session 4: Microbiome, Metabolites & Disease)
Professor, University of Alberta | Canada
Effects of lyophilized fecal filtrate compared to lyophilized donor stool in the treatment of recurrent Clostridioides difficile infection: a multi-center randomized trial
D Kao, K Wong, C Lee, T Steiner, R Franz, C McDougall, M Silva, H Xu, Walter J, Loebenberg R, M Yaskina, T Louie
Background: Fecal microbiota transplantation (FMT) is the most effective therapy in the treatment of recurrent Clostridioides difficile infection (rCDI), and can be offered in fresh, frozen or lyophilized formulation with similar efficacy. Microbial engraftment is thought to be one of the mechanisms mediating its efficacy. However, the importance of microbes has been questioned by two small preliminary studies showing sterile fecal filtrate could prevent C. difficile recurrence.
Aims: To establish whether lyophilized sterile fecal filtrate (LSFF) is noninferior to lyophilized FMT (LFMT) in efficacy
Methods: In this double-blind, non-inferiority study conducted in 4 academic centers , we randomized rCDI patients to either LSFF or LFMT (1:1). Sterile fecal filtrate was manufactured by filtering fecal slurry through a series of filters, down to a pore size of 0.2 um. The non inferiority margin was 10%. Each treatment dose consisted of 15 oral capsules. Should a participant fail the assigned treatment, an open label LFMT treatment was offered. An interim analysis was planned after reaching 50% of recruitment target (124/248 participants). Primary outcome was the proportion of participants without CDI recurrence 8 weeks following assigned treatment. Secondary outcomes included 1) the proportion of participants without CDI recurrence 24 weeks following assigned treatment, 2) serious adverse events, and 3) minor adverse events.
Results: 137 participants were randomized to LFMT group (66) and LSFF (71) group. Prevention of rCDI 8 weeks after assigned treatment was achieved in 63.8% of participants in LSFF group and 86.9% in LFMT group (difference, -23.1%), resulting in trial termination. Serious adverse events were infrequent: 4 hospitalizations deemed not related and 1 hospitalization possibly related, and 1 death from COVID deemed not related to assigned treatment. Minor adverse events were self-limited and infrequent: nausea (6), vomiting (2), abdominal discomfort (15) and fever (2).
Conclusions: Among adults with rCDI, LSFF was inferior to LFMT by oral capsules in preventing recurrent infection over 8 weeks. The result emphasizes the importance of microbes in mediating FMT efficacy.
Dr. Larisa Kovtonyuk (Session 5: Microbiome Regulation in Cancer & Cancer Therapy)
Postdoctoral Fellow, University of Calgary | Canada
The role of intestinal microbiome in CD19 directed CAR-T cell therapy
Larisa V. Kovtonyuk, Amy Shupe, Sacha Benaudia, Robert Puckrin, Mona Shafey, Doug Mahoney, Kathy D. McCoy
Adoptive T cell therapies have been under intense development for the past decades. Despite the success of chimeric antigen receptor (CAR)-T cell therapy, nearly 50% of patients with B cell malignancies either do not respond to the treatment or relapse. In addition, almost 50% of patients undergoing CAR T cell therapy experience severe adverse effects due to cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). The microbiome and microbial metabolites have been found to modulate the anti-cancer function of T cells in immune checkpoint inhibitor therapy. Similarly, differences in the patient’s microbiome composition may be one of the key extrinsic underlying factors determining responsiveness to CAR-T cell therapy. We hypothesize that specific intestinal microbial species can influence CD19CAR-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 CD19CAR-T cells transferred into germ free NSG (KbDb)null mice that received luciferase expressing Nalm6 cells can eliminate tumor cells in vivo. However, CD19CAR-T cells perform better and eliminate tumor cells more efficiently in NSG (KbDb)null mice harboring a diverse specific pathogen-free (SPF) microbiota. Moreover, CD19CAR-T cells perform better in mice mono-colonized with Bifidobacterium pseudolongum or Akkermansia muciniphila, compared to germ free mice. In addition, CD19CAR-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 metabolites involved.
Dr. Ann Gregory (Session 5: Microbiome Regulation in Cancer & Cancer Therapy)
Assistant Professor, University of Calgary | Canada
Viral predation guilds of the human gut
Ann C. Gregory, Olivier Zablocki, Tao Long, Guillaume Meric, Teemu Niiranen, Igor Segota, Jeramie Watrous, FINRISK Consortium, Michael Inouye, Mohit Jain, Rob Knight, Veikko Salomaa, Matthew B. Sullivan
The human gut virome is highly diverse and composed mostly of phages. Yet, despite the vast diversity of gut viral species identified so far, of which >90% are phage, there are a relatively low number of bacterial species (~4,500). This high ratio of phage to bacterial species suggests that many phage species share the same host. Here, we introduce the concept of using viral predation guilds, groups of viruses that exploit the same hosts, instead of taxonomy-based analyses to study the gut virome. We hypothesized that given high virome inter-individuality and low numbers of bacterial species in the human gut, individuals most likely share a high proportion of viral guilds despite distinct viral species. To test our hypothesis, we analyzed 7,230 gut metagenomes from the FINRISK 2002 Cohort.
Flash Talk & Poster Presenters
Shirin Afroj (Poster 1)
Laboratory Technician, University of Calgary | Canada
A massively parallel culturing platform to identify antibiotic potentiators from the natural products of microbiota
Rahgavi Poopalarajah, Andrew J. Flewelling, Shirin Afroj, Ben Rowley, Kevin C. Sule, Nathan McCartney, Yanqi Li, Carina Thomas, Shannon Pyke, Emma J. Griffiths, Craig Brideau, Barbara Waddell, Michael D. Parkins, John M. Conly, Ayush Kumar, Pina Colarusso, Elmar J. Prenner, William Hsiao, Kathy D. McCoy, Douglas J. Mahoney, Darren J. Derksen, Joe J. Harrison
Antibiotic potentiators are bioactive substances that enhance antibiotic activity. Many antibiotics have originated from microbial natural product screens; however, analogous tactics to find antibiotic potentiators seldom have been developed. Here we report a microbial natural product platform based on massively parallel culturing to identify antibiotic potentiators. Using a carbapenem-resistant Pseudomonas aeruginosa strain exhibiting resistance due to reduced drug uptake, we demonstrate the utility of this pipeline by identifying a multitude of species producing spent media that potentiate last-resort antibiotics. Bioassay-guided fractionation and chemical analyses identified that a group of bioactive molecules were cis-unsaturated fatty acid primary amides (FAPAs). These substances are produced by diverse bacterial and eukaryotic microbiota as well as plants, and are even found in human serum and tear film. Biochemical, genetic, and structure-activity relationship analyses indicate that cis-unsaturated FAPAs increase small molecule uptake by fluidizing bacterial membranes at micromolar concentrations, compromising the permeability barrier of the bacterial cell envelope. Taken together, we propose that our microbial natural product platform can be used to inform a basic understanding of biotic factors influencing antimicrobial resistance, and to inspire the discovery and design of antibiotic potentiators.
Jacqueline Barnett (Poster 2)
PhD Candidate, University of British Columbia, Okanagan | Canada
Inhereting the Toxic Effects of our Ancestors Diet: Prenatal Glyphosate Exposure Induces Colonic Inflammation, Immune Metabolic Dysfunction and Behavioural Deficits in Mice
Jacqueline A. Barnett, Jessica K. Josephson, Natasha Haskey, Carson McComb, Minseon Jung, Kiran K. Soma, Andrea Verdugo-Meza, Julien Gibon, Sanjoy Ghosh and Deanna L. Gibson
Glyphosate, commonly known as Roundup®, is heavily used in Canada, with over 50 million kilograms purchased annually. It is frequently applied pre-harvest, leading to high residues in many foods consumed within Canada, including wheat, cereals and legumes. Glyphosate can inhibit bacterial growth and promote bacterial dysbiosis, which is known to be causative in inflammatory bowel disease (IBD). Canada has the highest incidence of new cases of IBD in the world. This research aimed to determine if there are harmful effects on the gut bacteriome, resulting in colitis associated with altered metabolic health and behavioural changes resulting from prenatal glyphosate exposure.
Two physiological doses of glyphosate were chosen for the study: the acceptable daily intake set by the Environmental Protection Agency (EPA) (1.75mg/kg bw/day) and the Average American dose calculated with a dietician based on reported values of glyphosate in foods found within the average Canadian diet (0.01mg/kg bw/day). Breeding pairs of colitis-resistant and colitis-susceptible mice were exposed to glyphosate during pregnancy, and two generations of animals were raised.
The study found that healthy mice whose ancestors were exposed to glyphosate at both doses developed colitis, metabolic dysfunction, and behavioural deficits, including impaired memory and decreased locomotor activity. These effects persist two generations after exposure. Similarly, colitis-susceptible offspring of animals exposed to glyphosate display increased inflammation, impaired immune function, and alterations in critical metabolites implicated in the gut-brain-microbiome axis, including neurotransmitter levels and microbial community structures.
This study highlights the profound and enduring impact of glyphosate exposure, even at previously considered safe levels, on the gut bacteriome, metabolic health, and behaviour over multiple generations. Remarkably, healthy mice suffered more adverse effects than genetically susceptible mice, suggesting that glyphosate-induced detrimental effects may be an environmental factor contributing to the increased prevalence of IBD and related metabolic and behavioural comorbidities.
Dr. Francisco Daniel Davila Aleman (Poster 3)
Postdoctoral Researcher, University of Calgary | Canada
Novel oil-associated bacteria in Arctic seawater exposed to different nutrient biostimulation regimes
Francisco D. Davila Aleman, María A. Bautista, Janine McCalder, Kaiden Jobin, Sean M. C. Murphy, Brent Else, Casey R.J. Hubert
The Arctic Ocean is an oligotrophic ecosystem facing escalating threats of oil spills as ship traffic increases owing to climate change-induced sea ice retreat. Biostimulation is an oil spill mitigation strategy that involves introducing bioavailable nutrients to enhance crude oil biodegradation by endemic oil-degrading microbes. For bioremediation to offer a viable response for future oil spill mitigation in extreme Arctic conditions, a better understanding of the effects of nutrient addition on Arctic marine microorganisms is needed. Comprehensive population tracking of controlled oil-spill microcosms using cell counting and microbial biodiversity screening revealed a significant decline in community diversity together with changes in microbial community composition. These shifts were also indicative of changes in prevailing genomic traits as inferred from 16S rRNA taxonomy of resulting communities. In addition to well-recognized hydrocarbonoclastic bacteria, differential abundance analysis highlighted significant enrichment of unexpected genera Lacinutrix, Halarcobacter and Candidatus Pseudothioglobus. These groups have not been associated with hydrocarbon biodegradation until now, even though genomes from closely related isolates confirm the potential for hydrocarbon metabolism. These findings broaden understanding of marine oil spill bioremediation and how Arctic marine microbiomes and their novel lineages can respond to nutrient biostimulation as a strategy for oil spill mitigation.
Hans Ghezzi (Poster 4)
PhD Candidate, University of British Columbia | Canada
PUPpy: a primer design pipeline for substrain-level microbial detection and absolute quantification
Hans Ghezzi, Michelle Y. Fan, Katharine M. Ng, Juan C. Burckhardt, Deanna M. Pepin, Xuan Lin, Ryan M. Ziels, Carolina Tropini
Characterizing microbial communities at high-resolution and with absolute quantification is crucial to unravel the complexity and diversity of microbial ecosystems. This can be achieved with PCR assays, which enable highly selective detection and absolute quantification of microbial DNA. However, a major challenge that has hindered PCR applications in microbiome research is the design of highly specific primer sets that exclusively amplify intended targets. Here, we introduce Phylogenetically Unique Primers in python (PUPpy), a fully automated pipeline to design microbe- and group-specific primers within a given microbial community. PUPpy-designed primers enable the detection of individual microbes and quantification of absolute microbial abundance in a defined community below the strain level, requiring only coding sequence files of the community members as input. We experimentally evaluated the performance of PUPpy-designed primers using two bacterial communities as benchmarks. Each community was comprised of 10 members, exhibiting a range of genetic similarities that spanned from different phyla to substrains. PUPpy-designed primers also enabled the detection of groups of bacteria in an undefined community, such as the detection of a gut bacterial family in a complex stool microbiota sample. Taxon-specific primers designed with PUPpy showed 100% specificity to their intended targets, without unintended amplification, in each community tested. Lastly, we show absolute quantification of microbial abundance using PUPpy-designed primers in ddPCR, benchmarked against 16S rRNA and shotgun sequencing. Our data shows that PUPpy-designed microbe-specific primers can be used to quantify substrain-level absolute counts, providing more resolved and accurate quantification in defined communities than short-read 16S rRNA and shotgun sequencing.
Connor Gianetto-Hill (Poster 5)
PhD Candidate, University of Guelph | Canada
Assessing Gut Microbiota Differences in Adolescents at Risk of Type 2 Diabetes Using a Colonic Bioreactor Model
Connor Gianetto-Hill, Alessandra Granato, Jill Hamilton, Jayne Danska, and Emma Allen-Vercoe
The global incidence of Type 2 Diabetes (T2D) is on the rise, posing an escalating concern particularly among adolescents. Addressing this surge necessitates a deeper understanding of the risk factors contributing to the disease, including the involvement of the gut microbiome. Existing literature on the gut microbiome’s association with T2D predominantly focuses on populations over 30 already diagnosed with the condition. To bridge this gap, we utilized stool samples obtained from adolescents at risk of developing T2D to inoculate bioreactors designed to model the colonic environment. In parallel, stool from healthy young participants were inoculated in bioreactors to discern differences between the healthy and the at-risk of T2D gut microbiomes. Given the established influence of dietary nutrients on gut microbiome composition, bioreactors were initially supplied with nutrient-rich media, transitioning to a nutrient-poor medium after 28 days to investigate dietary impacts on community composition and function. Shotgun metagenomic sequencing of stool samples revealed some differences in bacterial composition and predicted functions between healthy and T2D risk groups. Furthermore, bacterial community composition in bioreactors was assessed using 16S rRNA marker gene sequencing, while metabolites were identified and quantified using proton nuclear magnetic resonance (1H-NMR), facilitating a comparative analysis between healthy and at-risk adolescents. This approach aims to identify absent bacterial taxa or functions in at-risk adolescent microbiomes, with the ultimate goal of designing potential gut therapeutics to restore these microbial communities.
Dr. Rebecca Jeffery (Poster 6)
Postdoctoral Fellow, University of Oxford | United Kingdom
Delineating the causal role of the gut microbiota in experimental colitis
Rebecca Jeffery, Lilian Lam, Claire Pearson, India Brough, Elizete Araujo, Andrew Macpherson, Fiona Powrie and Nicholas Ilott
Maladapted interactions between the mucosal immune system and gut microbiota are implicated in the pathogenesis of inflammatory bowel disease (IBD). Alterations in gut microbial communities are often seen in IBD patients, although the functional consequences of these shifts are not understood. Multiple mouse models of IBD fail to develop pathology when housed in germ-free conditions, suggesting an active role for the microbiota in disease pathogenesis. Helicobacter hepaticus is a murine pathobiont that induces colitis in interleukin-10 (IL-10) deficient mice, thus encompassing a role for both host and microbial factors in disease pathogenesis. Interestingly, IL-10 deficient mice monocolonized with H. hepaticus do not develop disease, suggesting other commensals are required to facilitate H. hepaticus-induced colitis. We have established that gnotobiotic mice stably colonized with the 12-member Oligo-Mouse-Microbiota (OMM12) are susceptible to H. hepaticus-induced colitis, facilitating the interrogation of commensal involvement in colitis development. Utilising longitudinal approaches, we have determined that the development of H. hepaticus-induced inflammation coincides with OMM12 dysbiosis, including an expansion of Enterococcus faecalis and reduction of Enterocloster clostridioformis and Akkermansia muciniphila, and alterations in intestinal metabolite composition. Through monocolonization experiments, we have determined that E. clostridioformis supports H. hepaticus-induced colitis, an ability that is not shared by A. muciniphila, E. faecalis, Lactobacillus reuteri and Clostridium innocuum. Work is ongoing to understand the differential abilities of OMM12 members to support H. hepaticus-induced colitis. Ultimately, this work will identify microbiota interactions that promote colitis representing novel therapeutic strategies.
Jessica Josephson (Poster 37)
PhD Candidate, University of British Columbia, Okanagan | Canada
The Alteration of the Gut-Brain-Microbiome Axis in a Spontaneous Colitis Murine Model
Jessica K. Josephson, Jacqueline A. Barnett, Deanna L. Gibson
Globally, incidence of IBD is projected to reach 0.5% of the population by 2030. Increases in IBD prevalence also see associated co-morbidities such as anxiety, depression and memory issues rising. The Mucin 2 knockout mouse has been utilized as a model for spontaneous colitis, however, the suitability of this model for modelling associated behavioural comorbidities microbiome influences and inflammation associated with ulcerative colitis (UC) remains unclear as multiple pathways influence the gut-brain axis (GBA) and contribute to their pathogenesis.
The bi-directional communication between the GBA implicates several neurometabolites (serotonin, kynurenine and dopamine) in anxiety, depression or memory deficits present in UC patients. This study aims to assess if alterations in neurometabolites from chronic inflammation correlate with behavioural changes and assess the role the microbiome plays in this interaction.
SPF and germ-free (GF) Muc2-/- and wild-type (C57Bl/6) mice (n=5/sex/genotype) mice underwent behavioural testing for anxiety, depression, locomotion and memory deficits. Significant sex-dependent differences were found in anxiety assessments (SPF female Muc2-/- more exploratory p=0.0728 as well as memory deficits were found to be present in SPF Male Muc2-/- p=0.002). While literature reports that GF mice have less depressive behaviour than SPF mice, GF Muc2-/- do not display this phenomenon (p>0.9999).
Serum was analyzed for dopamine, tryptophan, kynurenine, and serotonin concentrations. Data suggests that while no dopamine differences were present, serum tryptophan, kynurenine and serotonin were significantly decreased in the Muc2-/- model (p=0.0294, 0.0132 and 0.0054 respectively) but displayed a physiologically consistent pattern with wild-type mice suggesting physiological depletion. This was supported by an increased (p=0.0013) IDO1 gene expression in the Muc2-/- mice.
The Muc2-/- mouse has shown applicability as a behavioural comorbidity model for IBD patients with anxiety and memory deficits. As global IBD rates increase, this may be a useful model to test therapies and microbial influence on behavioural comorbidities.
Simarpreet Kaur (Poster 38)
Graduate Student, University of Calgary | Canada
Deciphering the Lung Microbiome during Pulmonary Exacerbations and Clinical Stability in Non-Cystic Fibrosis Bronchiectasis (NCFB)
Simarpreet Kaur, Jared Schlechte, Barbara Waddell, Michael Parkins, Braedon McDonald and Christina Thornton
Background
NCFB is characterized by cycles of inflammation and chronic infection. Pulmonary exacerbations are defined by periods of worsening symptoms and dropping lung function. Traditional dogma suggests these adverse events are due to overgrowth of canonical bacterial pathogens; however recent studies have shown this to not fully represent all events. The cause(s) of exacerbations are currently unknown, representing a knowledge gap. The central hypothesis of this research project is different pulmotypes in bronchiectasis, unique to the individual, can be used for future applications to delineate appropriate clinical therapy.
Methodology
A retrospective cohort of sputum samples and metadata (from 1980), including those with bronchiectasis (>20,000 samples) were analysed for different periods of clinical stability and acute exacerbation. Long-read DNA sequencing (MinION) was performed, Relative abundance, alpha and beta-diversity using an in-house developed R pipeline was done. Simultaneously, Fastq WIMP (What’s In My Pot) and Fastq Antimicrobial Resistance analysis was done using EPI2ME Desktop Agent.
Preliminary Findings
We evaluated 48 samples from 16 patients during periods of stability, acute exacerbation and recovery. Sequencing data demonstrated the majority of microbes were Pseudomonas, Staphylococcus and Escherichia. Anaerobic genera present included Prevotella, Gemella and Veillonella. We observed varying degree of inter and intra patient variability and heterogeneity with some patients that demonstrate relative stability and others showing considerable temporal variation.
Significance
Our preliminary data suggests that certain cohorts of patients with NCFB experience temporal change during periods of exacerbation, whereas others do not. This suggests pulmotypes exist and may be used to personalize therapeutic approaches. Expansion of our cohort across more subjects will be required to validate our initial findings and delineate clinical associations.
Dr. Maryam Kebbe (Poster 39)
Assistant Professor, University of New Brunswick | Canada
An immunomodulatory role of human milk components on the infant gut microbiota
Maryam Kebbe, Kartik Shankar, Leanne M. Redman, Aline Andres
Background: Human milk oligosaccharides have been shown to relate to the infant gut microbiome. However, the impact of other human milk components on infant gut bacterial colonization remains unexplored.
Objectives: Our cross-sectional analysis aimed to investigate associations between human milk components (energy, macronutrients, free amino acids, inflammatory markers, hormones) and infant gut microbiome diversity and composition (phylum, family, and genus) at 6 months of age.
Methods: Human milk and infant stool samples were collected at six months postpartum. The infant gut microbiome was profiled using 16S rRNA sequencing. Linear regression models were performed to examine associations, adjusting for pre-gravid body mass index (BMI), delivery mode, duration of human milk feeding, and infant sex, with q<0.2 considered significant.
Results: This analysis included a total of 54 mothers (100% exclusively feeding human milk) and infants (n=28 male; 51.9%). Total energy in human milk showed a negative association with α-diversity measures (Chao1 and Shannon). IL-8 in human milk was positively associated with Chao1 and observed OTUs. At the family level, human milk glutamine and serine levels showed a negative association with the abundance of Veillonellaceae, while isoleucine showed a positive association with Bacteroidaceae. Human milk IL-8 and IL-6 concentrations were positively associated with Bacteroidaceae abundance. IL-8 also had a positive relationship with Bifidobacteriaceae, whereas it had a negative relationship with Streptococcacea and Clostridiaceae. Human milk IL-8 was positively associated with the phylum Bacteroidetes, and negatively associated with Proteobacteria. At the genus level, human milk IL-8 exhibited a positive relationship with Bacteroides, whereas human milk isoleucine had a negative relationship with Bacteroides and Ruminococcus. Pre-gravid BMI and sex effects were observed.
Conclusions: IL-8 in human milk could potentially prepare the infant’s immune system to respond effectively to various microorganisms, potentially promoting the growth of beneficial gut bacteria and protecting against pathogens.
Kathryn Strayer (Poster 40)
MSc Student, University of Calgary | Canada
The role of Candida in airway microbiome dysbiosis and inflammation in critical illness
Kathryn A Strayer, Jared Schlechte, Nicole A Cho, Mark Gillrie, Christina Thornton, Braedon McDonald
The lungs harbour a distinct community of microorganisms that are important in lung health and homeostasis. In chronic lung diseases the lung microbiome shows significant alterations (dysbiosis) which is associated with adverse outcomes. Similarly, patients with acute lung injury in intensive care units (ICU) who require mechanical ventilation also suffer dysbiosis of the lung microbiome, which has been linked with adverse outcomes, including hospital-acquired infections and increased mortality. Currently, much of the work has focused exclusively on the bacterial microbiome and ignored the fungi that also colonize the lungs. In particular, Candida is commonly found in the lungs of ICU patients, and its presence is associated with an increased risk of bacterial ventilator-associated pneumonia (VAP) for unclear reasons. In this project, we hypothesize that lung colonization by Candida potentiates the development of bacterial VAP by augmenting the growth and virulence of bacterial pathogens and promoting airway inflammation and damage. We analyzed endotracheal aspirate samples from 49 ICU patients and performed 16S and ITS2 amplicon sequencing to characterize the lung microbiome composition. Approximately 60% of the patients have Candida colonization in the lungs, and we see that Candida colonization is associated with an increased risk for bacterial VAP or all-cause mortality. Candida colonization is also associated with higher relative abundance of common bacterial VAP pathogens (S. aureus, Enterobacteriaceae, Pseudomonas, Acinetobacter). In vitro co-culture of Candida albicans with these bacterial VAP pathogens demonstrated that Candida potentiates pathogen growth. Co-culture of Candida and bacterial pathogens induces more epithelial cell death than the Candida or the bacteria on their own. These data suggest that in ICU patients on mechanical ventilation, airway colonization by Candida spp. results in pathological cross-kingdom interactions to potentiate the growth and virulence of bacterial pathogens, culminating in an increased risk of bacterial VAP.
Athalia Voisin (Poster 41)
Graduate Student, University of Manitoba | Canada
Unfermented dietary β-fructan fibers induce P53 pathways associated with colorectal cancer in inflammatory bowel diseases
A. Voisin, S. Balko, R. Mahmood, O. Fedorova, E. Zoungrana Ouali, T. Jeanson, A. Raouf, C.N. Bernstein, H. Armstrong
Dietary fibers are not digested; they are fermented by gut microbes producing beneficial short-chain fatty acids (SCFAs). However, inflammatory bowel disease (IBD) patients have altered gut microbiota, reduced SCFAs, and can experience sensitivity to a high-fiber diet. We showed unfermented β-fructan fibers induce inflammation and gut damage via TLR2 and NLRP3 inflammasome in IBD patients. Chronic inflammation and gut damage in IBD increases the risk of developing colorectal cancer (CRC). Therefore, we hypothesize that diet-induced inflammation could create a mutagenic environment that promotes risk of CRC in IBD.
Bulk RNA sequencing was performed on biopsies taken from IBD patients before and after 6-month consumption of either 15g/day β-fructans or placebo (clinicaltrials.gov NCT02865707). To validate findings, biopsies from non-IBD (n=5) and IBD (n=5) patients were cultured ex vivo with β-fructans (inulin and oligofructose) or a combination of oligofructose+SCFAs reflective of levels following fermentation by non-IBD or IBD whole gut microbiota (Fig 3F PMID: 36183751). Changes in cancer pathway markers were examined (microscopy [p53, villin, axin2, NLRP3], mesoscale discovery, ROS production). Cell migration was examined in Caco-2 human gut epithelial cells exposed to β-fructans (scratch wound assay with mitomycin C).
From the clinical trial cohort, the CRC-related genes SLIT2/MAPK (p<0.0001) and SOS1 (P<0.001) were induced only in IBD patients who displayed reduced fiber-fermenting capacity (stool) and who had relapsed following 6-months consumption of β-fructans. In ex vivo cultures, unfermented β-fructans increased expression of axin2 and p53 compared to no fibre control (densitometry; fold change). β-fructans had no significant impact on cell migration in cell lines (p>0.05; one-way ANOVA).
My findings suggest that if β-fructan fibres are not fermented in IBD, they can induce p53-associated CRC pathways. Our observations suggest that such fibres should be avoided in a subset of IBD patients if their gut microbiota are unable to utilize these fibres.
Christopher Yau (Poster 42)
Graduate Student, University of Toronto | Canada
Host genetics and early life environment shape the trajectory of immune reactivity to intestinal bacteria associated with future autoimmunity in infants at risk for Type 1 Diabetes
Christopher Yau, Alexandra Paun, Shahab Meshkibaf, Taina Harkonen, Suvi M. Virtanen, Jorma Ilonen, Michelle C. Daigneault, Jordan T. Russell, Eric W. Triplett, Emma Allen-Vercoe, Mikael Knip on behalf of the DIABIMMUNE Study Group, and Jayne S. Danska
Type 1 diabetes results from autoimmune destruction of β cells in the pancreas. HLA class II alleles confer diabetes risk but the increased disease incidence during the past 50 years invokes causal environmental factors. Human fecal microbiome analyses suggest that bacterial composition is associated with T1D, but it has been challenging to identify relationships between defined microbes and development of islet-specific autoimmunity. We prospectively evaluated serum antibodies in healthy infants with diabetes high-risk HLA haplotypes that reacted with human gut bacteria. Antibody response patterns reflected diversification of the gut microbiota and breastfeeding duration in infancy. Antibodies against specific bacterial species were associated with HLA haplotypes and discriminated children who developed islet autoantibodies from those who did not. The longitudinal patterns of anti-bacterial antibodies predicted the appearance of islet autoantibodies. Thus, early life immune responses to intestinal microbes are conditioned by immunogenetic variants and environmental exposures and predict future islet autoimmunity.
Poster Presenters
Spencer Abbott (Poster 7)
MSc Student, University of Calgary | Canada
Investigating how bacterial expression of molecular mimics’ shapes islet-specific adaptive immunity and diabetes development in NOD mice
Spencer Abbott, Carolyn Thomson, Kathy D. McCoy
Autoreactive T cells recognize the islet autoantigen IGRP206-214 with varying avidities due to differences in T cell receptor (TCR) V-alpha regions, uniquely influencing type 1 diabetes (T1D) progression in non-obese diabetic (NOD) mice. CD8+ T cells recognizing IGRP with high avidity promote T1D by killing insulin-producing beta cells in the pancreatic islets, whereas those with low avidity suppress disease by eliminating autoantigen-loaded antigen presenting cells. These T cell clonotypes also recognize a microbial epitope, BacIYL36-44, from a Bacteroides-derived integrase enzyme. Integrase expression by gut commensals promotes the activation of these T cells, which then suppress colitis in IGRP-/- mice. Our aim was to investigate whether the presence of the molecular mimic integrase in the gut provides protection from T1D and if varying T cell avidities can differentially influence microbiome composition, thereby influencing T1D progression.
Germ-free NOD mice were monocolonized with an isogenic pair of integrase-positive or negative bacterial strains and monitored for T1D incidence. We found that under these conditions, bacterial expression of the molecular mimic did not alter T1D development. To establish if T cell recognition of a commensal-derived microbial mimic modulates composition in the microbiome, two cohorts of transgenic mice with monoclonal TCR’s that differ in their avidity to BacIYL36-44 were colonized with a specific-pathogen free microbiota. Fecal samples were collected weekly over twenty weeks and assessed by 16S sequencing. Differences in T cell avidity to BacIYL36-44 shaped microbiome community composition, resulting in significant differences in alpha and beta-diversity. Specifically, the relative abundance of Bacteroides species was significantly reduced in male mice when T cells recognized BacIYL36-44 with high affinity. Therefore, immune recognition of a bacterial microbial mimic can shape microbiome composition. Further work will determine if T1D is modulated following T cell mediated changes in the microbiota.
Dr. Shokouh Ahmadi (Poster 8)
Postdoctoral Fellow, University of Calgary | Canada
Exploring the effects of gut microbial and viral shared epitopes in Inflammatory Bowel Disease
Shokouh Ahmadi, Isla Skalosky, Carolyn A. Thomson, Simon A. Hirota, Markus B. Geuking
Nazanin Arjomand Fard (Poster 9)
PhD Student, University of Alberta | Canada
Exploring the Role of Klebsiella variicola and Klebsiella pneumoniae in Pediatric Ulcerative Colitis Pathogenesis
Nazanin Arjomand Fard, Michael Bording-Jorgensen, Christopher Cheng, Katie Kerr, Harmol Aujla, Sarah Mansour, Wael Elhenawy, Troy Perry, Eytan Wine
Background and Aims: Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells. We isolated these strains from pediatric ulcerative colitis (UC) patients’ appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC.
Methods: K. variicola and K. pneumoniae were isolated from two pediatric UC patients’ appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) chemokines and cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).
Results: Klebsiella strains exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression.
Conclusions: K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria.
Panuya Athithan (Poster 10)
Graduate Student, University of Guelph | Canada
The impact of human milk oligosaccharides on the gut symbiont, Ruminococcus gnavus, in relation to gastrointestinal and metabolic diseases
Panuya Athithan, Emma Allen-Vercoe
Breastmilk contains high concentrations of structurally diverse oligosaccharides that constitute the third most abundant component in human milk which are known as human milk oligosaccharides (HMOs). HMOs are best known for their prebiotic effects on the infant gut microbiome and confer several beneficial functions to infant development such as aiding in commensal microbe colonization and host immune cell modulation. Due to these benefits, there has been considerable interest in the impact of HMOs on the adult gut microbiome as recent studies have shown that HMOs have the potential to restore health-associated microbial communities in individuals with gut diseases. Dominant species of gut microbes capable of HMO utilization have a growth advantage over those lacking this ability. Recent findings have shown growth advantages employed by a wide diversity of resident gut bacteria isolated from infant fecal samples in the presence of HMOs. However, several strains of bacteria across a variety of species and genera experienced HMO-induced growth inhibition. Due to this novel antimicrobial property, there is considerable interest in investigating the range of HMO-induced inhibition across pathobiont species, including Ruminococcus gnavus (R.gnavus). R.gnavus is widely distributed at low abundance in the gut microbiomes of healthy individuals. However, it is disproportionately overrepresented in gut microbiomes of individuals with chronic metabolic diseases such as inflammatory bowel disease and type 1 diabetes. Thus, the aim of this study is to investigate the impact of HMOs on R.gnavus strains, the nature of HMO inhibition, and discern intra-species variation within R.gnavus. HMO utilization was assessed by treating R.gnavus strains with pooled HMOs(pHMOs) and measuring optical density(OD600). Strain specificity detected amongst R.gnavus through their different growth properties when exposed to pHMOs. While many of the strains demonstrated enhanced growth, some strains experienced a spectrum of pHMO-induced growth inhibition. These findings advance our understanding of HMO interactions with the gut microbiota at the strain level and opens new avenues for exploring the therapeutic potential of HMOs in modulating microbial composition.
Dr. Preethi Balasundaram (Poster 11)
Scientist II, BioAro Inc. | Canada
Therapeutic Impact of Phytonutrient in the Diet on Gut Microbiota and Microbial Markers – A Case Study
Balasundarm P, Dubli K, Chaudhari R, Davila Aleman FD, Kovalchuk I, Hubert CRJ, Singh R, Kapoor A, Tripathi MB
Dr. Preethi Balasundaram (Poster 11)
Scientist II, BioAro Inc. | Canada
Therapeutic Impact of Phytonutrient in the Diet on Gut Microbiota and Microbial Markers – A Case Study
Balasundarm P, Dubli K, Chaudhari R, Davila Aleman FD, Kovalchuk I, Hubert CRJ, Singh R, Kapoor A, Tripathi MB
Food is fundamental to the health and well- being of living organisms. Humans follow a mixed variety of diet like, vegetarian, non-vegetarian, keto, vegan etc. All the diet practices utilise plant-based phytonutrients as flavouring agent, spices, or condiments or as component of salads, smoothie etc. Recognising the magnitude of phytonutrients in variety of diet, we investigated the impact of phytonutrients like phenolic acids, flavonoids, catechins and anthocyanins before and after consumption of food in the diet rich in these elements (different time points – 1 and 2). We analysed their impact on gut microbiota and various gut conditions. Statistical analyses of the samples were performed based on two variables, 1. Time point based comparison (p=0.088) and 2. sample-based comparison. Variation was observed after consumption of food rich in flavonoids and catechins (p=0.16). All subjects had reduction in constipation symptoms and bowel discomfort after consuming food rich in phenolic acids. This can be corroborated with increase in gut beneficial species majorly like Akkermensia mucinphila, Bifidobacterium longum and Faecalibacterium prasunitzii. Variation in the abundance of top 10 species in gut microbiome was observed post consumption of foods (2nd time point) containing polyphenols, anthocyanins, catechins. Food rich in polyphenols led to increase of gut health supporting bacterial species like Akkermensia spp., Bacteroides spp., Bifidobacterium spp., Roseburia spp. etc. Our analysis of case study determined the potential of phytonutrients in food to influence the health of the gut.
Sabrin Bashar (Poster 12)
PhD Candidate, University of Alberta | Canada
Longitudinal Analysis of Postnatal Hospital Length-of-stay Effects on Gut Microbiota and Subsequent Atopic Sensitization in Canadian Infants
Sabrin Bashar, Hein M Tun, Joseph Ting, Matthew Hicks, Piushkumar Mandhane, Theo J. Moraes, Elinor Simons, Stuart Turvey, Padmaja Subbarao, James Scott, Anita Kozyrskyj
This study determined whether postnatal hospital length-of-stay (LOS) following different birth modes is associated with gut microbiota pathways in 3-and 12-months infants and predicts atopic sensitization at ages 1 and 3.
The gut microbiota of 1313 infants from the CHILD Cohort Study was analyzed through Illumina 16S rRNA sequencing of fecal samples collected at 3-and 12-months. The gut microbial profiles of infants hospitalized for >1-day in vaginal delivery (VD) and ≥3-days in cesarean delivery (CD) were compared with those of infants with shorter hospital stays. Associations between hospital LOS, gut microbiota composition, and atopic (food and inhalant) sensitization were evaluated using logistic regression and mediation analyses.
At 3 months, infants with prolonged hospitalization exhibited depletion of commensal microbes Bacteroides in VD infants (p=0.03) and Bifidobacterium in CD infants (p=0.025). In VD infants without maternal intrapartum antibiotic (IAP) exposure, a longer LOS was associated with higher abundance of pathobionts Enterococcus (aOR 1.41, 95%CI 1.04-1.93) and Citrobacter (aOR1.42, 95%CI 1.04-1.94) at 3-months, and lower abundance of Bacteroidaceae (aOR0.74, 95%CI 0.54-1.01) at 3-and 12-months. Sequential mediation analysis revealed higher abundance of Enterococcus or Citrobacter at 3-months with Bacteroidaceae depletion at 12-months as a microbiota pathway from LOS to atopic sensitization at 1-year (EffectEnterococcus0.0117 bootstrap 95%CI 0.0003-0.0307 or EffectCitrobacter0.0139 bootstrap 95%CI 0.0018-0.0346) and food sensitization at 1- (EffectCitrobacter0.0141 bootstrap 95% CI 0.0016-0.0355) and 3-years (EffectCitrobacter0.0205 bootstrap 95%CI 0.0016-0.0547) in VD-noIAP infants. No microbiota mediation paths were found for CD.
In conclusion, prolonged postnatal exposure to hospital microbial environments, even without antimicrobial treatment, may lead to an overrepresentation of pathogenic bacteria and depletion of beneficial microbiota, ultimately contributing to atopic sensitization in later life.
Diya Chandrasekhar (Poster 13)
Graduate Student, Burman University | Canada
Investigating the synergistic effects of Linum usitatissimum (flaxseed) and Lonicera caerulea (haskap) supplementation on stool microbiomes, cardiometabolic disease and cognitive function
Diya Chandrasekhar, Ethan Burgess, Richard Gonzalez, Naomi Akanmori, Eva Unger, Nahi Kim, Joyce Sekleh, Matthew Unger, Peter Wass, Catherine J. Field, Philip Sherman, Devin Holman, and Pekka Määttänen
Haskap berries have been shown to decrease triglycerides and LDL cholesterol and enhance HDL cholesterol in rodent studies. Flaxseeds are known from human trials to decrease systolic blood pressure and lower LDL. Haskaps have been shown to have acute vasodilatory and episodic memory benefits in humans, and can improve endurance running performance after 6 days of feeding. Longer term trials of haskaps in humans have not been done. Flaxseeds are the highest known source of dietary lignans which when metabolized to enterolactone and enterodiol by the gut microbiota improves cardiovascular health. Additionally, flaxseeds are very high in n-3 PUFA alpha linolenic acid which has been shown to increase BDNF levels in the brain which might improve cognitive function when consumed. Both haskaps and flaxseeds contain prebiotics, and their health promoting effects may in part be mediated by their impacts on the microbiota. Here we sought to investigate possible synergies between haskaps and flaxseeds in an open-label pilot human food-based clinical trial targeting cardiometabolic disease for 8 weeks. Participants between ages 31-85 were recruited by advertising in central Alberta and screened for disease risk factors; at least 3 of the following 6 risk factors: >140mm Hg SBP, >90 mm Hg DBP, Triglycerides >200 mg/dL, HDL cholesterol <40 mg/dL, LDL cholesterol >129 mg/dL, BMI >28 kg/m2. Eligible participants were randomly assigned into three groups: Oat milk (vehicle), Oat milk and Haskap berries, and Oat milk, Haskap berries and Flaxseed. Our primary endpoints were weight, systolic and diastolic blood pressure, LDL Cholesterol, HDL Cholesterol, triglycerides, other blood metabolites including TMAO, cognitive assessments including the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and Trail Making tests, and stool microbiome and metabolomes including short chain fatty acids. Preliminary results of the trial will be presented.
Dr. Rinku Chaudhari (Poster 14)
Quality Manager & Lab Technician, BioAro Inc. | Canada
Interplay of Subgingival Microbiome, Lifestyle, Periodontal Disease and Health condition
Balasundarm P, Chaudhari R, Dubli K, Davila Aleman FD, Kovalchuk I, Hubert CRJ, Singh R, Kapoor A, Tripathi MB
Dr. Rinku Chaudhari (Poster 14)
Quality Manager & Lab Technician, BioAro Inc. | Canada
Interplay of Subgingival Microbiome, Lifestyle, Periodontal Disease and Health condition
Balasundarm P, Chaudhari R, Dubli K, Davila Aleman FD, Kovalchuk I, Hubert CRJ, Singh R, Kapoor A, Tripathi MB
Background: In close proximity with the gut, the oral cavity harbors a highly diverse microbial ecosystem due to its solid and malleable anatomical structures. Oral health is preserved by the oral microbiota in a delicate equilibrium. Poor oral hygiene and lifestyle practices that result in oral dysbiosis contribute to a variety of oral diseases and overall health of an individual. Besides, it may influence the intricate etiology of diabetes and other metabolic disorders. The underlying mechanism which links the oral microbiome dysbiosis to different diseases at molecular level remains largely unexplored.
Method: Using shotgun next-generation sequencing, we compared the oral microbiota of 20 distinct samples that varied by gender, age, and health condition to that of 50 healthy subjects from the human microbiome project.
Result: The current study suggested the observation of 533 different species representing 117 genera in 20 samples. The findings revealed that people with metabolic disorders such as obesity and diabetes had higher abundance of Veillonella puarvula, Prevotella sp., the presence of Porphyromonas gingivalis and lower abundance or absence of beneficial bacteria Streptococcus salivarius and Lactobacillus sp. Individuals with smoking habits and poor oral hygiene were also found to have Veillonella sp., which might have an impact on respiratory infections.
Conclusion: Our investigation found that few bacterial species are linked to the risk of oral disease and other metabolic disorders. According to this study on the relationship between periodontal health, diseases and the oral microbiome, the consumption of sugar, oral hygiene and diet may alter the presence of microorganisms, subsequently influencing the metabolic processes. Further investigation with larger dataset shall provide more insightful conclusion.
Christopher Cheng (Poster 15)
Research Technician, University of Alberta | Canada
Effects of indole-3-propionic acid on pathogenicity of potential pathobionts derived from pediatric patients with ulcerative colitis
Christopher C Cheng, Nazanin Arjomand Fard, Harmol Aujla, Wael Elhenawy, Troy Perry, Eytan Wine
Introduction: Indole-3-propionic acid (IPA) is a microbiome-derived metabolite of tryptophan that has been shown to elicit beneficial effects for the host. However, the mechanism of action and effects on pathogenic microbes remain unknown. Here, we assessed the potential therapeutic properties of IPA in an in vitro setting, utilizing an intestinal epithelial cell line, Caco-2, challenged with potential pathobionts isolated from non-inflamed sections of pediatric patients afflicted with ulcerative colitis (UC). We hypothesized that IPA exposure decreases the virulence of pathobionts and reduces inflammation.
Methods: Caco-2 cells were infected with Klebsiella variicola and Klebsiella pneumoniae (isolated from pediatric UC patients). Both Caco-2 and bacterial cells were maintained with or without 0.5 mM IPA for 20 hours. Gentamicin protection assay was used to quantify bacterial invasion. Gene expression of IL-8, IL-10, and TNF-α in Caco-2 cells during infection was assessed by quantitative polymerase chain reaction (qPCR). The impact of infection on barrier integrity was measured using a transepithelial electrical resistance (TEER) assay.
Results: IPA-exposed Caco-2 cells had significantly reduced invasion of UC isolates, which was further reduced when bacteria were also exposed to IPA. qPCR revealed a significant decrease in the expression of IL-8 with IPA-treated cells during infection. However, IL-10 expression had a non-significant decrease and variable TNF-α expression. A beneficial trend in tight junction integrity was observed with IPA supplementation in the TEER assay, but without significant differences.
Conclusion: These results demonstrate the relevance of IPA as a potential modifier of immune reaction and bacterial pathogenesis in Caco-2 cell lines. IPA can potentially influence multiple aspects of host cell immune functions and attenuate bacterial infection in this experimental setting. Continued study on the efficacy of IPA should be performed before the compound is to be used as a treatment for individuals with Inflammatory Bowel Diseases.
Sonia Czyz (Poster 16)
Medical Student, University of Calgary | Canada
Investigating the Role of Maternal Microbiota and Antibodies in Development of the Neonatal Immune System
Sonia Czyz, Aline Ignacio, Markus B. Geuking, Kathy D. McCoy
Introduction. Our group previously showed that the mother’s Immunoglobulin G (IgG) can bind to metabolites and products derived from the maternal microbiota, which are transferred to the offspring across the placenta and through breastmilk. These IgG-bound bacterial metabolites/products drive neonatal immune development, including the induction of group 3 innate lymphoid cells (ILC3). However, the underlying mechanisms of this transfer remain unclear. The present study aims to investigate (1) the requirement of bacterial specificity and (2) the role of the neonatal Fc receptor (FcRn), a transcytosis receptor found at maternal-offspring interfaces, for this transfer.
Methods. (1) To determine whether maternal microbial-induced IgG shows specificity to bacterial-derived metabolites/products, germ-free mice were injected systemically with auxotrophic Escherichia coli (HA107) to induce anti-E. coli IgG. Since HA107 cannot survive for more than 48 hours in vivo, the mice reverted to germ-free status. The germ-free primed mice were then monocolonized with various bacterial species to generate metabolites/products that could potentially bind to the previously induced anti-E. coli IgG. Serum from these mice was injected into germ-free pregnant dams and their offspring were analyzed for intestinal immune cell changes. (2) Germ-free FcRn-heterozygous mice breeding pairs were set up. Dams were transiently colonized with HA107 throughout gestation. Intestinal immune cell changes were analyzed in wild-type and FcRn-knockout pups.
Conclusion. The transfer of anti-E. coli IgG induced intestinal ILC3 populations in pups, even when it was associated with metabolites/products generated by different bacterial species like Enterococcus faecalis, Bacteroides caecimuris and Bifidobacterium animalis. This indicates that bacterial molecules may bind maternal IgG in a non-specific manner and that this is sufficient to transfer associated bacterial molecules to induce offspring immune cell changes. FcRn-knockout pups born to HA107-colonized dams lacked the induction of ILC3s, suggesting the FcRn facilitates the transfer of maternal IgG-bound bacterial metabolites/products to the offspring.
Dr. Vanessa DeClercq (Poster 17)
Research Associate, Dalhousie University | Canada
Body site-specific microbiomes hold predictive value for lung cancer outcomes
Vanessa DeClercq, André M. Comeau, Nidhi R Parmar, Rowan Murphy, Robyn Wright, Alison Wallace, Morgan GI Langille
Background and aim: There is a critical need to improve lung cancer outcomes and survival. Unique microbial profiles have been described in tumours and the gut microbiome has been shown to influence late-stage lung cancer survival. However, site-specific microbial implications for early-stage lung cancer outcomes and the interaction of known risk factors are largely unknown. This work aims to identify microbial biomarkers that can be used to predict lung cancer outcomes in early-stage non-small cell lung cancer (NSCLC) while considering known risk factors.
Methods: This is an observational prospective study of patients undergoing curative surgery for early-stage NSCLC with a subset treated with neoadjuvant chemotherapy plus an immune checkpoint inhibitor. In total, 600 patients will be recruited to provide detailed questionnaire data, samples from multiple body sites (stool, saliva, tumour, adjacent tissue, blood, and bronchoalveolar lavage fluid) and be followed for up to 5 years. Taxonomic composition and functional profiles will be generated using 16S rRNA gene sequencing, metagenomic sequencing, and metabolomics.
Results: Using 16S rRNA gene sequencing, our pilot data demonstrates our ability to characterize overall bacterial diversity and composition of multiple sample types and distinguishes the genera Massilia, which has recently been associated with lung cancer. Recruitment and data/sample collection is ongoing and will allow for the examination of specific microbial signatures (e.g., specific strains and functions) associated with better survival.
Conclusions: This study will provide important insights into the site-specific microbiomes and predictability of the microbiome as a robust biomarker of treatment outcomes in patients with NSCLC.
Dr. Marcel de Leeuw (Poster 18)
Head of Bioinformatics, MRM Health | Canada
Investigating the role of the small intestinal microbiome in Parkinson’s disease (SMILE study)
Marcel de Leeuw, Jean-Michel Muhlinghaus, Ludo Haazen, Katja Conrath
MRM Health, a clinical stage live biotherapeutics company, has solicited the U. of Calgary and Nimble Science (Calgary, AB, Canada) to conduct an observational cross-sectional clinical study in Parkinson’s Disease, called “SMILE” for Small Intestine and Blood fingerprint, NCT06003608, investigating the relation between the small intestinal microbiome and the disease. The study involves the use of Nimble’s investigational medical device consisting of a passive small intestine microbiome aspiration (SIMBA) capsule that is ingested orally and recovered from stool, to sample the small intestinal- and paired stool microbiome for whole metagenome shotgun (WMS) sequencing. Also, blood is collected for semi-targeted metabolomics and extensive phenotypic data is collected. The U. of Calgary and its platform involvement is triple, the PI for the study is Davide Martino, MD PhD, the W21C is managing the study and the IMC will be performing the DNA sequencing. The study has started up and encompasses patients (n=75) and neurotypical controls (n=25), who are recruited from Calgary through the Calgary Parkinsons Research Initiative (CAPRI). The study rationale and outline will be presented, as well as technological choices, with illustration of the use of large scale non-redundant gut microbe genome catalogs for WMS based microbiome analytics and exploratory statistics in discovery.
Dr. Zerihun Demissie (Poster 19)
Scientific Evaluator, Health Canada | Canada
Meta-omics based identification and functional profiling of microbial mixtures – a new frontier in risk assessment of microbial based products of biotechnology
Zerihun Demissie, Stéphane Bernatchez, Eric Cardenas-Poiré, Pedro Dimitriu, Malcolm Kendall, Azam Tayabali, Phil Shwed, Derek D.N. Smith, and Valar Anoop
Biotechnology products containing microbial mixtures (MMs) perform novel and complex tasks in a wide variety of biotechnology applications. Under the New Substances Notification Regulations (Organisms)[1], micro-organisms that are not currently on the Domestic Substances List must be tested individually for pathogenicity and toxicity. It would be cost effective to test MMs, however, it is unknown how microbial interaction affect their adverse effect to human health and the environment.
In collaboration with Innovative Solutions Canada program and Microbiome Insights, an “omics” based analysis tool was developed to identify and characterize the hazard profile of micro-organisms in commercial and mock MMs. A bioinformatic pipeline was created to analyze shotgun metagenomic datasets to determine the taxonomic composition and relative abundance of micro-organisms in MMs. The pipeline accurately identified all micro-organisms of simple mock MMs, containing up to five members, and identified 10 out of the 12 micro-organisms (83%) of the complex MMs while estimating their abundance with relative accuracy.
In vitro metatranscriptomic analysis of complex MMs exposed to different environmental stresses revealed that genes associated with antimicrobial resistance, virulence factors and toxin production were differentially expressed. In addition, co-expression analysis and module detection indicated that exposure to environmental stress tended to activate genes involved in protein production and carbohydrate metabolism. Further analysis will be conducted to determine the correlation between key genomic features and metabolomic signatures. Potential limitations (e.g. limits of detection, misclassification of metagenome assembled genomes, etc.) in using these approaches in testing MMs were also identified. Upon further validation of these results by in vitro and in vivo toxicity and pathogenicity testing methods, omics-based approaches will advance our microbial risk assessments by incorporating in silico hazard prediction outputs and, reducing reliance on animal testing and the pathogenicity/toxicity testing costs for companies.
Dr. Kirti Dubli (Poster 20)
Postdoctoral Fellow, University of Lethbridge | Canada
Understanding Impact of Skin Care Routine and Ethnicity on Skin Microbiome
Dubli K, Balasundaram P, Chaudhari R, Davila F, Kovalchuk Igor, Hubert C, Singh R, Kapoor A, Tripathi BM
Human skin and its microbiome are incessantly exposed to multiple external factors. Skin microbiome contributes to immunity and well being of the organism. The health of skin is determined by multiple factors like food habits, skin care, cosmetics, skin type, ethnicity etc. Thus, it is impertinent to understand the influence of different factors on skin microbiome. In the current investigation, we have studied 60 subjects including healthy subjects from Human Microbiome Project. Our work analysed the impact of different skin care routine – use of sunscreen and moisturisers on skin microbiome. Variation was noticed in the skin microbiome of individuals using no skin care, moisturiser (p=3.1e-5) and moisturizer with sunscreen (p=3.2e-13). Cutibacterium acnes was found to be dominant in all subjects irrespective of their skin care routine. Cutibacterium granulosum and Staphylococcus saccharolyticus were observed in skin microbiota of subjects using only moisturizer, while Staphylocccus capitis was observed in subjects using both sunscreen and moisturizers. Analysis was also conducted to compare male and female skin microbiome with respect to use of sunscreen. In females, higher variation in diversity including higher abundance of bacteria like Staphylococcus epidermidis, Malassezia restricta, and Cutibacterium acnes was observed in comparison to males which is highly significant (p=0.034). This may be attributed to enhanced skin care routine in females. Additionally, we also studied the diversity of skin microbiome with respect to ethnicity and skin type. Higher diversity of microbiota was observed in dry and Asian skin type (dry and oily). The present analysis revealed that females skin care routine with sunscreen and moisturiser use exhibits a healthier skin microbiota. Moreover, it allowed to understand the influence of these habits on skin disease microbial markers for cancer and eczema. The present evaluation shall aid in developing products to provide personalised skin care.
Saly El Salti (Poster 21)
Clinical Research Specialist, Lallemand Health Solutions | Canada
The effects of probiotics on maintenance of health in pregnancy and infants: a randomized, double-blind, placebo-controlled study (MOM study)
Saly El Salti, Marie-Laure Oula, Isaac-Jacques Kadoch, Stéphane Bronner, Sylvie Binda
Rose-Marie Fortin (Poster 22)
MSc Student, Université Laval | Canada
Investigating the composition and role of bacteriophages in an in vitro digestion system
Rose-Marie Fortin, Charlène Roussel, Vincenzo Di Marzo, Elsa Rousseau
Bacteriophages, the viruses of bacteria, are present in the same amount as their bacterial hosts in the gastrointestinal tract. They are recognized as an important modulator of the microbiome because of their obligate interaction with their bacterial hosts. Yet, they are still overlooked in most studies of the gut microbiota, which generally only focus on bacteria. In this project, we aim to characterize bacteriophage populations in an in vitro digestion system, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), and study their role on the dynamics of the microbiota. A longitudinal SHIME experiment was conducted simulating the lumina colon of two individuals for 3 weeks. An Ahiflower (omega-3) supplement was administered twice a day. To characterise the viral population, samples were taken every few days, and were ultracentrifugated before DNA extraction. We found that bacteriophage populations differ from one individual to another. There is also an important variation in time of viral relative abundances. Most bacteriophages found in our samples have not been studied before as they do not have taxonomic information, only identifier numbers. Our results show that bacteriophages are cultivated in in vitro systems. Further investigation of our ongoing project could also lead to a better understanding of the dynamics between bacteriophages and their hosts as well as the role they play in the gut microbiota.
Arash Ghasemi (Poster 23)
Graduate Student, Dalhousie University | Canada
Analyzing the Blood Microbiome’s Role in Canadian Cancer Cases
Arash Ghasemi, Vanessa Declercq, Robyn J. Wright and Morgan G.I. Langille
The human microbiome has been implicated in several types of cancer. To date, most research has focused on microbes within the gut, but more recently, the role of the blood microbiome in human health has been reported. These microbes may translocate from one region of the body to another, including the tumour site. Further research is needed to assess blood microbes as cancer prediction tools across different cancer types. In this study, we have sequenced blood microbiome samples from Atlantic Canadians who have previously been diagnosed with breast, prostate, colon, and lung cancer with the aim of assessing the relationship between the blood microbiome and cancer. Participant health data and blood samples were accessed from the Atlantic Partnership for Tomorrow’s Health (PATH) project. Cancer cases were matched 1:1 to healthy individuals based on sex, age, and BMI (n=1,014). Blood samples were analyzed by 16S rRNA gene sequencing and a QIIME2 pipeline was used for the processing, filtering, and taxonomic classification of raw reads. Current analyses being performed will account for low biomass samples, compositional data, and technical biases so that blood microbiome profiles of specific cancer types can be compared to healthy controls. Machine learning approaches will then be employed to identify bacterial biomarkers in the blood that are associated with cancer. We hope to identify robust biomarkers within the blood microbiome that may be pursued in future translational studies for early cancer diagnoses and/or a predictor of clinical outcomes.
Dr. Thaís Glatthardt (Poster 24)
Postdoctoral Fellow, University of Calgary | Canada
Malassezia restricta gut colonization effect on lung immune responses to Respiratory Syncytial Virus and post-infection asthma development
Thaís Glatthardt, Kaetlyn Phillips, William Nguyen, Ellen Ren, Emily Mercer, 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. Neonate gnotobiotic Balb/c mice colonized with bacterial consortium Oligo-MM12 alone or in combination with M. restricta were infected intranasally with RSV (Line 19 strain). A mice cohort was euthanized seven days post-infection and lungs harvested for immune response profiling. No significative differences on the immune response against RSV were noticed when mice were colonized with M. restricta. When the remaining mice reach eight weeks-old they will be submitted to the airway inflammation model, which consistis in three intranasal challenges with 100ug HDM (house dust mite) weekly. Inflammatory cells will be harvested by bronchoalveolar lavages (BAL) and lungs harvested to evaluate inflammation through flow cytometry and histology. This second timepoint will evaluate the impact of M. restricta on susceptibility to airway inflammation post RSV infection. Our results might 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.
David Good (Poster 25)
PhD Student, University of Guelph | Canada
Developing Protocols for Ethical Microbiome Research and Stewardship Among the Yanomami
David Good, Emma Allen-Vercoe, Sarah Vancuren
Microbiome research has emerged as a promising field with profound implications for human health and disease. However, its application within indigenous communities such as the Yanomami of the Amazon rainforest raises complex bioethical considerations on exploring the tensions between scientific inquiry, cultural sensitivity, protection of indigenous rights, stewardship, and equitable return of benefits. Furthermore, there are immense logistical and economic challenges to carrying out field work among communities far remote and isolated within the Amazon rainforest. The Yanomami inhabit the border regions of Venezuela and Brazil and possess unique and highly diverse microbiomes shaped by millennia of symbiotic relationships with their environment. Since many communities have limited to no foreign contact, they have maintained much of their traditional lifestyles, which include their food system of hunting-gathering and small-scale horticulture. Researchers posit that the Yanomami may possess key microbial species that play a role in preventing chronic inflammatory diseases and autoimmune disorders within their population. However, increased foreign contact and intensifying globalization may perturb the Yanomami microbiome. Understanding these microbial ecosystems and uncovering microbial species hold potential for advancing global healthcare. Yet, the pursuit of such knowledge must navigate ethical challenges rooted in colonial legacies, power differentials, and cultural considerations. Central to the bioethical discourse is the principle of respect for Yanomami sovereignty and self-determination.The objective of this study is to develop protocols for ethical research among the Yanomami in order to further investigate Yanomami gut-derived microbial strains and their potential impact on Western gut microbiota. During three major expeditions to remote Yanomami communities in Brazil and Venezuela, we have prioritized Yanomami autonomy, ensuring meaningful engagement, informed consent processes, and equitable partnerships. Throughout the research process, we have included Yanomami knowledge systems, consent protocols grounded in community values, and engaged in community-based participatory research.
Dr. Riadh Hammami (Poster 26)
Associate Professor, University of Ottawa | Canada
Probiotic extracellular vesicle-mediated inhibition of enteric pathogens
Saba Miri, Mariem Chiba, Basit Yousuf, Galal Ali Esmail, Luana Leao, Yingxi Li, Maxwell Hincke, Zoran Minic, Walid Mottawea, Riadh Hammami
This study aimed to explore the ability of probiotic candidates to control enteric pathogen infection through novel antimicrobial mechanisms. Bacterial probiotic candidates were screened in vitro against Salmonella and Campylobacter jejuni. Interestingly, extracellular vesicles (EVs) isolated from the cell-free supernatant (CFS) of strains UO.C109, UO.C121, and UO.C249 were found to selectively inhibit Salmonella or C. jejuni. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that EV production is a distinct inhibitory signaling mechanism used by these probiotic strains to control enteric pathogens.
Jerry He (Poster 27)
PhD Student, University of British Columbia | Canada
Exploring the Role of Commensal Biofilms in Gut Microbial Community Dynamics
Jerry He, Carolina Tropini
Biofilms are a sessile bacterial lifestyle prevalent in a wide range of ecological niches. Traditionally defined as multicellular aggregates immersed in a self-produced matrix of extracellular polymeric substances, these aggregates experience direct cell-to-cell contact and molecular concentration gradients, resulting in distinct phenotypes compared to planktonic cultures. In human health, gut biofilms are of particular interest due to their high microbial density and role in human health and development. While current knowledge regarding commensal gut biofilms is limited, studies have alluded to possible roles of commensal biofilms in colonization resistance, highlighting the importance of understanding how biofilms affect gut community dynamics. We hypothesize that expression of biofilm genes in the gut promotes bacterial colonization and increases community resilience to changes in the gut environment. Here, we investigate gut biofilm formation using Enterocloster bolteae, a prevalent commensal biofilm-forming bacterium. To identify potential biofilm-associated genes in E. bolteae, transcriptomics was performed on the bacterium, grown as a biofilm or planktonically. Gene frequency analysis was then used to assess for consistently differentially expressed genes within a biofilm, representing candidate biofilm-associated genes in E. bolteae. Top candidate genes were associated with processes including quorum sensing, phosphotransferase systems, sugar utilization, and carbohydrate-associated ABC transport, all of which possess known ties to biofilm formation. These genes will be validated for differential biofilm formation using knockout/knockdown mutants in E. bolteae. Altogether, this work will provide novel insights into possible roles of biofilm formation in the gut microbiome.
Chadabhorn Insuk (Poster 28)
PhD Candidate, McMaster University | Canada
DNA Metabarcoding Analyses Reveal Fine-Scale Microbiome Structures on Western Canadian Bat Wings
Chadabhorn Insuk, Naowarat Cheeptham, Cori Lausen, and Jianping Xu
Healthy wings are vital for the survival and reproduction of bats and microbiome is a key component of bat wing health. However, relatively little is known about the wing microbiomes of bats in western Canada where the white nose syndrome has become an increasing threat. Here we used DNA metabarcoding to investigate the bacterial and fungal communities on the wings of three bat species: Big Brown bat (Eptesicus fuscus), Yuma myotis (Myotis yumanensis), and Little Brown myotis (M. lucifugus) from four field sites in Lillooet, British Columbia, Canada. The bacterial 16S rRNA metabarcoding revealed a total of 4,167 amplicon sequence variants (ASVs) belonging to 27 phyla, 639 genera, and 533 known and 2,423 unknown species. The wing bacteria were dominated by phyla Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria and the most common genera were Delftia, Phyllobacterium, Bordetella, Ralstonia, Sphingomonas, Bosea, Bradyrhizobium, and Pseudomonas. The fungal ITS metabarcoding revealed a total of 11,722 ASVs belonging to 16 phyla, 806 genera, and 1,420 known and 10,302 unknown species. The wing fungi were dominated by phyla Ascomycota, Basidiomycota, and Motierellomycota and the most common genera were Cladosporium, Aspergillus, and Mycosphaerella. Principal coordinates analysis showed that both bat species and field sites contributed significantly to the diversity and distribution of bacterial and fungal communities on bat wings. Interestingly, both positive and negative correlations were found among several groups of taxa. We discuss the implications of our results for bat health, including the management of P. destructans infection and white-nose syndrome spread.
Fatimoh Kasaba (Poster 29)
PhD Student, University of Calgary | Canada
Associations between early-life gut microbiome and risk of developmental delays in infants born preterm
Fatimoh Kasaba, Emily M. Mercer, Belal Alshaikh, Thierry Lacaze-Masmonteil, Laura Sycuro, Gerald F. Giesbrecht, and Marie-Claire Arrieta
Preterm birth and the associated complications constitute a tremendous public health burden. Survivors are predisposed to neonatal conditions that increase their vulnerability to brain injury. Consequently, there is a greater risk of neurodevelopmental impairments (NDI), which persist and have long-lasting impacts on learning and activities of daily living. Some survivors, however, escape impairments for largely unknown reasons. Understanding the underlying factors could help mitigate the public health burden of preterm birth.
Early-life head circumference (HC) growth is marker of neurodevelopmental outcomes. Persistently low HC growth in early life is associated with suboptimal neuro-cognitive development. The reasons for this association and the variations in HC recovery are not well understood; but they are likely multifactorial, including biological and environmental factors.
There is growing evidence of the microbiome mediation of the bi-directional gut-brain axis, i.e. microbiome-gut-brain axis (MGBA). The MGBA is important in early life when the brain and gut microbiome undergo concurrent and interrelated developments, and it has been proposed to impact neurodevelopment. Thus, the increased risk of gut dysbiosis and brain injury following preterm birth could result in NDI. The mechanisms that drive the observed association between early-life immature microbiota and NDI in this cohort need to be elucidated. HC trajectories have been shown to differ by microbiome composition, weight gain, and nutrition, and the association between the gut microbiome and nutrition could explain the impact on neurodevelopment. Therefore, we hypothesize that the gut microbiome mediates the association between early-life HC and neurodevelopmental outcomes in early childhood. Preliminary evidence shows that attaining microbiome maturation is associated with improved HC growth trajectories. We will further scrutinize the association between the gut microbiome maturational patterns and the risk of developmental delays in preterm infants participating in the Alberta BLOOM study, as well as the factors, such as nutrition, that influence this association.
Dr. Wei Wei Thwe Khine (Poster 30)
Postdoctoral Fellow, SickKids | Canada
The environmental factors influencing the nasal microbiome in the first year of life
Wei Wei Thwe Khine, Yu Chen Qian, Jonathan Thorsen, Myrtha E Reyna, Ruixue Dai, Kelsey Fehr, Charisse Petersen, Anastasia Teterina, Maria Medeleanu, Wendy Lou, Stuart E Turvey, Piush J Mandhane, Michael G Surette, Marek Smieja, Elinor Simons, Meghan B Azad, Theo J Moraes, Jakob Stokholm, Padmaja Subbarao
The nasal microbiome during early-life is shaped by environmental exposures, maternal influences, and child characteristics. These factors impact the composition and diversity of the nasal microbiome during infancy, affecting health outcomes like asthma and allergy.
Using data from the Canadian CHILD Cohort study, we investigated the impact of older siblings, rhinovirus, and breastfeeding on the nasal microbiome of infants. We analyzed 4671 nasal swabs from 2934 children at 3 and 12 months using 16s rRNA amplicon sequencing and viral quantification.
After adjusting for other variables, alpha diversity of the nasal microbiome was significantly lower in children with older siblings, those with rhinovirus presence, and those exclusively or partially breastfed at 3 months. Sex and the Vancouver study site were positively associated with Chao1 and Shannon diversity at 3 months, respectively. Maternal education negatively impacted both diversities at 3 months. The Winnipeg study site and summer sampling were positively associated with both alpha diversities at 12 months. Other variables, including mode of delivery and prenatal/first-year antibiotic use, showed no association with alpha diversity.
Differences in beta diversity were noted in children with older siblings, rhinovirus presence, exclusive breastfeeding at 3 months, and any breastfeeding at 12 months. Moraxella species were consistently higher in children with older siblings, rhinovirus, and breastfeeding. Older siblings were positively associated with Moraxella and Haemophilus species but negatively associated with Streptococcus, Staphylococcus, and Corynebacterium species. Rhinovirus presence at 12 months was negatively associated with 10 species. The Vancouver and Winnipeg sites, with summer sampling, maternal education, antibiotics use at 12 months, and ethnicity were associated with certain few species.
During early life, the diversity and composition of nasal microbiota are influenced by older siblings, rhinovirus presence, and breastfeeding at 3 months.
Grace Kim (Poster 31)
MSc Student, McMaster University | Canada
Human Milk Oligosaccharides Utilization by Bifidobacterium: Linking Microbiota and Asthma Risk in CHILD
Grace Kim, Charisse Peterson, Kristija Sejane, Kamand Rahimi, Kennedy Spann, Stuart Turvey, Theo Moraes, Piush Mandhane, Elinor Simons, Padmaja Subbarao, Meghan Azad, Lars Bode, Michael Surette
The gut microbiota in early life significantly influences host health and the risk of chronic diseases such as atopy and asthma. The composition of the infant gut microbiota is influenced by multiple determinants such as birth mode and breastmilk feeding practices, antibiotic usage, and the environment. Numerous studies have identified specific taxa associated with protection from atopy and asthma including Bifidobacterium. Human milk oligosaccharides (HMOs) are an abundant component of breastmilk; however, they cannot be digested by the infants themselves. Bifidobacterium produce specific glycoside hydrolases (GHs) that can break down HMOs leading to the production of short chain fatty acids, which improve intestinal barrier function and immune cell development. The colonization of B. longum subsp. infantis (B. infantis) in the first year of life is predicted to play a protective role against developing asthma, intermediate wheezing, and permanent atopy (PMID: 36603585). However, there are numerous Bifidobacterium species and other B. longum subspecies that colonize the infant gut as well as strain level differences in in GHs. I hypothesize that HMO utilization will be strain specific and a mapping of GH proteins to specific HMOs degradation activities coupled with metagenomic data will identify specific protective genes. To investigate this, both phenotypic screens (HMO utilization assays) with 78 strains of Bifidobacterium and comparative genomics have been performed. Sequence similarity networks of GH proteins are being mapped to specific reactions in the degradation of different HMOs. This will allow us to use metagenomic reads from the CHILD cohort study to identify specific GH proteins (and specific strains) associated with protection against asthma. Identifying Bifidobacterium strains associated with HMO utilization and determining Bifidobacterium pathways linked to protection against asthma will provide a better understanding of the association between the early life microbiome and host health.
Meggie Kuypers (Poster 32)
PhD Student, University of Toronto | Canada
Exposome attrition leads to transgenerational alterations in adaptive immune landscape
Kuypers M, Li Z, Bayer G, Liu Z, de Amat Herbozo C, Sephton V, Copeland J, Guttman D, Philpott D, Mallevaey T
The microbiota encompasses all of the commensal microorganisms living on and within the host. It is greatly impacted by the exposome, which is the cumulative sum of all environmental exposures encountered throughout an individual’s lifetime. Beyond genetic makeup, each person’s unique and diverse history of microbial exposure shapes their immune landscape. Importantly, recent advances have shown that laboratory mice raised in environmentally restricted, specific pathogen-free (SPF) conditions possess an underdeveloped immune phenotype equivalent to human newborns, while “dirty” pet store (PS) mice that are colonized with a greater array of microbes exhibit a more mature immune system reminiscent of human adults.
SPF mice released into the natural environment are able to efficiently acquire a phenotype closely resembling free-living animals, suggesting that exogenous microbial exposure is sufficient to induce immune system maturation. Conversely, PS mice housed under environmental restriction experience a transgenerational loss of their CD44hiCD62Llo (activated/effector memory) T cell population despite possessing a diverse endogenous microbiome, with a corresponding increase in CD44loCD62Lhi (naïve) T cells. Under exposome attrition, the immune phenotype of PS mice reverts to SPF levels within four generations (PS F0 – PS F3). This is also reflected in their functional response, with diminished cytokine production from PS F3 T cells following in vitro PMA/ionomycin stimulation when compared to newly purchased PS (NPS) mice that have rich exposome exposure. This effect can be mitigated through short-term co-housing of PS F3 mice with NPS mice, providing further evidence of the importance of exogenous microbial stimulation to immune system maturation. 16S rRNA sequencing suggests that these differences may not be bacterially driven, which is further supported by introducing either SPF or wild mouse-derived bacterial communities into germ-free C57BL/6 mice. Overall, this work will enrich our understanding of microbe-immune interactions and ultimately improve the translatability of preclinical research using animal models.
Dana Lowry (Poster 33)
PhD Candidate, University of Calgary | Canada
Changes in the milk lipidome of maternal microbiota-altering diets
Dana E. Lowry, Melissa Biddle, Erin W. Noye Tuplin, Gabriel Venegas, Kate M. Sales, Adriana Zardini Buzatto, Raylene A. Reimer
Maternal consumption of highly processed foods overabundant in fat and lacking in fiber compromises maternal gut microbiota and predisposes offspring to obesity. We have shown that lactating rats consuming low-fiber diets produce offspring with metabolic propensity to obesity, whereas maternal prebiotic supplementation was protective. We hypothesize that changes in maternal microbiota and milk lipidome synergistically mediate the metabolic phenotype of offspring exposed postnatally to diets differing in fat and fiber content. Pregnant Sprague-Dawley rats were randomized to: 1) Chow control (CT), 2) low-fat, low-fiber (LF), 3) high-fat, low-fiber (HF) or 4) low-fat, inulin-enriched [IE] (20% prebiotic inulin) diets throughout lactation. Milk was collected at days 5, 10 and 21 of lactation and pup milk intake was assessed at day 14. Gut microbiota was assessed in fecal samples and the milk lipidome was characterized using liquid chromatography mass spectrometry. A total of 2171 lipid species were identified in the milk samples and both time and maternal diet altered the lipidome. At all time points, the lipidome of milk from HF and CT dams was significantly separated from LF and IE dams. Compared to CT dams, 1504 lipids significantly differed in HF dams, 887 in LF dams and 717 in IE dams. Sphingolipids, especially sphingomyelins, glycosphingolipids, and ceramides, were increased in milk from HF dams. Compared to CT dams, triacyl and diacylglycerols were altered in milk from all groups, while glycerophospholipids were down-regulated in LF and IE milk, and up-regulated in HF milk. As both energy substrates and complex signalling molecules, the lipid species present in milk may mediate the differential susceptibility of offspring to obesity due to maternal diets that alter gut microbiota. Further analysis will include milk miRNA and hormone quantification, alongside milk and gut microbiota sequencing to determine whether transferred gut microbiota or maternal milk confer an obesity risk.
Christine Macpherson (Poster 34)
MSc Student, University of Guelph | Canada
The “BumbleBiome”: Developing a Bioreactor-Based Model for the Bumble Bee Gut Microbiome
Christine V. Macpherson, Brendan A. Daisley, Sarah J. Vancuren, Dylan J. L. Brettingham, and Emma Allen-Vercoe
Bumble bees (Bombus spp.) are highly beneficial pollinator insects who, similar to humans, possess a core gut microbiota consisting of host-adapted symbionts consistently present across spatiotemporal divides. Along with similar microbiota compositions, both bumble bees and humans exhibit similar social structures and encounter the same environmental stressors inhabiting anthropogenic environments, such as antimicrobials and pollutants. However, they notably differ in their species richness, as bumble bees possess a microbiota consisting of 50-100 bacterial species, whereas humans host approximately 1000. Given this magnitude of difference, the bumble bee holobiont can serve as a model of microbe-microbe and microbe-host interactions relevant to health that are less complex to decipher than those of a human. To model these interactions, we are working to establish the “BumbleBiome”, a novel bioreactor-based model which supports the bumble bee gut microbiota. Building upon the framework of the “Robogut”, a similar human colon-based system created in the Allen-Vercoe lab, we adapted a single stage chemostat bioreactor to replicate the conditions of the bumble bee gut. Preliminary results demonstrate promising outcomes, including the establishment of a stable microbial community and the presence of core bumble bee-associated microbes. As well, analysis of 42 small molecule compounds relevant to bumble bee health suggested the presense of pollen-utilizing microbes and confirmed the fermentive capacity of microbes within the BumbleBiome system. The success of our model is promising for the advancement of microbiome research relevant to a wide variety of species. Work is currently underway to optimize a model that will be used for future studies focused on, for example, the impact of xenobiotics and nutrition on the microbiota. Overall, the establishment of the BumbleBiome offers a tractable model with potential to help with the development of novel, microbiome-based treatments to safeguard pollinators, and, therefore, ecological wellbeing and global human food security.
Isabelle Mainville (Poster 35)
Platform Manager, St-Hyacinthe Research and Development Centre | Canada
In vitro tools to study the effects of a novel probiotic on Salmonella in microbiota derived from piglet intestinal contents
Isabelle Mainville, Amély Grandmont, Mohamed Rhouma, Marie-Pierre Létourneau-Montminy, William Thériault, Yves Arcand and Alexandre Thibodeau
In vitro digestion models serve as a non-invasive tool for testing multiple hypotheses and developing new products, including feed supplements or probiotics, significantly diminishing the necessity for animal studies. Salmonella in pigs poses a significant concern for both the agricultural and healthcare sectors worldwide. Consumption of contaminated pig products could result in humans contracting salmonellosis, while certain Salmonella serotypes may induce illness in swine, causing economic losses for pig farmers. This study aimed to assess the anti-Salmonella properties of a new Bacillus-based probiotic using two in vitro models: One to generate the digested piglet feed or chyme, the second containing an intestinal microbiota derived from piglets. Two administration methods of the probiotic were evaluated: a daily dose and a continuous dose. Salmonella enumeration was conducted using BGS-rifampicin agar at time points T24, T48, T72, T96, and T120. Simultaneously, DNA extraction from bioreactor samples was carried out to analyze the composition and diversity of the intestinal microbiota using Illumina Miseq sequencing. Results showed that the chyme used to maintain the piglet microbiota was effective. Stabilization in the bioreactor was achieved within 48 hours, and could be maintained stable, in control conditions, for at least 120 hours. Probiotic addition demonstrated a reduction in Salmonella enumeration at T96 for the daily dose and at T120 for the continuous dose. Both doses of the probiotic influenced the piglet-derived microbiota significantly (p < 0.05). In summary, the Bacillus-based product tested exhibited the potential to modulate gut microbiota and diminish Salmonella colonization in a piglet-derived intestinal microbiota.
Sarah Mansour (Poster 36)
MSc Student, University of Alberta | Canada
Metabolic trade-off promotes the fitness of Crohn’s Disease-associated Escherichia coli in the gut
Mansour S, Ortiz J, Chen X, Stojic A, Tollenaar S, Bhavsar AB, Marchant DJ, Willing BP, Elhenawy W
Crohn’s disease (CD) is a chronic inflammatory condition that diminishes the quality of life of the affected patients. While the etiology of CD is undetermined, the gut microbiome is known to contribute to disease development. Adherent-invasive strains of Escherichia coli (AIEC) are frequently isolated from CD patients, and are known to drive a strong inflammatory response, propelling disease progress. Elhenawy et al. previously showed that the host environment drives the evolution of AIEC, leading to the emergence of lineages that can utilize short chain fatty acids (SCFA) more efficiently than commensals, promoting their fitness in the murine gut. Using genomics, we identified several genetic mutations in the lineages proficient in consuming SCFA. This includes a non-sense mutation in the ATPase required for importing maltose, resulting in a truncated variant of the protein, and impairing bacterial growth on this sugar. Upon restoration of maltose utilization, we observed a reduced efficiency in SCFA consumption as a carbon source. This finding suggests a trade-off between the two metabolic pathways. Using biochemical assays, we show that the evolved ATPase variant can bind to and promote the function of a key regulator of metabolism in E. coli. As a consequence, this complex drives the expression of the genes required for the import and metabolism of SCFA. Furthermore, we show that this evolved metabolic node exacerbates gut inflammation in a pre-clinical model of colitis. This work not only provides key insights into how AIEC strains promote inflammation in the gut, but also furthers our understanding of how the host environment drives the evolution of novel protein functions. This information is key to inform the search for new antimicrobial therapies that can eliminate CD-associated pathogens.
Giselle McCallum (Poster 43)
PhD Candidate, University of British Columbia | Canada
Genetic tools for the spatial characterization of gene expression in the commensal gut bacterium Bacteroides thetaiotaomicron
Giselle McCallum, Juan Burckhardt, Jerry He, Charlotte Clayton, Alice Hong, Laurent Potvin-Trottier, Carolina Tropini
The human gut hosts a diverse and dynamic collection of bacteria that are critical to our health. Changes in community composition are associated with many diseases, yet the role of individual species’ spatiotemporal dynamics remains relatively unexplored. The gut is comprised of diverse and heterogenous ecological niches ranging in oxygen levels, pH, antimicrobial peptides, and other factors that affect bacterial growth and function. Characterizing spatial differences in bacterial abundance and function is critical to understand the link between health and microbiota composition and will be critical for the implementation of emerging technologies such as engineered probiotics or targeted therapeutics. As tools to measure bacterial function in situ with spatial resolution are lacking, we aim to develop a biosensor that reports host cell gene expression levels via fluorophore expression to characterize spatial differences in gene expression in the human commensal bacterium Bacteroides thetaiotaomicron (B. theta) using confocal microscopy. To this end, we have engineered several repressible promoters that can drive fluorophore expression when activated, which form the basis of a modular B. theta-compatible transcriptional reporter to quantify expression from native B. theta promoters. Preliminary biosensors reporting expression from an oxidative stress-induced promoter (PahpC) have shown detectable changes in GFP (~5-fold change) in response to peroxide-induced oxidative stress in vitro. We are now validating this reporter in an in vivo mouse model, in which we will characterize differences in oxidative stress response throughout regions of the gut, as a function of distance from the epithelium, and in models of inflammation. In the future, this modular reporter will allow the interrogation of B. theta gene expression levels throughout the gut and under various perturbations to the environment, ultimately helping to characterize the bacterial microbiome’s dynamic response to the diverse and varying biogeography of the gut.
Ailidh McGonigle (Poster 44)
MSc Student, University of Calgary | Canada
The Impact of Intestinal Microbiome-Derived Metabolites on T cell development
Ailidh McGonigle, Isla Skalosky, Shokouh Ahmadi, Markus Geuking
Many mucosal surfaces, such as the skin and intestines, are colonised by microbes. These microbes are known as our microbiota and can have both beneficial and harmful effects. The intestinal microbiota is the most well studied microbiota. Intestinal mucosal surfaces are colonised from birth, and this microbiota has been shown to have effects on the immune system. For example, immune maturation during early life and immunoregulatory activity have been demonstrated to be affected by the intestinal microbiota. Microbiota-derived metabolites mediate effects of the microbiota on the immune system. A major component of the adaptive immune response are antigen specific T cells. These cells develop in the thymus. Abnormalities during this development in the thymus can result in, for example, autoimmunity, which contributes to the development of autoimmune disorders. T cells which have most recently left the thymus are known as recent thymus emigrants (RTEs). RTEs have been shown to be the preferential precursor of regulatory T cells (Tregs), which are important for preventing the development of autoimmune disorders. The effect of the intestinal microbiota on T cell development and RTE activity is not well understood. In this project, the effect of microbiota-derived metabolites on thymic function, T cell ontogeny, and the ability of RTEs to acquire a regulatory phenotype will be examined. To achieve this, mouse models with either no intestinal microbiota (germ-free) or intestinal microbiotas of varying complexity (OMM12, SPF, and re-wilded) will be used in combination with in vitro organotypic thymic cultures and T cell differentiation cultures. Understanding the effect of the intestinal microbiota on T cell development and RTE activity may uncover novel targets for the prevention or therapeutic intervention of autoimmune disorders.
Emily Mercer (Poster 45)
PhD Candidate, University of Calgary | Canada
Development of the bacterial and fungal gut microbiome in premature infants in relation to experiences of early-life stress
Emily M. Mercer, Sarah Piché-Choquette, Shirin Moossavi, Belal Alshaikh, Thierry Lacaze-Masmonteil, Laura Sycuro, Gerald F. Giesbrecht, and Marie-Claire Arrieta
Introduction: The gut microbiome encompasses a multi-kingdom population of microbes that communicate bi-directionally with the brain and have broad influences on development. Animal models have revealed a causal relationship between the microbiome and dysregulated stress physiology in early life, but clinical research remains lacking. Preterm infants exhibit distinct microbiome profiles and experience significant early-life stress in the NICU, suggesting altered gut-brain communication may play a unique developmental role in this population.
Methods: Gut microbiome maturation patterns, cortisol levels, and exposure to early-life stress were evaluated in 105 preterm infants from the Alberta BLOOM Study using stool and urine samples collected over the first 8 weeks of life. Clinical factors (e.g., nutrition, medications) were recorded from hospital charts. Bacterial and fungal microbiome composition were evaluated by Shallow shotgun and ITS2 sequencing, respectively. Urine cortisol was measured by ELISA. Ecological and multivariate analyses were performed using RStudio.
Results: The gut microbiome and cortisol release in preterm infants exhibits distinct maturational patterns depending on gestational age at birth, with infants born extremely preterm exhibiting lower bacterial alpha diversity, higher relative abundances of opportunistic microbes (e.g., Klebsiella, Escherichia, and Candida), and elevated urine cortisol levels relative to very and moderate-late preterm infants. However, differences in alpha diversity metrics and urine cortisol were no longer detected between prematurity categories by weeks 7-8 of life. Generalized linear mixed modeling revealed bacterial alpha diversity was positively associated with antenatal corticosteroids and probiotics use and inversely associated with stress-inducing procedures, whereas fungal alpha diversity was positively associated with stress-inducing procedures.
Discussion: Our findings suggest a dynamic relationship exists between gut microbiome maturation and factors that may influence stress response physiology in preterm infants, with the number of stress-inducing procedures experienced in the first 8 weeks of life being linked to both bacterial and fungal microbiome composition.
Fynn Mitchell (Poster 46)
Graduate Student, University of Calgary | Canada
Microbial Metabolites as Mediators of Regenerative Homeostasis in Inflamed Intestinal Epithelium
Sara Zbinden, Fynnis Mitchell, Sarah Vancuren, Alexander Botschner, Emma Allen-Vercoe, Arshad Ayyaz
Crohn’s Disease (CD) and Ulcerative Colitis (UC), collectively known as inflammatory bowel disease (IBD), significantly impact the global population, affecting over 300,000 Canadians and approximately 10 million people worldwide. IBD not only diminishes the quality of human life but also increases the risk of developing colon cancer. Unfortunately, current therapies merely alleviate symptoms without offering a long-term cure. Fecal microbiota transplantation (FMT) has emerged as a potential therapeutic approach to induce remission in IBD patients, yet it carries safety concerns, necessitating a deeper understanding of its impact on the metabolic and cellular processes affected by transplanted microorganisms. We analyzed publicly available single-cell RNA-seq datasets from both inflamed and non-inflamed intestinal regions of two human IBD patient cohorts and compared them with those from healthy individuals. This analysis revealed fundamental differences in the epithelia of IBD patients and healthy volunteers, regardless of inflammatory status. Notably, the gut epithelia of IBD patients exhibit enhanced transcriptional activity and replication stress, and a fetal-like regenerative program that we previously found can regenerate the intestinal epithelium following inflammation. To further explore this, we adopted a recently developed 3D organoid-based model that replicates the hypertranscription, hyperproliferation and colitis-associated regenerative program seen in IBD. We then assessed how these organoids are impacted by the metabolites produced by Robogut, an artificial gut simulator developed by Dr. Allen-Vercoe to culture and study complex microbial communities under conditions that closely mimic the human gastrointestinal tract. Our results show that supernatants of healthy gut microbiota significantly reduce hypertranscription and replication stress while maintaining the stem cell compartment. We are currently generating metabolic and single-cell transcriptomic profiles of both mock and supernatant-treated organoids, to identify novel therapeutic targets that could mitigate IBD and enhance existing treatment modalities. An update on these ongoing studies will be provided at the IMPACTT 4 symposium.
Hana Olof (Poster 47)
MSc Student, University of Manitoba | Canada
Dietary fiber physiochemical properties are impacted by exposure to cooking temperatures, resulting in reduced inflammatory response to unfermented fibres in inflammatory bowel disease
Hana Olof, Athalia Voisin, Linda Ho, Christopher Song, Jun Gao, Thava Vasanthan, Heather K. Armstrong
High-fiber diet is thought to promote gut health in inflammatory bowel disease (IBD). However, some IBD patients express intolerance of fibers. We found if gut microbiota are unable to ferment fibres, unfermented β-fructan fibers can induce gut damage and inflammation, while select b-glucans and pectins have null or anti-inflammatory effects. The traditional healthful Ethiopian beverage, Kineto, is made from straining (room temperature) or cooking barley (b-glucan) and citrus fruits (pectin). Temperature exposure is known to impact dietary fibre physiochemical properties and dietitians observe cooking high-fibre foods improves tolerance in IBD. I hypothesize exposing Kineto to varying temperatures alters the physiochemical properties of its fibre components, impacting its effects on gut immunity.
Kineto (K) was produced at varying temperatures (room temp, 70°C, 150°C). Fiber concentrations and physiochemical properties were determined using AOAC methods and Megazyme kits. Gut biopsies (non-IBD n=5; IBD n=5) were cultured ex vivo with KRT, K70, and K150 or the separate fibre components (barley, citrus peel, individual fibres). Inflammatory secretions were measured in supernatants (ELISA and Olink). Biopsies were used to examine gut damage (H&E), and inflammatory markers associated with response to fibres in our prior studies (microscopy; e.g., CD45, TLR2, mitochondrial dysfunction, NLRP3 inflammasome).
Inflammatory secretions (e.g., IL1B, IL23) and tissue markers (H&E, microscopy) were significantly decreased in response to K70 and K150 compared to no fibre control. When constituents of K were analyzed, components containing greater β-glucan (1-3.5 pg/ml), and lower phenolic content (0.14-0.17 pg/ml), induced pro-inflammatory response (p<0.05; one-way ANOVA), while anti-inflammatory effects were associated with higher pectin and phenolic content, and lower arabinoxylan (P>0.05).
Exposing high-fibre foods to temperatures reflective of cooking can reduce inflammatory response, due to changes in fibre physiochemical properties. Cooking high-fibre foods may improve health benefits in IBD patients whose gut microbiota does not support fibre fermentation.
Dr. Mona Parizadeh (Poster 48)
Postdoctoral Fellow, University of Calgary | Canada
The Early-life Ecological Dynamics of a Rural Infant Microbiome
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 at birth, displaying high heterogeneity until age 2-3. Social geography significantly influences the gut microbiome impacts on human development. Research shows infants raised in rural areas have greater microbial richness than urban infants, yet we know little about early-life gut microbes in less industrialized regions. The composition of non-bacterial communities is also poorly understood despite their importance in host crosstalk. To address these gaps, we used shotgun metagenomic and 18S rRNA sequencings to characterize bacterial and eukaryotic microbiomes in fecal samples collected monthly – from birth to 18 months – from a longitudinal human cohort of nine children in Morelos, Mexico (average 14 samples/ child). Our results showed significant temporal changes in both bacterial and eukaryotic composition and alpha diversity. Proximity to animals also emerged as a significant determinant shaping the early-life gut microbial community composition. Additionally, the mode of birth notably affected the infants’ gut bacterial composition and diversity over time, with vaginally born infants exhibiting higher gut bacterial alpha diversity than those born via C-section. The abundance of bacterial genera Bifidobacterium and Blautia significantly increased with age, while the fungal genus Malassezia temporally decreased. This study provides insights into the contribution of environmental factors and temporal dynamics to shaping the early-life gut microbiome in rural areas. These findings advance our understanding of gut microbiome development, which could have implications for human health. Further research is required to understand the early-life gut microbial interactions and the ecological processes that drive them.
Kaetlyn Phillips (Poster 49)
Graduate Student, University of Calgary | Canada
The Role of Preterm Infant Gut Bifidobacterial Abundance on Neurodevelopment
Kaetlyn Phillips, Simrit Rai, Dr. Van Ortega, Dr. Marie-Claire Arrieta
Preterm birth makes up 8% of live births in Canada; A proportion of these infants display slower rates of head growth clinically associated with neurodevelopmental disabilities. The gut microbiome is a proposed contributor to head growth rates through bidirectional communication networks between the gut, the microbiome, and the central nervous system. Preliminary evidence from our lab suggests that preterm infants with an immature microbiome (<60% bifidobacterial relative abundance) display slower head growth, and that microbiome maturation (shifting towards >60% bifidobacterial relative abundance) driven by probiotic administration is associated with accelerated head growth. However, the causal mechanisms of these connections are unknown, partly due to a lack of randomized control trials and experimental evidence in animal models. Here, germ-free C57Bl/6 dams were inoculated with pooled preterm fecal slurries characterized as either mature (>60% bifidobacterial relative abundance) or immature (<5% bifidobacterial relative abundance). After delivering F1 litters, dams received nipple spreads of the same fecal slurry on post-natal day 2 (P2) and P4 to encourage engraftment of the communities of interest in the pups. The dams also received nipple spreads of a commercial probiotic consortium containing Bifidobacterium longum sbsp. infantis or PBS every 48h from P4 to P19. At P21, F1 fecal samples, body weights and forebrains were collected. Fecal samples will be analyzed by qPCR and 16S sequencing to quantify relative and total abundances of key taxa. Mouse body weight will be a proxy for human head growth to compare growth trajectories. Immunofluorescent imaging of cerebral cortex and hippocampal brain tissue will explore patterns in neuronal maturation, synapse distribution and myelination. This work will establish a novel bifidobacterial colonization model in mouse pups and develop mechanistic insights into the interactions between microbiome maturity and brain development in the preterm population, which could inform future NICU intervention strategies,
Dr. Leila Rezaei (Poster 50)
Postdoctoral Fellow, University of Calgary | Canada
Investigation of Steroid Metabolism in the Gut Microbiome
Leila Rezaei, Marcel van de Wouw, Marie-Claire Arrieta, Lianne Tomfohr-Madsen, Catherine Lebel, Gerald Giesbrecht
Introduction: Current evidence suggests that most human gut microbiomes can metabolize steroids like cortisol. This finding is significant as stress exposure elevates stool cortisol levels, implying a potential avenue for host-microbe interactions in stress-related diseases. Enzymes that are associated with cortisol and are encoded by bacteria, such as Clostridium scindens, are not typically abundant or are found in limited quantities. Consequently, the mechanisms enabling most microbiomes to metabolize steroids remain unclear. This project aims to further identify and characterize steroid metabolizing enzymes.
Method: Data were collected from the Pregnancy During the COVID-19 Pandemic Cohort, where stool samples were obtained during the 3rd trimester of pregnancy using stool collection kits (n=502). Metagenomic shotgun sequencing was employed to assess gut microbiome composition and enzymes, while untargeted metabolomics analysis was conducted to quantify cortisol, the upstream metabolite 11-deoxycortisol and downstream metabolite cortisone. To investigate if stress exposure was linked to stool cortisol levels, we quantified Pandemic Objective Hardship (POHI) exposure, which measures stressful events that were caused by the pandemic.
Results: POHI correlated positively with stool cortisol levels (rs = 0.133). We observed that only Blautia obeum correlated with cortisol (rs = 0.179), whereas cortisone correlated with 27 different bacterial taxa. EC number: 1.3.99.4 which has been shown to degrade steroids like cortisone was detected in some of the bacterial taxa that correlate with cortisone (e.g., Eggerthella lenta; rs = -0.135).
Conclusion and future directions: The results of our work provide novel insights into the ability of the gut microbiome to metabolize steroids such as cortisol and its related metabolites. This significantly contribute to our understanding of host-microbe interactions, particularly in stress-related contexts. Our ongoing research suggests that there are numerous other less-established gut microbial enzymes that potentially capable of metabolizing steroids.
Dr. Jennifer Ronholm (Poster 51)
Associate Professor, McGill University | Canada
Optimizing the Bovine Udder Microbiome using Probiotics to Reduce Agricultural use of Antibiotics
Jennifer Ronholm, Soyoun Park, Dongyun Jung, Bridget O’Brien, Daryna Kurban, Zhangbin Cai, Sam Nguyen, Simon Dufour
Bovine mastitis is one of the most common and costly diseases in the dairy industry resulting in decreased milk production and quality, expensive antibiotic-based treatments, and culling of chronically infected cows. This disease causes an annual economic loss of $665 million CAD in Canada. It is an extremely complex disease and 137 different species of bacteria have been demonstrated to cause mastitis. To control mastitis, on average, cows on Canadian dairy farms receive 3.3 defined course doses/year of antibiotics – including third generation cephalosporins. In this project we aim to define the optimal mammary microbiome composition to prevent mastitis and isolate potential pathogen-antagonists from healthy cattle, so that probiotics can be developed to mitigate antibiotic use. We collected about 27,000 milk samples from 689 dairy cattle every 2 weeks for 16 months. If a cow developed clinical mastitis from Staphylococcus aureus, Escherichia coli, or Klebsiella pneumoniae each milk sample collected from that individual was analyzed via 16S rRNA targeted amplicon sequencing. Some samples were also selected for shot-gun metagenomics. Network analysis determined that the presence of Aerococcus urinaeequi and Staphylococcus xylosus were negatively correlated with S. aureus clinical mastitis. Aerococcus spp., UCG-005, and Lachnospiraceae were negatively correlated with K. pneumoniae clinical mastitis; and the presence of non-aureus staphylococci, A. urinaeequi, and Serratia marcescens were negatively correlated with E. coli clinical mastitis. Several A. urinaeequi and S. xylosus isolates from healthy cattle inhibited the growth of S. aureus in co-culture. A probiotic product with the strains above may mitigate the use of antibiotics in the dairy industry. In addition, this could be a proof of concept that could lead to development of additional preventive products.
Dr. Elsa Rousseau (Poster 52)
Assistant Professor, Université Laval | Canada
Artificial intelligence to predict gut bacteria and phages biomarkers of host nutrition and health
Alexandre Boulay, Sasha-William Ménard-Mérette, Marie-Claude Vohl, Elsa Rousseau
The gut microbiome is recognised as an important modulator of the effects of nutrition on health. Many nutritional factors influence the balance of the gut microbiota, leading in some cases to its deregulation, called a dysbiosis, impacting host’s health. Up to now, bacteria were the most studied organisms within the microbiome. Their viruses, called phages, outnumber them in any environment and are increasingly recognized to shape the microbiome because of their obligate interaction with bacteria. Nevertheless, phages are still one of the most poorly understood members of the microbiota. My research program aims to shed light, through artificial intelligence approaches, on the role of phages in the microbiome and the complex interrelations between bacteria, phages, and host’s nutrition and health. In this presentation, I will show recent results from my research team on the prediction of phages and bacteria biomarkers of host’s nutrition. We have analysed several whole-metagenomics shotgun sequencing datasets from the Institute of Nutrition and Functional Foods (INAF) of Université Laval, coming from nutritional intervention studies. We have identified gut bacteria and phages in these datasets through their processing in bioinformatic pipelines, and trained interpretable machine learning algorithms to predict the host’s status (pre- or post-nutritional intervention). Interpretability is key for the prediction of bacteria and phages biomarkers of a host’s phenotypic signature, as well as in the context of applications in health, where experts must understand and validate the reasons behind a model’s predictions. Our first results show common bacteria and phage biomarkers between the best models, indicating potential robust signatures of the effects of the nutritional interventions on the gut microbiome.
Mahana Sabachvili (Poster 53)
PhD Student, University of Tübingen | Germany
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 the gut microbiota. Indeed, a subgroup of IBD patients responds to microbial manipulation therapies such as fecal microbiota transplantation (FMT). We hypothesized that specific bacteria drive inflammation in a subset of IBD patients. We aim at identifying these bacteria and understanding the underlying mechanisms how they do so. In the future, this may lead to microbiome-based precision therapies in IBD.
To tackle our hypothesis, we first recruited IBD patients that showed reduced gut inflammation post-FMT. Germ-free IBD susceptible mice were then colonized with stool samples collected from patients pre- and post-FMT. Six weeks after colonization, gut inflammation was assessed by histology. Moreover, immune responses were analyzed in the mesenteric lymph nodes and the large intestine by flow cytometry, and the intestinal content was collected to culture and identify 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 and identified four putative pathobionts.
To determine their pathogenic potential, IBD susceptible mice were monocolonized with each bacterium and gut inflammation was assessed six weeks later as described before. Our data indicates that two of these bacteria induce an inflammation in the large intestine as assessed by histology and flow cytometry.
Together, our data suggest that patient-derived microbiomes have different inflammatory potentials, and we developed an approach to identify specific inflammation-inducing bacteria.
Amy Shupe (Poster 54)
MSc Student, University of Calgary | Canada
Unravelling the mechanisms of microbiome modulation in cancer immunotherapy
Amy Shupe, Larisa Kovtonyuk, Fabien Franco, Kathy McCoy
The microbiome has been shown to modulate the efficacy of cancer immunotherapy, such as immune checkpoint blockade therapy (ICBT) and chimeric antigen receptor (CAR)-T cell therapy. Gut microbiota signatures have also been associated with immune related adverse events (irAEs) in response to ICBT and cytokine release syndrome in CAR-T cell therapy. This highlights the potential for harnessing the microbiome to improve cancer immunotherapy and reduce adverse events. Although the mechanisms by which the microbiome modulates the efficacy of cancer immunotherapy are still under study, microbiome-secreted metabolites have been found to play critical roles. Our aim is to elucidate the mechanisms by which microbial produced metabolites modulate cancer immunotherapy and irAEs.
We have previously found that the bacterial metabolite inosine enhances ICBT efficacy in a context dependent manner. We are now investigating whether inosine can also modulate the efficacy of CAR-T cell therapy. Pre–treatment of human CAR-T cells with inosine, but not the short chain fatty acids butyrate or pentanoate, was found to improve the killing of a Nalm6 human lymphoma cancer cell line in vitro. We will now determine whether CAR-T cells treated with inosine in vitro also show enhanced killing of tumor cells in vivo in a Nalm6 lymphoma bearing germ free xenograft mouse model.
Mice housed under specific pathogen-free (SPF) conditions do not develop irAEs in response to ICBT. As mice colonized with a wild microbiome have been shown to better model the human immune system, germ-free mice will be colonized with a wild or SPF microbiome and the development of irAEs will be evaluated after treatment with ICBT. This project will uncover the interplay between the microbiome, metabolites, and cancer treatments, aiding novel microbial therapeutic design for enhanced efficacy and reduced toxicity of cancer immunotherapies.
Claire Sie (Poster 55)
PhD Student, University of British Columbia | Canada
Early life inflammation affects endocrine and microbiota development in a mouse model
Claire Sie, Olivia Sullivan, Katharine M. Ng, Sophie A. Cotton, Cal Rosete, Jordan E. Hamden, Jatin Choudhary, Annie Vogel Ciernia, Carolina Tropini
Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions that affect over 7 million people worldwide and currently have no cure. Alarmingly, 25% of diagnoses occur during childhood or early adolescence, with these young patients experiencing a more severe disease course than patients diagnosed in adulthood. Patients with IBD also often experience extraintestinal manifestations of disease – particularly endocrine comorbidities such as decreased serum hormone levels, hormone-related mood disorders, and delayed puberty. Although it is known that the gut microbiome is one of the drivers of IBD pathogenesis, it is only recently that the role of the microbiome in regulating hormone levels has been investigated in the context of human health and disease. Likewise, the role of the pediatric IBD microbiome in driving endocrine comorbidities has yet to be fully understood. In this study, we utilize the chemical colitogen, dextran sodium sulphate (DSS), to model repeated cycles of early life gastrointestinal inflammation and remission, as well as associated extraintestinal comorbidities. First, we confirmed that DSS administration resulted in colitis by observing weight loss and histological changes to the intestinal epithelium. We then demonstrated that administration of DSS during early life developmental periods in mice decreases seminal vesicles weight, serum androgen concentrations, and alters specific mate-seeking behaviours. Additionally, DSS-induced early life inflammation resulted in both short- and long-term changes to the gut microbiome – specifically, decreases in the relative abundance of anti-inflammatory short-chain fatty acid-producing bacteria. Strikingly, several of these taxa correlated with serum androgen concentrations and are known to express β-glucuronidases, which are bacterial-derived enzymes that can re-activate hormones tagged for excretion. Overall, this study serves as a critical first step into defining the gut microbiome’s role in hormone-related comorbidities in pediatric IBD.
Isla Skalosky (Poster 56)
PhD Candidate, University of Calgary | Canada
Impact of Intestinal Microbiota Complexity and Antigen Mimicry of a Viral Epitope on Anti-viral T Cell Responses
Isla Skalosky, Shokouh Ahmadi, Marcela Davoli Ferreira, Regula Burkhard, and Markus B. Geuking
It is well known that the intestinal microbiota, mainly via its products and metabolites, impacts the development and regulation of not only mucosal immunity but systemic immunity. Diverse commensal colonization of the gut is essential to health, with dysbiosis linked to various immune-mediated diseases. The microbiota has also been linked to anti-pathogen immunity, affecting host susceptibility to infectious diseases. However, what role the intestinal microbiota plays during systemic anti-viral immunity is less clear. To investigate the intestinal microbiota’s contribution to anti-viral T cell immunity, we have established a systemic lymphocytic choriomeningitis virus (LCMV) infection model in germ-free and gnotobiotic mice colonized with a precisely defined microbiota. Anti-viral T cell immunity is characterized by high throughput flow cytometry with tetramer staining to measure epitope-specific T cell responses and intracellular cytokine staining to measure cellular activity. Preliminary results reveal that anti-viral T cell responses of germ-free mice are significantly improved with gnotobiotic colonization.
Moreover, microbial metabolites are not the only microbe-derived factor influencing anti-viral T cell responses. Due to the large diversity of bacterial species present in the gut microbiota, there is a large repertoire of antigens expressed, particularly T cell epitopes, that are similar or identical to host auto-antigens. These shared epitopes are referred to as antigen mimicry. Microbial mimicry of self-antigens can be either beneficial or detrimental to autoimmunity. So far, investigations of antigen mimicry have focused on self-antigens, leaving events of viral epitope mimicry and consequences to T cell immunity relatively unstudied. We therefore have monocolonized mice with either a wild-type or genetically modified Escherichia coli strain expressing an LCMV-derived epitope. Preliminary data shows that the presence of a molecular mimic within the microbiota significantly attenuates systemic anti-viral T cell activity at the level of interferon production, possibly through the induction of epitope-specific anergic T cells.
Dr. Matthew Sorbara (Poster 57)
Assistant Professor, University of Guelph | Canada
Mechanistic analysis and genetic manipulation of non-model gut Lachnospiraceae reveals pathways to produce key microbiota metabolites
Sim M., Fitzgerald B., Moore A., Sorbara M.T.
Dr. Yves St-Pierre (Poster 58)
Professor, INRS-Centre Armand-Frappier | Canada
Exploring the Microbiome: Bridging Marine Biology and Human Dysbiosis
Yves St-Pierre, France Caza, Sophia Ferchiou, Fanny Fronton, Dominique Robert
Recent studies have highlighted intriguing parallels between the marine and human microbiomes, suggesting potential cross-kingdom interactions and shared mechanisms governing microbial community dynamics. Thus, investigating the marine microbiome has the potential to shed light on the factors influencing human dysbiosis.
In recent years, our team has established a new research program to study the microbiomes of various aquatic and marine species. We are particularly interested in intricate interactions between microbes, their hosts and their ecosystems. Understanding these interactions in natural habitats and controlled settings can provide valuable insights into how the changing environment impacts dysbiosis-associated diseases and the emergence of pathogens. Changes in the microbiome composition can indicate stress, disease, or environmental disruptions. Early detection of these changes can warn of potential problems within the ecosystem before they become severe.
In this communication, we will present key findings that enhance our understanding of the response of marine species to their environment. For our research, we have adapted the concept of liquid biopsy to marine species, with particular attention to cell-free microbial DNA. Specifically, we will delve into the circulating microbiome of ecologically significant marine organisms and present our findings on the influence of environmental factors and pollutants, including endocrine disruptors, on the circulating microbiome. Additionally, we will highlight the development of logistically friendly, low-cost, and ethical methodologies tailored for studying circulating microbiomes in remote polar regions and discuss their adaptability to different high-throughput multi-omics platforms.
Our results advocate integrating marine biology and human microbiome research to advance our understanding of dysbiosis. By embracing interdisciplinary approaches, we can harness the knowledge and techniques developed in marine microbiome studies to unravel the complexities of dysbiosis in humans and vice-versa. This, in turn, can pave the way for prevention approaches and innovative therapeutic strategies.
Dr. Marcel van de Wouw (Poster 59)
Postdoctoral Fellow, University of Calgary | Canada
The Impact of Blue and Green Spaces on the Infant Gut Microbiome and Developmental Outcomes
Marcel van de Wouw, Samarpreet Singh, Leila Rezaei, Marie-Claire Arrieta, Anita Kozyrskyj, Lianne Tomfohr-Madsen, Catherine Lebel, Gerald Giesbrecht
Introduction: Contact with natural environments like native vegetation is linked to a reduced infant gut microbial diversity or dominance of key species and subsequent reduction in allergy sensitization. However, it is unclear if other types of natural environments (e.g., water bodies – blue spaces) confer similar effects on child development, and whether natural environments affect other child outcomes. This project aims to investigate how exposure to different types of natural environments (i.e., blue & green spaces) associate with the infant gut microbiome and subsequently developmental outcomes.
Methods: Data are from the pan-Canadian Pregnancy During the COVID-19 Pandemic Cohort. Green space (normalized difference vegetation index (NDVI) & tree canopy coverage (TCC)) and blue space (distance to closest water body) data were obtained from CANUE and linked to participants via 6-digit postal codes. Stool samples were collected at 3 months of age to analyze gut microbiome composition using metagenomic shotgun sequencing (n=311). Child development was measured with a multi-trait approach using the Ages & Stages Questionnaires (ASQ-3) at 12 months of age.
Results: Microbial Chao1 diversity correlated negatively with green space measures (NDVI: rs=-0.172; TCC: rs=-0.184), but positively with blue space proximity (rs=0.118). Even though no taxa correlated with blue spaces following a correction for multiple testing, green spaces were negatively associated with 15 bacterial taxa. Of these taxa, Ruminococcus bromii, Faecalibacterium prausnitzii and Prevotella copri were negatively linked with Communication scores (rs=-0.156; rs=-0.151; rs=-0.140, respectively) and Problem-Solving scores (rs=-0.133; rs=-0.124; rs=-0.127, respectively), whereas Alistipes putredinis and Fusicatenibacter saccharivorans were only negatively linked to Communication scores (rs=-0.120; rs=-0.119, respectively).
Conclusion: These data reveal that the previously established link between green spaces and reduced infant gut microbial diversity does not extend to blue spaces. Furthermore, green spaces may positively impact Communication and Problem-Solving scores by reducing the relative abundance of specific bacterial taxa.
Dr. Joongjae Kim (Poster 60)
Chief Technology Officer, Nimble Science Ltd. | Canada
Multi-Omics Mapping of Spatial Differences Along the Gastrointestinal Tract with the SIMBA Capsule: Benchmarking Against Endoscopy and Clinical Applications
Gang Wang, Cedoljub Bundalovic-Torma, Oksana Lukjancenko, Sharanya Menon, Heidi H. Hau, Sabina Bruehlmann, Lynn Wilsack, Renata Rehak, Sahar Bagheri, Mohammad M. Banoei, Lawrence Lou, Yasmin Nasser, Matthew Woo, Ian Lewis, Kathy D. McCoy, Christopher N. Andrews
Dr. Joongjae Kim (Poster 60)
Chief Technology Officer, Nimble Science Ltd. | Canada
Multi-Omics Mapping of Spatial Differences Along the Gastrointestinal Tract with the SIMBA Capsule: Benchmarking Against Endoscopy and Clinical Applications
Gang Wang, Cedoljub Bundalovic-Torma, Oksana Lukjancenko, Sharanya Menon, Heidi H. Hau, Sabina Bruehlmann, Lynn Wilsack, Renata Rehak, Sahar Bagheri, Mohammad M. Banoei, Lawrence Lou, Yasmin Nasser, Matthew Woo, Ian Lewis, Kathy D. McCoy, Christopher N. Andrews
OBJECTIVE: The small intestine (SI) plays a central role in uptake of nutrients and dysbiosis of its microbiome has been implicated with significant impairments in normal gastro intestinal (GI) function. However, owing to its relative inaccessibility and lack of a widely-applicable standard sampling techonology, the SI microbiome still remains relatively little unexplored.
METHOD: The Small Intestinal MicroBiome Aspiration Capsule (SIMBA) was developed as an effective means for sampling SI luminal content and platform for multi-omic microbiome analysis. SIMBA capsule SI targeting, sampling sealing, and preservation performance was evaluated in a two-visit observational clinical study of healthy and Irritabe Bowel Syndrome (IBS) patients using X-ray tracking. Detailed assessment of SIMBA capsule sample quality was also performed against paired-fecal and ‘gold-standard’ duodenal endoscopy sampling using 16S amplicon and shotgun metagenomic sequencing, and metabolomics.
RESULTS & CONCLUSIONS: X-ray monitoring confirmed all capsules sampled from the distal small intestine with a few (5 of 49) sealing in the proximal colon. 16S amplicon and metabolomics analysis demonstrated that SIMBA capsules were significantly different to fecal samples, and similar to endoscopic aspirate and cytological brush sampling. Shotgun metagenomics demonstrated that despite the low-biomass of SI samples, SIMBA capsules retained a minimal level of host contamination, in comparison to endoscopic aspirate and cytological brush samples (~5 % of vs. ~ 93 – 96% of average total reads per sample). Increased resolution and sequencing depth enabled GI regional-specific differences in known probiotic strains to be traced, as well as functional differences in microbiome-mediated bile acid metabolism and short-chain fatty acid production, demonstrating the great potential for the SIMBA capsule for high-resolution spatial characterization of the GI microbiome with a wide range of applications from disease to nutraceutical research.
Ray Wang (Poster 61)
MSc Student, Western University | Canada
sc-MC: Single-cell RNA Profiling for Complex Microbial Community
Ray Wang, Teddy Lee, Alice Deng, Binh Tran, Vanessa Dumeaux
Host-adapted microbial communities (or microbiomes) are assemblies of commensal, symbiotic, and pathogenic microorganisms present at specific body sites. The population of bacteria, viruses, archaea, and fungi that comprise the human microbiome depends on functional interactions dictating the community’s composition, stability, and resilience, such as the positive symbiotic and negative antagonistic relationship existing between members of the microbial community. The human gut microbiome, estimated to express 3.3 million genes, plays a critical role in host health. Single-cell RNA profiling has recently been developed to investigate cell-specific gene expression and identify different cell types and states in complex tissues. Our lab and others have developed such methods to profile microbial monocultures, revealing significant heterogeneity even in isogenic cell populations. We extend this method to profile complex microbial communities such as the human gut microbiome, optimizing cell permeabilization for different microbial cell walls and capturing small amounts of microbial RNA, including those from low-abundant microbes. Validation involves real-life stool samples collected from sedentary individuals who participated in an 8-week physical training program. This research presents a unique and effective technique to functionally characterize individual microbes’ heterogeneous behaviour in complex microbial communities. As our understanding of microbial communities evolves, so will the applications to modulate them for a diverse range of biomedical, environmental, food sciences, and synthetic biology applications.
Dr. Amber Fedynak (Poster 62)
Postdoctoral Fellow, University of Guelph | Canada
The Microbiome of Kettle Lake in Norther Ontario Contains a Diversity of Pathogens
Anastasia A. Fedynak, Opeyemi U. Lawal, Noah Bryan, Valeria R. Parreira, Mitra Soni, Melinda Precious, Yanhong Chen, Lawrence Goodridge
The health of our freshwater systems are crucial to ensuring food quality and safety of food products intended for human consumption. A multitude of factors can affect microbiological safety of our food sources such as contaminated water sources. The aim of our study was to detect pathogens, such as Salmonella spp, from Lake Wilcox, a kettle lake in Northern Ontario, Canada (43° 56′ 59.1″ N -79° 26′ 11.5″ W). We performed shotgun metagenomic sequencing using Oxford Nanopore Technology, GridION, on the sample and use established bioinformatics workflows including Kraken2 for taxonomic classification of bacterial species present in the sample. We found a proportion of the microbiome is Gammaproteobacteria (15% of all bacteria identified in our sample), a phylum that contains several well-known human pathogens including Salmonella, Eschericha, and Vibrio, all of which were identified in our sample. We also found a Microcystis aeruginosa a member of the phlum Cyanobacteriouta (5%) and further confirmed with in silico identification of M. aeruginosa plasmids. Previous studies have shown M. aerignosa to be associated with algeal blooms in freshwater systems and can impact the overall health of the lake. Another intriguing finding was the identification of Paenibacillus larvae, the largest single species present representing 2.72% of the total bacteria in the sample. P. larvae has been found worldwide and in a variety of sources including freshwater. It has been well studied and established as a lethal pathogen of honeybee larvae causing the devastating disease of Americian Foulbrood. Paenibacillus species have also been identified in opportunistic infections of humans. Overall, our analysis has identified a diverse set of pathogenic bacteria causing illness in humans and lethal disease honeybee larvae impacting food quality and food protection.
Noor Alsmadi (Poster 63)
PhD Candidate, University of Toronto | Canada
Utilizing Genome-Scale Metabolic Modelling Strategies to Study the Metabolism of Defined Gut Bacterial Communities
Noor W. Alsmadi, Giuliano Bayer, Trisha Shankara Subramanian, Emma Allen-Vercoe, Radhakrishnan Mahadevan, Dana J. Philpott
Studying the metabolism of interacting bacteria in gut microbial communities is difficult to perform using purely experimental means due to the complexity and diversity of the gut microbiome. Genome-scale metabolic models are mathematical representations of the reaction networks of an organism that enable the study of cross-feeding interactions and metabolic pathways in microbial communities. My work focuses on defining best practices to apply in silico genome-scale metabolic modelling strategies to study the metabolism of a minimal defined human gut bacterial community called Human Defined Community 1 (HDC1) as a gnotobiotic mouse model in vivo. Whole genome sequencing and assembly was performed using DNA samples from bacterial isolates from HDC1. Taxonomic characterization using genome assemblies showed that the community represents the major phyla in the human gut microbiome, making it a suitable model for further metabolic characterization. Functional genome annotation was performed followed by genome-scale metabolic model reconstruction. To further curate the individual metabolic models, the metabolic capacity of the individual bacteria will be determined in culture via untargeted metabolomics strategies. Moreover, substrate uptake profiles will be determined using microplate culture growth-based experiments. The experimental data will then be used to validate and curate the individual models prior to integration into a community model. The community model will then be used to simulate and study the metabolism of our community towards metabolic optimization of the production of metabolites of interest, such as short chain fatty acids. All in all, my work highlights the utility of using in silico metabolic modelling tools to mechanistically study and further optimize the metabolism of defined gut microbial communities in vivo.
Dr. Marcela Davoli Ferreira (Poster 64)
Postdoctoral Fellow, University of Calgary | Canada
Exploring the Influence of Human Commensal Bacteria on Autism Spectrum Disorder Development
Marcela Davoli-Ferreira, Lukas Mager, Aline Ignacio, Kirsty Brown, Fernanda Castanheira, Carolyn Thomson, Jenine Yee Pardis Kiani, Linda Ward, Thomas Louie, Markus B. Geuking, Kathy D. McCoy
Clinical trials using fecal microbiota transplantation (FMT) in pediatric autism spectrum disorder (ASD) patients induce long- term benefits, ameliorating both behavioral and neurological symptoms associated with ASD, thus implicating the gut microbiota in ASD pathogenesis. 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.
Employing a gnotobiotic ASD murine model (BTBR) colonized with ASD or neurotypical individual-derived C. innocuum strains, we evaluated ASD-like gastrointestinal symptoms and microglial phenotype via flow cytometry and confocal microscopy, as well as ASD-like behaviors utilizing sociability and social novelty tests. Colonization with ASD-derived C. innocuum induced an altered immune phenotype, characterized by increased monocyte frequencies and CD11b+CD103- and CD11b+CD103+ dendritic cells frequencies,as well as exacerbated Th1 cells within the gut compared to mice colonized with neurotypical-derived strains. ASD-derived strains also elicited reduced ileal tight junction expression and increased intestinal permeability, resembling gastrointestinal symptoms prevalent in ASD individuals. Furthermore, mice colonized with ASD-derived strains displayed ASD-like behaviors and exhibited alterations in brain metabolomics, with microglial cells showing higher CSF1R levels and diminished CX3CR1 expression, indicative of an ‘immature’ microglial phenotype classically associated with ASD.
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.
Lucas de Figueiredo Soveral (Poster 65)
Graduate Student, University of Calgary | Canada
Reconstitution of pasteurized donor’s milk microbiota as an immune interventional strategy for premature newborns: molecular benefits revealed by a multiomic approach of a clinical trial
Lucas de Figueiredo Soveral, Thaise Cristina Brancher Soncini, Izadora Borgmann Frizzo, Isis Maia Apolinário de Mello, Lívia Budziarek Eslabão, Dany Mesa, Jussara Kasuko Palmeiro, Thaís Cristine Marques Sincero, Aline Ignacio, Carlos Arterio Sorgi, Maria Marlene de Souza Pires, Oscar Bruna-Romero, Kathy McCoy, Carlos Rodrigo Zárate-Bladés
Human milk, along with its associated microbiota and metabolites, plays a crucial role in the development of premature newborns (PNs). When a mother’s own milk (MOM) is insufficient to meet the needs of the PN, pasteurized donor’s milk (PDM) is often the preferred alternative. However, pasteurization diminishes the levels of beneficial microorganisms in the MOM. In our clinical study (ReBEC:RBR-729kr8x), we investigated the microbiota composition and metabolomic profile of reconstituted PDM. PDM were spiked with 10% MOM and incubated at 37°C for 4 hours. Eighty PNs (<32 weeks) were randomly assigned to receive PDM or microbiota-reconstituted milk (RM). Milk microbiota (PDM, RM, and MOM) and PN fecal microbiota were analyzed using 16S rDNA sequencing. Metabolomic profiles of PDM and RM were obtained through liquid chromatography-mass spectrometry. Remarkably, microbiota analysis revealed that most RM samples closely resembled MOM microbiota after incubation, diverging from PDM. Fecal microbiota of RM-receiving PNs showed increased Acinetobacter, Enterobacter, Novosphingobium, and Veillonella levels compared to PDM. Metabolomic analysis showed significant enrichment in glycosphingolipid metabolism, tryptophan metabolism, and lipid beta-oxidation pathways in RM. Specifically, RM displayed increased levels of Oxidized Lysophospholipids (LPA O-16:1, LPE O-16:1), Sphingomyelin, L-Tryptophan, and Indoleacrylic Acid. Notably, these metabolites play critical roles in regulation of immune cell functions and inflammation, evidencing the importance of gut microbiota-host crosstalk during immune development. Our results highlight the potential use of RM to enhance immunological support for newborns and endorse a strategy to potentially improve the quality of PDM in neonate care units.
Dr. Erik Bakkeren (Poster 66)
Postdoctoral Fellow, University of Oxford | United Kingdom
Ecology of the human microbiome: how diversity in the microbiome protects against pathogens
Erik Bakkeren, Frances Spragge, Martin T. Jahn, Elizete B. N. Araujo, Claire F. Pearson, Xuedan Wang, Louise Pankhurst, Olivier Cunrath, Kevin R. Foster
A diverse human gut microbiome plays an important role in resisting colonization of the host by pathogens. While many mechanisms have been found to influence the ability of the microbiome to provide colonization resistance, these mecahnsisms are often context-specific and dependent on individual species. Therefore, we currently lack general principles to predict which microbiota communities will be protective versus those that will allow a pathogen to colonize and cause disease. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. We then applied our findings to successfully predict communities that resist a novel target strain. Our results support that more diverse microbiomes protect better against pathogens. Crucially, although increased microbiome diversity increases the probability of protection against pathogens, the overlap in nutrient-utilization profiles between the community and the pathogen is key. Our work suggests a route to optimize the composition of microbiomes for protection against pathogens.
Juan Camilo Burckhardt (Poster 67)
PhD Candidate, University of British Columbia | Canada
With a little help from our [tiny] friends: modifying gut bacteria to detect malabsorption
Juan C. Burckhardt, Giselle McCallum, Jerry He, Charlotte Clayton, Alice Hong, Carolina Tropini
Gut osmolality, reflecting particle concentration in intestinal fluid, is vital in gut physiology, governing functions such as water absorption and cell structure. Gut osmolality imbalances frequently occur in conditions such as inflammatory bowel disease, food intolerances, and laxative use. These imbalances arise as impairments in nutrient absorption that lead to an accumulation of particles in the gut lumen. Despite its importance, human intestinal osmolality remains understudied. Existing stool-based methods cannot capture the osmolality of different gut regions, limiting their use as a diagnostic tool. Furthermore, gut disease diagnosis usually necessitates invasive endoscopic procedures. Previous research from our lab revealed that certain bacterial families persist during gut osmotic upshifts. These osmotically-tolerant bacteria sense the gut osmotic environment and respond to its changes by regulating the transcription of specific sets of genes. Thus, we proposed to detect malabsorption by leveraging the genetic mechanisms that these commensals employ when responding to changes in the gut’s physical environment. We repurposed regulatory sequences of osmolality-responsive genes from the genetically tractable, osmotically-tolerant, and prevalent commensal Bacteroides thetaiotaomicron to create a biosensor for localized and fine-scale osmolality measurements. Through transcriptomics analyses, we identified B. theta genes selectively expressed across increased osmolality both in vitro and in vivo. Subsequently, we cloned the promoters from the identified genes into a synthetic reporter fluorophore circuit, creating osmolality biosensors in B. theta. We selected biosensors with a graded response to osmolality, and through further optimization, we achieved a 15- to 25-fold fluorescence difference in high osmolality conditions compared to baseline. Currently, we are validating our optimized biosensor in vivo by measuring its signal in the intestines of mice experiencing disrupted gut osmolality. Through this work, we are creating a platform for commensal-based microbial diagnostics that respond to disease-induced gut changes, improving our understanding of gut physical conditions.
Dr. Dina Kao (Poster 68)
Professor, University of Alberta | Canada
Effects of lyophilized fecal filtrate compared to lyophilized donor stool in the treatment of recurrent Clostridioides difficile infection: a multi-center randomized trial
D Kao, K Wong, C Lee, T Steiner, R Franz, C McDougall, M Silva, H Xu, Walter J, Loebenberg R, M Yaskina, T Louie
Background: Fecal microbiota transplantation (FMT) is the most effective therapy in the treatment of recurrent Clostridioides difficile infection (rCDI), and can be offered in fresh, frozen or lyophilized formulation with similar efficacy. Microbial engraftment is thought to be one of the mechanisms mediating its efficacy. However, the importance of microbes has been questioned by two small preliminary studies showing sterile fecal filtrate could prevent C. difficile recurrence.
Aims: To establish whether lyophilized sterile fecal filtrate (LSFF) is noninferior to lyophilized FMT (LFMT) in efficacy
Methods: In this double-blind, non-inferiority study conducted in 4 academic centers , we randomized rCDI patients to either LSFF or LFMT (1:1). Sterile fecal filtrate was manufactured by filtering fecal slurry through a series of filters, down to a pore size of 0.2 um. The non inferiority margin was 10%. Each treatment dose consisted of 15 oral capsules. Should a participant fail the assigned treatment, an open label LFMT treatment was offered. An interim analysis was planned after reaching 50% of recruitment target (124/248 participants). Primary outcome was the proportion of participants without CDI recurrence 8 weeks following assigned treatment. Secondary outcomes included 1) the proportion of participants without CDI recurrence 24 weeks following assigned treatment, 2) serious adverse events, and 3) minor adverse events.
Results: 137 participants were randomized to LFMT group (66) and LSFF (71) group. Prevention of rCDI 8 weeks after assigned treatment was achieved in 63.8% of participants in LSFF group and 86.9% in LFMT group (difference, -23.1%), resulting in trial termination. Serious adverse events were infrequent: 4 hospitalizations deemed not related and 1 hospitalization possibly related, and 1 death from COVID deemed not related to assigned treatment. Minor adverse events were self-limited and infrequent: nausea (6), vomiting (2), abdominal discomfort (15) and fever (2).
Conclusions: Among adults with rCDI, LSFF was inferior to LFMT by oral capsules in preventing recurrent infection over 8 weeks. The result emphasizes the importance of microbes in mediating FMT efficacy.
Dr. Larisa Kovtonyuk (Poster 69)
Postdoctoral Fellow, University of Calgary | Canada
The role of intestinal microbiome in CD19 directed CAR-T cell therapy
Larisa V. Kovtonyuk, Amy Shupe, Sacha Benaudia, Robert Puckrin, Mona Shafey, Doug Mahoney, Kathy D. McCoy
Adoptive T cell therapies have been under intense development for the past decades. Despite the success of chimeric antigen receptor (CAR)-T cell therapy, nearly 50% of patients with B cell malignancies either do not respond to the treatment or relapse. In addition, almost 50% of patients undergoing CAR T cell therapy experience severe adverse effects due to cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). The microbiome and microbial metabolites have been found to modulate the anti-cancer function of T cells in immune checkpoint inhibitor therapy. Similarly, differences in the patient’s microbiome composition may be one of the key extrinsic underlying factors determining responsiveness to CAR-T cell therapy. We hypothesize that specific intestinal microbial species can influence CD19CAR-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 CD19CAR-T cells transferred into germ free NSG (KbDb)null mice that received luciferase expressing Nalm6 cells can eliminate tumor cells in vivo. However, CD19CAR-T cells perform better and eliminate tumor cells more efficiently in NSG (KbDb)null mice harboring a diverse specific pathogen-free (SPF) microbiota. Moreover, CD19CAR-T cells perform better in mice mono-colonized with Bifidobacterium pseudolongum or Akkermansia muciniphila, compared to germ free mice. In addition, CD19CAR-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 metabolites involved.
Mursal Nader (Poster 70)
PhD Student, University of Toronto | Canada
Delineating Molecular Crosstalk Between Gut Microbiota and Hematopoietic System
Mursal Nader, Meggie Kuypers, Ximing Li, Alicia Aguilar, Gibran Edun, Minerva Fernandez, Soheil Jahangiri, Igor Dolgalev, Armin Gerbitz, Thierry Mallevaey, Anastasia N. Tikhonova
Adult hematopoietic stem cells (HSCs) give rise to immune and blood cell lineages in a process termed hematopoiesis. Following inflammatory stress, HSCs exit quiescence, and skew their differentiation towards the myeloid lineage – a process known as emergency hematopoiesis. It has been well established that intestinal commensal bacteria play a crucial role in maintaining overall health and influencing disease outcomes. In the last decade, the gut microbiota has emerged as a key regulator of steady state and emergency hematopoiesis. Additionally, clinical studies have shown that loss of microbial diversity in the gut serves as a predictor of mortality in HSC transplantation. Strikingly, antibiotic treatment prior to HSC transplantation is associated with a 27% increase in transplant-related mortality. Yet, the molecular mechanisms underlying the crosstalk between the gut microbiome and the hematopoietic system are poorly understood. Our preliminary data demonstrates that reductions in commensal bacteria achieved through the administration of broad-spectrum antibiotics results in a significant loss of hematopoietic stem and progenitors (HSPCs) at steady-state, attenuated emergency hematopoiesis, and impaired HSC engraftment compared to controls. This finding suggests that gut microbiota regulates hematopoiesis at steady-state and under different stress conditions. To delineate the molecular crosstalk between the gut microbiota and hematopoietic system, we are conducting transcriptomics and metabolomics analysis of HSPCs from antibiotic-treated and control animals. Ultimately, we envision that these studies will lead to the development of novel microbiome-based therapeutics to improve hematopoietic regeneration.
Dr. Xiaofan Jin (Poster 71)
Assistant Professor, University of Calgary | Canada
An integrated experimental-bioinformatic workflow to track spatial eco-evolutionary dynamics in synthetic microbial communities
Xiaofan Jin, Katherine Pollard
The behavior of microbial communities such as the human gut microbiome depends on both their ecological composition as well as physical structure. Metagenomic sequencing of fecal samples has greatly revealed the rich ecological diversity in the gut with instances of coexistence between evolutionarily similar taxa, yet our understanding remains limited in terms of how these complex communities are spatially organized between different gut regions, such as lumen and mucosa. Here, we apply in vitro culture incorporating mucin hydrogel carriers to simulate mucosal surface adhesion over time, comparing supernatant (i.e. planktonic) and carrier-attached culture fractions analogous to spatial organization of microbes between the lumen and mucosa in vivo. Our approach uses a complex defined community to enable strain level resolution measurements of microbial abundances within the context of a realistically diverse gut community, encompassing several instances of strains with recent evolutionary divergence. We demonstrate that incorporation of mucin carriers yields a more diverse community than traditional liquid culture, allowing co-existence of these closely related strains. Distinct enrichment patterns emerge with specific strains enriched on mucin carriers over supernatant and vice versa, highlighting cases where closely related strains exhibit divergent spatial enrichment phenotypes. We also develop bioinformatic workflows to evolutionary changes in these communities based on metagenomic sequencing data, revealing mutations and genomic inversions that are associated with spatial structure. These findings indicate that spatial structure and surface adhesion in gut microbial communities directly modulates their eco-evolutionary trajectories.
