Having IMPACTT 2:
Advancing Microbiome Research
June 2 & 3, 2022
Selected Abstracts
Selected Abstracts for Oral and Poster Presentation
See below for the abstracts selected for oral and poster presentations for our Having IMPACTT: Advancing Microbiome Research Symposium on June 2 & 3, 2022 at the Malcolm Hotel in Canmore, AB, Canada.
Presenters
Oral Presenters
Flash Talk & Poster Presenters
Poster Presenters
Ayodeji Samuel Ajayi
Dr. Vugar Azizov
Arshpreet Bhatwa
Dominique Bihan
Kirsty Brown
Diana Changirwa
Dr. Olabisi Coker
Thibault Cuisiniere
Sonia Czyz
Dr. Marcela Davoli Ferreira
Michelle Dean
Dr. Vanessa DeClercq
Dollina Dodani
Sharon Dong
Dr. Marija Drikic
Yi (Edward) Fan
Zhi Yi Fang
Claire Gerkins
Chanel Ghesquiere
Dr. Thaís Glatthardt
Dr. Alessandra Granato
Mackenzie Gutierrez
Diana Haider
Dr. Alya Heirali
Dr. Aline Ignacio Silva
Baofeng (Justin) Jia
Dr. Tingting Ju
Dr. Larisa Kovtonyuk
Hannah Lantz
Sukyoung (Suzie) Lee
Dr. Karen Lithgow
Sijie (Nicole) Liu
Dr. Pekka Määttänen
Emily Mercer
Dr. Chunlong Mu
Dr. Van Ortega
Stuti Patel
Roxanne Plante
Tausha Prisnee
Rajibur Rahman
Dr. Hena Ramay
Laura Rojas
Isla Skalosky
Erin Strachan
Dr. Marcel van de Wouw
Erik van Tilburg Bernardes
Dr. Yan Wang
Nadia Yorke
Kassandra Zachos
Abstracts
Oral Presenters
Dr. Kyle Burrows (Session 4: Microbiome & Influences on Disease)
Postdoctoral Fellow, University of Toronto (Canada)
A gut commensal protozoan tunes pulmonary disease severity
Kyle Burrows, Louis Ngai, Pailin Chiaranunt, Eric Cao, Catherine J. Streutker, Brian Forde, Liam O’Mahony, and Arthur Mortha
Commensal protozoa, which are an integral part of the vertebrate microbiome, are understudied compared to their conventional pathogenic counterparts, thus the impact of protozoan colonization on mucosal immune homeostasis remains poorly understood. Modulating diversity of the gut microbiota can influence immunity throughout the host via various tissue-axes. The gut-lung axis, for example, has linked dysbiosis to chronic inflammation of the airways. While protozoa colonization can modulate local intestinal immune responses, its potential contributions to extra-intestinal immunity has not been characterized. Here, we show that the gut commensal protozoa, Tritrichomonas musculis (T.mu), remotely imprints a pro-inflammatory immune phenotype in the lung following colonization. Although the permanent engraftment of T.mu into the microbiome has no impact on the overall health status of mice, colonization mediates local eosinophilia, via gut-derived inflammatory group 2 innate lymphoid cells (ILC2) that migrate to the lung to interact with tissue-resident T cells and B cells. This tripartite immune network serves as a tissue specific niche for lung eosinophilia in protozoan colonized mice. Critically, the T.mu induced remodeling of the lung immune landscape exacerbated house dust mite-induced allergic airway inflammation. Further, a survey for human associated Tritrichomonas internal transcribed spacer (ITS) sequences on metagenomic DNA sequences from human sputum samples revealed an enrichment of protozoan DNA sequences in patients with severe allergic asthma. Collectively, this data indicates that commensal protozoa tune pulmonary disease severity by remotely shaping a lung immune network that potentiates local eosinophilia.
Dr. Alissa Cait (Session 4: Microbiome & Influences on Disease)
Post Doctoral Fellow, Malaghan Institute of Medical Research (New Zealand)
Microbiome and Immune profiling of a 1-year controlled human hookworm infection
Alissa Cait, Francessco Vacca, Brittany Lavander, Sophia-Louise Noble, Kate McLean, John Mamum, Bibek Yamnam, Tama Te Kawa, Tom Mules, Sam Old, Laura Ferrer, Graham Le Gros, Mali Camberis, Stephen Inns, Olivier Gasser
There is a well-established immunoregulatory role for helminths in animal models of allergic disorders. In humans, several studies have identified an inverse correlation between helminth burden and allergic and autoimmune disease. Despite this, the field is still lacking knowledge on the immunological interplay between the human host and parasite infection in a controlled infection setting. In this study, healthy volunteers were recruited and infected with Necator americanus. Infection was monitored for one year. Immune responses were studied in peripheral blood and serum, and the microbiome was analysed by 16S rRNA amplicon sequencing and shotgun metagenomics from stool samples. All participants were successfully infected as demonstrated by detectable eggs in the feces and adult worms in the intestine. Smart-pill analysis allowed for monitoring of gut physiology and worm behavior. High dimensional analysis of peripheral blood mononuclear cells, inflammatory cell subsets and cytokines was carried out and correlated to symptom scores and microbiome profiles. Our data shows the detailed effects of a controlled hookworm infection in healthy volunteers and highlights the interplay between the microbiome, gut health, and systemic immunity. Future studies will trial helminths such as N. americanus in the treatment of autoimmune and inflammatory disorders, such as inflammatory bowel disease.
Dr. Brittany Duggan (Session 6: Microbiome Short Talks)
Post-doctoral Fellow, McMaster University (Canada)
Gut microbiota-based vaccination uses NOD-RIPK2 immunity to improve long-lasting blood glucose control in obese mice
Brittany M. Duggan, Akhilesh K. Tamrakar, Nicole G. Barra, Fernando F. Anhê, Gabriella Paniccia, Jessica G. Wallace, Hannah D. Stacey, Matthew S. Miller, Deborah M. Sloboda, Jonathan D. Schertzer
Postbiotics such as bacterial cell wall components derived from the gut microbiota engage innate and adaptive immunity and can have opposing effects on blood glucose and insulin sensitivity. Specific postbiotics are higher in circulation during obesity and promote inflammation and insulin resistance during metabolic disease while others, such as the NOD2 ligand muramyl dipeptide (MDP), have been shown to alleviate inflammation and improve blood control during obesity. We have shown that vaccination-style injection of a crude mixture of postbiotics derived from either small intestine or cecum can lower blood glucose in lean mice in a concentration dependent, sex-independent manner without altering blood insulin levels. We also found that repeated administration/vaccination using crude preparations of intestinal postbiotics can lower blood glucose levels without altering body mass or body composition in obese mice. We demonstrated a requirement for intact NOD2-RIPK2 immunity in vaccination-induced glucose improvements, however MDP alone was insufficient to produce long-lasting decreases in blood glucose after vaccination-style injection of upper gut postbotics. Interestingly, NOD1-RIPK2 immunity protected against vaccination-induced increases in blood glucose. Improved blood glucose control was associated with an IgG response directed against the postbiotics that was compartmentalized to the lower small intestine (SI), and with an altered microbiota composition in the lower small intestine and colon of vaccinated mice. Vaccination with postbiotics to improve blood glucose control may overcome some of the limitations of prebiotics or probioitcs. Our simple microbiota-based vaccination procedure shows that the net effect of injecting a specific concentration of a segment-specific intestinal postbiotic mixture promotes durable improvements in blood glucose control in lean and obese mice, and where NOD1-RIPK2 versus NOD2-RIPK2 immune responses dictate the net glycemic effect of vaccination.
Dr. Roy Hajjar (Session 2: Microbiome & Cancer)
PhD Candidate & Resident Physician, Université de Montréal (Canada)
The gut microbiota modulates colonic healing after surgery in patients with colorectal cancer
Roy Hajjar, Gabriela Fragoso, Manon Oliero, Annie Calvé, Hervé Vennin Rendos, Thibault Cuisiniere, Claire Gerkins, Ahmed Amine Alaoui, Emmanuel Gonzalez, Nicholas J B Brereton, Souad Djediai, Borhane Annabi, Khoudia Diop, Bertrand Routy, Patrick Laplante, Jean-François Cailhier, Nassima Taleb, Hefzi Alratrout, François Dagbert, Rasmy Loungnarath, Herawaty Sebajang, Frank Schwenter, Ramses Wassef, Richard Ratelle, Éric Debroux, Carole Richard, Manuela M Santos
Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer, and the third in terms of cancer-related deaths. CRC treatment requires a surgical resection of the colon or rectum, and a reconnection, or anastomosis, of the remaining bowel ends to re-establish intestinal continuity. Poor healing of the anastomosis, or anastomotic leak (AL), is a major complication in colorectal surgery that may affect up to 20% of patients, and significantly increases morbidity and mortality. Our objective is to investigate the possible role of the gut microbiome in anastomotic healing.
Methods: Preoperative fecal samples and intraoperative mucosal samples were collected from a cohort of patients with CRC. The gut microbiota of patients with AL and of other patients that presented optimal healing was analyzed, and compared using the Anchor 16S rRNA gene amplicon pipeline. Fecal microbiota transplantation (FMT) was performed in mice using fecal samples from patients with and without AL. Transplanted mice underwent surgical anastomosis. Anastomotic healing and gut barrier integrity were evaluated 6 days later. Additionally, the gut microbiota composition was assessed to detect potential differences.
Results: After surgery, mice transplanted with the fecal microbiota of donors with AL displayed poorer macroscopic healing of the anastomosis. They also displayed higher gut permeability as judged by increased bacterial translocation to the spleen. Lower concentrations of collagen and fibronectin in these mice indicated poor extracellular matrix (ECM) formation at the wound site. This was accompanied by higher expression of collagenolytic enzymes involved in ECM degradation. Gut microbiota beta-diversity was significantly different between the two groups and bacterial species were shown to be associated with the healing process.
Conclusion: The gut microbiota in patients with poor postoperative healing induces poor healing in mice. These results suggest that the gut microbiota in patients with CRC may play a role in anastomotic healing after surgery.
Rebecca Jeffery (Session 6: Microbiome Short Talks)
DPhil Student, University of Oxford (United Kingdom)
Understanding host-bacterial and bacterial-bacterial interactions that support intestinal inflammation
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.
Dr. Kevin McGregor (Session 5: Bioinformatics & Multi-Omics)
Assistant Professor, York University (Canada)
Proportionality-based association measures in count-based compositional data
Kevin McGregor and Nneka Okaeme
Compositional data comprise vectors of quantitative measures that describe the constituent parts of a whole. Data arising from various -omics platforms are compositional in nature. For instance, 16S sequencing and single-cell RNA-sequencing are both examples of platforms that yield compositional data. However, correlations between features on raw counts have no meaningful interpretation. Measures of association based on proportionality have previously been proposed, and have been shown to outperform other association metrics in single-cell sequencing data. These metrics were designed for continuous compositional data and do not account for the additional sampling variability in sequencing data. In this presentation I will discuss the pitfalls of using standard proportionality metrics in count-based platforms and show that models accounting for sequencing variability (in particular the multinomial logit-normal model) improve estimates of association.
Manon Oliero (Microbiome & Cancer)
PhD Student, CRCHUM, Université de Montréal Hospital Research Centre (Canada)
Tumorigenic effect of inulin in ApcMin/+ mice depends on the presence of colibactin-producing bacteria
Manon Oliero, Roy Hajjar, Thibault Cuisiniere, Gabriela Fragoso, Annie Calvé, and Manuela M. Santos
Purpose: Inulin has been suggested to protect against intestinal carcinogenesis. However, some studies have shown that inulin may promote tumor formation in ApcMin/+ mice, a model for colorectal cancer (CRC). Colibactin-producing gut bacteria, such as Escherichia coli, promote carcinogenesis and facilitate the progression of CRC through cellular senescence and chromosomal abnormalities. Therefore, colibactin-producing E. coli may change the protection conferred by inulin against tumor formation.
Experimental design: ApcMin/+ mice received a 2% dextran sodium sulfate solution in the drinking water for one week, followed by oral gavage with 108 CFU of the pathogenic, polyketide synthetase (pks)-containing E. coli strain, NC101. Mice were then fed a diet supplemented with 10% cellulose as control or 10% inulin for four weeks. Colonic tumors were counted and evaluated by histology, and DNA double-stranded breaks (DSBs) and cell proliferation were assessed using immunohistochemistry to detect phosphorylated-H2AX and Ki67, respectively. Stool samples from CRC patients (n=82) and healthy controls (n=48) were also collected, and detection of pathogenic pks+ bacteria was performed by PCR on the colibactin A gene.
Results: E. coli NC101 presence was enhanced in the intestine of ApcMin/+ mice fed the inulin diet compared to mice receiving the control diet, resulting in a higher number of DSBs, cell proliferation, tumor burden, and tumor development. However, supplementation with the pasteurized probiotic E. coli Nissle 1917 inhibited the colonization of E. coli NC101 enhanced by inulin, which overall slowed CRC development. Significantly, 51% of CRC patients and 41% of healthy controls were colonized by pks+ E. coli.
Conclusions: The presence of pks+ E. coli influences the outcome of inulin supplementation in a CRC context. Due to the high prevalence of E. coli pks+ in both healthy and CRC populations, the carcinogenic effect of inulin should be further investigated.
Ana Popovic (Session 1: Microbiome & Early Life)
PhD Candidate, The Hospital for Sick Children (Canada)
Intestinal protozoan Tritrichomonas induces transcriptional remodeling of the gut microbiome, which is accelerated in immunocompromised mice
Ana Popovic, Eric Y. Cao, Nirvana Nursimulu, Michael E. Grigg, Arthur Mortha, John Parkinson
Intestinal colonization by protozoa can lead to asymptomatic infection or manifest in colitis-like symptoms. Emerging evidence suggests that these organisms are more prevalent in the gut than previously appreciated, with a capacity to modulate host immunity and induce shifts in resident bacterial composition. To gain a better understanding of their impact on the gut environment, in this study we explore the interactions between the model protozoan Tritrichomonas musculis (Tmu) and gut bacteria during the first four weeks following infection, in healthy and IgA-deficient mice (i.e. those compromised in immune control of gut microbiota). Using 16S sequencing and metatranscriptomics, we show that Tmu induces diversification of bacterial communities, with increased abundances of Bifidobacterium, Lachnospiraceae and Proteobacteria, but gene expression increases predominantly in Helicobacter and several taxa of the Bacteroidetes clade. Protist colonization led to upregulation of bacterial metabolism, cell division and iron transport machinery (a possible consequence of competition for the mineral) across both host genotypes, with changes accelerated in IgA-deficient mice. Protist metabolism and expression of virulence genes increased in parallel, with expression notably higher in IgA-deficient mice relative to WT early in the infection (at day 2), possibly reflecting faster proliferation in the absence of IgA. Single cell transcriptional profiling of Tmu, isolated from mouse caeca, revealed 15 distinct protist subpopulations, capturing cells at various stages of cell cycle, with variable patterns of virulence gene expression and evidence of transition from active trophozoite cells to dormant cyst-like structures. Through fluorescent labelling of uniquely expressed genes, we validated this cell state transition both in situ and in vitro. Our findings demonstrate dynamic ecological remodeling in the gut in response to a new protozoan resident, the timing of which may be determined by the integrity of the immune system.
Sara Stickley (Session 1: Microbiome & Early Life)
PhD Student, Queen’s University (Canada)
Host genomics and exposure to human milk oligosaccharides are associated with the gut microbiota of infants with asthma in the CHILD Cohort Study
Sara A. Stickely, Zhi Y. Fang, Amirthagowri Ambalavanan, Yang Zhang, Charisse Petersen, Darlene Dai, Bianca Robertson, Chloe Yonemitsu, Stuart E. Turvey, Piushkumar J. Mandhane, Elinor Simons, Theo J. Moraes, Malcolm R. Sears, Padmaja Subbarao, Lars Bode, Meghan B. Azad, Qingling Duan
Asthma is the most prevalent chronic disease and a leading cause of hospitalizations among Canadian children. Although numerous risk factors have been reported, the mechanisms underlying asthma remain poorly understood. Earlier studies suggest that the gut microbiota may play a crucial role in asthma pathogenesis through bidirectional crosstalk along the gut-lung axis. In this study, we integrate host genomics with early-life exposures, including breastfeeding practices and human milk composition, to study their main and interaction effects on the gut microbiota and risk of asthma. We leverage data from the CHILD Cohort Study including gut microbiota data generated from 16S rRNA sequencing of stool samples collected at two ages: 3 months (N=747) and 1 year (N=766). We also utilize genomic profiles obtained from the Illumina HumanCoreExome Bead Chip, breastfeeding practices determined by repeated questionnaires, and concentrations of the 19 most abundant human milk oligosaccharides (HMOs) quantified from breastmilk samples collected 3-4 months postpartum. We identified gut microbial shifts such as increased abundance of Clostridioides difficile at 3 months associated with increased risk of recurrent wheeze (ages 2-5 years; P=0.028) and asthma (diagnosed by age 5; P=0.023). This microbe was decreased in infants who were breastfed until at least 3 months compared to those who were not (P=2.2×10-6) and among breastfed infants with low genetic risk of recurrent wheeze who were exposed to increased concentrations of the HMO fucosyl-disialyllacto-N-hexose (P=5.2×10-3). Moreover, our genome-wide association study of C.difficile abundance in the gut at 1 year identified associations with single nucleotide polymorphisms on chromosome 3 (e.g. rs73218890, P=3.5×10-9). Our findings suggest host genomics and exposure to variable human milk components impact the infant gut microbiota, which may influence asthma and wheeze susceptibility. A better understanding of the link between gut dysbiosis and asthma will help identify early-life asthma biomarkers, preventative strategies, and therapeutic targets.
Dr. Robyn Wright (Session 5: Bioinformatics & Multi-Omics)
Postdoctoral Research Fellow, Dalhousie University (Canada)
From defaults to databases: parameter and database choice dramatically impact the performance of metagenomic taxonomic classification tools in low microbial biomass samples
Robyn J. Wright, André M. Comeau and Morgan G.I. Langille
In metagenomic analyses of microbiomes, one of the first steps is usually the taxonomic classification of reads by comparison to a database of previously taxonomically classified genomes. While different studies comparing metagenomic taxonomic classification methods have determined that different tools are “best”, there are two tools that have been used the most to-date: Kraken2 and MetaPhlAn 3. Common criticisms of these tools are that they identify lots of false positive or false negative taxa, respectively, which can be particularly problematic when aiming to examine samples that are expected to have low microbial biomass, such as blood or tumour samples. With the aim of determining a method that may be suitable for the taxonomic classification of reads within low microbial biomass samples, we systematically evaluated the parameters and databases used to run the different taxonomic classification tools. We investigated which of these tool-parameter-database combinations would give classifications closest to the real composition of metagenomic samples using a range of simulated and mock samples, revealing that there may not be a one-size-fits-all “best” tool choice. While Kraken2 can achieve better overall performance, with higher precision, recall and F1 scores, as well as alpha and beta diversity measures closer to the known composition, than MetaPhlAn 3, the default database and parameters should not be used. We will discuss how we are applying the knowledge gained from these mock samples in order to taxonomically classify reads in blood samples that were initially collected for human genome sequencing (Personal Genome Project Canada [PGPC]). Preliminary results suggest that human blood consistently contains DNA from the genera Mycobacterium, Escherichia and Staphylococcus, while ongoing work is aimed at associating the taxa present with participant metadata.
Flash Talk & Poster Presenters
Giuliano Bayer (Poster 1)
PhD Student, University of Toronto (Canada)
Generation of a Humanized Gnotobiotic Mouse Model to Study Host Gene-Microbiota Interactions
Giuliano Bayer, Bana Samman, Amy Cao, Boyan K Tsankov, Grace V Visser, Tapas Mukherjee, Elisabeth G Foerster, Nathaniel J Winsor, Emma Allen-Vercoe, Dana J Philpott
Studies of the gut microbial ecosystem and its role in disease are often complicated by the intricate network of interactions between its microbial members and the host. Therefore, mouse models in which the microbiota diversity can be strictly controlled, and the functional potential be easily assessed, are needed.
To generate a gnotobiotic mouse model, we inoculated germ-free mice with a defined bacterial community isolated from the stool of a healthy human donor. Analysis of fecal samples using 16S rRNA gene sequencing revealed reproducible colonization of inoculated mice with 8 bacterial species, collectively termed Human Defined Community 1 (HDC1). The HDC1 is vertically transmitted from mothers to offspring and the relative abundances remain stable in the colony over a period of 14 weeks in a gnotobiotic isolator. We recently established a species-specific qPCR approach that allows rapid absolute quantification of 16S rRNA gene copy numbers, omitting the need for 16S sequencing. Characterization of the colonic lamina propria immune cells in HDC1 mice revealed T cell frequencies closely resembling those of mice harboring a complex microbiota (specific pathogen-free [SPF] mice), including normal proportions of Treg, TH1, and TH17 cells. In addition, staining of Carnoy-fixed colon sections with Alcian Blue periodic acid-Schiff showed that HDC1 mice had a mucus layer comparable to SPF mice. Finally, we showed that HDC1 mice can be transplanted with human gut commensals and Crohn’s disease (CD)-associated bacteria, demonstrating the tractability of this defined microbiota.
In summary, HDC1 gnotobiotic mice are stably colonized with a human defined bacterial community and display normal intestinal physiology. Therefore, HDC1 mice represent a reliable and tractable tool for host-microbe interaction studies. Ultimately, we will employ the HDC1 model to decipher the role of Nod2 – the strongest genetic risk factor for CD development – in shaping the microbiota and regulating intestinal inflammation.
Dr. Andrew Forgie (Poster 2)
Postdoctoral Fellow, University of Alberta (Canada)
Changes to the resident gut microbiota from high dose vitamin B12 supplementation promotes Citrobacter rodentium colonization in mice
Andrew J. Forgie, Deanna M. Pepin, Tingting Ju, Stephanie Tollenaar, Consolato M. Sergi, Samantha Gruenheid, Benjamin P. Willing
Vitamin B12 is a modulator of microbial ecology. In the mammalian gastrointestinal tract, competitive and syntrophic interactions for B12 exist between microbes, and to what extent these interactions contribute to the overall microbiota community and host health is poorly understood. The objective of this study was to evaluate the effect of excess B12 on microbial ecology by challenging the B12-induced dysbiosis to resist pathogen colonization. Mice were fed a standard chow diet and received either water or water supplemented with B12 (cyanocobalamin; 120 μg/day) for two weeks, equivalent to a megadose of approximately 20 mg/day in humans. Mice were subsequently challenged with Citrobacter rodentium, a mouse specific pathogen. Infection severity was determined by body weight, pathogen load, histopathological scoring and biomarkers of inflammation. Cyanocobalamin supplementation led to enhanced pathogen colonization at day 1 (P < 0.05) and day 3 (P < 0.01) post-infection. The impact of cyanocobalamin on gut microbial communities was minor but characterized by reduced alpha diversity with specific changes in the low abundance Lachnospiraceae populations. Cyanocobalamin treatment enhanced the amount of IL-12/23p40 (P < 0.001) and IL-17A (P < 0.05) cytokines in the colon of naïve mice. The production of IL-12/23p40 in colon tissues was dependent on a gut microbiota but varied based on starting microbiota, which suggest specific microbes may be inducing this immune phenotype. Metatranscriptomics revealed that B12 supplementation decreased glucose utilizing genes by C. rodentium, a metabolic attribute previously associated with greater virulence. High dose oral vitamin B12 supplementation created a dysbiosis that activated IL-12/23p40 in colon tissues, which encouraged the early onset of C. rodentium pathogenesis in mice. This research suggests that high B12 concentrations in the gut can negatively impact the gut microbiota’s ability to resist pathogen colonization.
Ali Mirza (Poster 3)
PhD Candidate, University of British Columbia (Canada)
Metagenomic Analysis of the Pediatric-Onset Multiple Sclerosis Gut Microbiome
Ali I Mirza, Feng Zhu, Natalie Knox, Jessica D Forbes, Gary Van Domselaar, Charles N Bernstein, Morag Graham, Ruth Ann Marrie, Janace Hart, E. Ann Yeh, Douglas L Arnold, Amit Bar-Or, Julia O’Mahony, Yinshan Zhao, William Hsiao, Brenda Banwell, Emmanuelle Waubant, Helen Tremlett
Background and Objectives: Little is known of the functional potential of the gut microbiome in pediatric-onset multiple sclerosis (MS). We performed metagenomic analyses using stool samples from individuals with pediatric-onset MS and unaffected controls.
Methods: Persons ≤21 years old enrolled in the Canadian Pediatric Demyelinating Disease Network providing a stool sample were eligible. Twenty MS patients (McDonald criteria) with symptom onset <18 years were matched to 20 controls by sex, age (±3 years), stool consistency, and race. Microbial taxonomy and functional potentials were estimated from stool sample-derived metagenomic reads and compared by disease status (MS vs controls) and disease-modifying drug (DMD) exposure using alpha-diversity, relative abundance, and prevalence using Wilcoxon rank-sum, ALDEx2 and Fisher’s exact tests, respectively.
Results: Individuals with MS were aged 13.6 years (mean) at symptom onset and 8 were DMD naïve. Mean ages at stool sample were 16.1 and 15.4 years for MS and control participants, respectively; 80% were girls. Alpha-diversity of enzymes and proteins did not differ by disease or DMD status (p>0.20), but metabolic pathways, gene annotations and microbial taxonomy did. Individuals with MS (vs controls) exhibited higher methanogenesis prevalence (odds ratio=10, p=0.044), and Methanobrevibacter abundance (log2 fold-change[LFC]=1.7, p=0.0014), but lower homolactic fermentation abundance (LFC=-0.48, p=0.039). Differences by DMD status included lower phosphate butyryltransferase for DMD-naïve vs exposed MS patients (LFC=-1.0, p=0.033).
Discussion: The gut microbiome’s functional potential and taxonomy differed between individuals with pediatric-onset MS versus controls, including higher prevalence of a methane-producing pathway from Archaea and depletion of the lactate fermentation pathway. DMD exposure was associated with butyrate-producing enzyme enrichment. Together these findings indicate that the gut microbiome of individuals with MS may have a disturbed functional potential.
Jacob Nearing (Poster 4)
PhD Candidate, Dalhousie University (Canada)
Examining the salivary microbiome for biomarkers of breast, prostate and colon cancer
Jacob T. Nearing, Vanessa DeClercq, Morgan. G.I. Langille
Several works have indicated that microbial communities found within or on the body may act as microbial biomarkers of disease. Indeed, work within the fields of pancreatic and colorectal cancer detection have shown strong microbial signatures within fecal or oral samples after disease diagnosis. This has led to an interest in identifying whether microbial biomarkers exist for other types of cancer, including breast and prostate cancer. To answer this question, we accessed salivary samples from two Canadian cohorts, the Atlantic Partnership for Tomorrow’s Health project and Alberta’s Tomorrow Project. Sample selection was then divided into two study designs focusing on breast, colon, and prostate cancer. The first study examined samples from individuals who have been diagnosed with cancer up to 25 years before salivary collection (retrospective) and the other focusing on samples from individuals that were diagnosed 5-7 years after salivary collection (prospective). Samples were matched in a 1:5 or 1:1 case control design for retrospective (n=508) and prospective analyses (n=581). We used amplicon sequencing to generate both diversity metrics and taxonomic compositions. Alpha and beta diversity were compared for each cancer grouping and study design, revealing only minor differences. Differential abundance analyses showed several taxa that were differentially abundant in all three case groupings in the retrospective design. Additionally, a small number of low abundance taxa were identified as differentially abundant in the prospective study across the three different cancers. Random forest modeling was used to classify samples for each respective cancer and study design. Classification showed varying results with retrospective cases of colon cancer showing the strongest accuracy (AUROC: ~0.8). Overall, our results indicate that the salivary microbiome does not contain strong microbial biomarkers for prospective detection these cancers. However, in retrospective cases there exist several microbial taxa that differ in relative abundance.
Jared Schlechte (Poster 5)
MSc Student, University of Calgary (Canada)
Dysbiosis of the gut microbiome and immune paralysis in critical illness: a multi-omics and systems-based analysis
Jared Schlechte, Amanda Zucoloto, Katrina Yu, and Braedon McDonald
Patients in the intensive care unit (ICU) with critical illness have been found to harbour profound dysbiosis of their gut microbiome. This dysbiosis has been associated with adverse outcomes including increased susceptibility to secondary infections (hospital-acquired infections), worsening organ dysfunction, and increased mortality. Critical illness is also marked by disruptions of the immune system, with many patients displaying signatures of immune paralysis and dysfunction. Currently, the mechanisms by which intestinal dysbiosis contributes to adverse outcomes in the ICU have not been defined. Given the fundamental role of the microbiome in immune development and homeostasis, we hypothesized that dysbiosis is mechanistically linked to adverse outcomes in the ICU through the induction of immune dysfunction and impaired host defense. To test this hypothesis, we performed a prospective and longitudinal multi-omics and systems-based analysis of the gut microbiome and immune landscape of 30 critical ill adults admitted to the ICU, compared to 15 healthy controls. 16S rRNA sequencing of the gut microbiome was then integrated with the single-cell analysis data using multifactor analysis tools. Using this approach, we reveal notable associations between dysbiosis and signatures of immune paralysis. Furthermore, these features of dysbiosis are associated with a significantly increased risk of secondary infection and death in the ICU. These data suggest that intestinal dysbiosis is a key pathological driver of systemic immune dysfunction and increased susceptibility to secondary infections in critical illness, and therefore may be an important target for therapeutic development to prevent the high burden of hospital-acquired infections in the ICU.
Dr. Caitlin Simopoulos (Poster 6)
Postdoctoral Associate, University of Ottawa (Canada)
A novel computational approach for large-scale microbiome screens using mass spectrometry profiling
There is an increasing need to characterize how drugs may be altering the human gut microbiome, and consequently, affecting human health. Recently, metaproteomics has been used to explore the gut microbiome and its taxonomic and functional shifts when challenged drugs. However, acquiring metaproteomic data by tandem mass spectrometry (MS/MS) can be time consuming and resource intensive, and there is demand for computational methods that can be used to reduce these resource requirements. We present MetaProClust-MS1, a computational framework for rapid microbiome screening developed to prioritize samples and treatments for follow-up MS/MS. In this proof-of-concept study, we tested MetaProClust-MS1 on microbiome data acquired using short 15-minute MS1-only chromatographic gradients, MS1 spectra from longer 60-minute gradients using MS/MS, and reanalyzed data from a clinical MS/MS diagnostic study of pediatric patients with inflammatory bowel disease.
We found that MetaProClust-MS1 identified robust microbiome responses caused by drugs using MS1 profiles only. In addition, clusters of comparable drug-microbiome interaction datasets were significantly correlated, suggesting similar results are acquired by both MS1-only and MS/MS. MetaProClust-MS1 could also distinguish between inflammatory bowel disease diagnoses (ulcerative colitis vs Crohn’s disease) using MS1 features from microbiome samples. In addition, MetaProClust-MS1 clusters were used to guide feature selection and resulted in an accurate model for inflammatory bowel disease prediction.
Through our example applications, we demonstrate that MetaProClustMS1 can screen microbiomes using MS1 profiles to guide future and follow-up experiments. We also show that our approach can be used to reanalyze previously acquired MS/MS by considering only MS1 features for novel applications. MetaProClust-MS1 may be especially useful in large-scale microbiome screens for prioritization of samples for further deep metaproteomic characterization, and may be promising as a novel approach for clinical diagnostic screening.
Anita Singh (Poster 7)
MSc Student, McMaster University (Canada)
Microbiota-derived D-lactate alters macrophage inflammation
Singh, A.M., Anhe, F.F., Schertzer, J.D.
Obesity-induced inflammation is a factor involved in the risk and progression of type 2 diabetes and fatty liver disease. These diseases are associated with changes in gut microbiota composition and metabolites. Pathogen-associated molecular patterns can cooperate with non-immunogenic metabolites derived from the gut microbiota to alter host immunity. We aimed to determine how microbial derive lactate cooperates with bacterial well wall components to engage host immunity in macrophages.
Gut bacteria produce almost all D-lactate in the host, whereas L-lactate is host-derived. Higher D-lactate in blood could promote inflammation characteristic of obesity, type 2 diabetes, and fatty liver disease. The liver and adipose tissue are key sites of inflammation in these diseases. It is known that microbial-derived D-lactate in the portal circulation programs hepatic-resident macrophages to help combat bacterial infections. We hypothesize that microbiota-derived D-lactate alters macrophage inflammation differently than L-lactate and can drive metabolic inflammation typical of type 2 diabetes and fatty liver disease.
Our data shows that D-lactate alters macrophage inflammation differently than L-lactate. We show that transcript levels of IL-1beta and IL-18, which are effectors of the NLRP3 inflammasome, are higher in adipose tissue of mice colonized with gut microbes that produce high levels of D-lactate. In clonal and primary macrophages from mice, we also show that equimolar and physiological levels of D-lactate are more inflammatory than L-lactate by altering Nos2 transcription. Further, D-lactate prevents L-lactate-induced lowering of NO production and NLRP3 inflammasome-mediated release of IL-1beta from primary macrophages.
Future work will use co-culture of macrophages and hepatocytes or adipocytes to investigate how this immune-metabolic cell interaction affects lipolysis and lipogenesis. This research may lead to the identification of gut microbiota-based approaches to limit liver and adipose inflammation in metabolic disease.
Andrea Verdugo Meza (Poster 8)
PhD Candidate, University of British Columbia, Okanagan (Canada)
Two live biotherapeutic products protect Muc2-/- and DSS-exposed mice from severe colitis
Andrea Alejandra Verdugo-Meza, Sandeep K. Gill, Jiayu Ye, Jacqueline A. Barnett, Natasha Haskey, Geet Hans, Deanna L. Gibson
As we gain insight into the role of the gut microbiome in health and disease, the application of microbiome-based therapies becomes more rationalized. Live biotherapeutic products (LBPs) include beneficial microorganisms with known therapeutic effects. BioColoniz and BioPersist are two LBPs with known beneficial effects in gut mucosal healing but improved to thrive under the hostile gut environment that characterizes ulcerative colitis (UC). To address if these two LBPs provide further therapeutic effects in comparison to their parental strains L. reuteri and E. coli Nissle 1917, we performed preclinical studies in a mouse model of spontaneous colitis. Muc2-/- mice received BioColoniz, BioPersist, or both LBPs and were monitored for signs of disease. During the acute phase of the disease, mice were euthanized, and the colons were collected for macroscopic, histopathological, and molecular analysis. We found that mice receiving BioColoniz, BioPersist, or the two LBPs were protected against severe colitis as they did not develop rectal prolapses and presented lower clinical scores. Furthermore, BioPersist showed the most protective effect, as mice treated with BioPersist presented greater colon length, lower histopathological scores, and decreased relative mRNA expression of RELMβ, a driver of colitis in Muc2-/- mice, when compared to mice treated with the parental strains. To validate the protective role of BioPersist and BioColoniz in UC, we also evaluated them in the DSS acute model of colitis, and this time we compared its prophylactic effect versus 5-ASA treatment, a drug commonly used to treat mild to moderate UC. We found that pre-treatment with BioPersist or BioColoniz delayed and dampened the colitic phenotype in mice exposed to DSS, including lower histopathological damage. These results confirm that BioColoniz and BioPersist are more protective than the parental strains. Future studies will focus on describing the host-LBPs interactions at the mucosal level.
Poster Presenters
Ayodeji Samuel Ajayi (Poster 9)
PhD Student, Université de Montréal (Canada)
Dietary supplementation with inulin slows the progression of extracolonic colorectal tumors in mice
Ayodeji Samuel Ajayi, Roy Hajjar, Manon Oliero, Thibault Cuisiniere, Claire Gerkins, Gabriela Fragoso, Annié Calve, Hervé Vennin-Rendos, Manuela M. Santos
Introduction: The role of dietary fibers in shaping the gut microbiota towards a more beneficial composition for the host has recently gained attention. Oligosaccharides are fermented by the colonic gut microbiota to produce short chain fatty acids (SCFAs), including butyrate, acetate, and propionate. SCFAs, especially butyrate, have been reported to alleviate inflammation and inhibit the proliferation of colorectal cancer cells in the gut. Data on their systemic effect in the context of metastatic CRC is scant.
Objective: To assess the effect of dietary fiber supplementation on the progression of distance colorectal tumours in a mouse model of subcutaneous CRC.
Methods: BALB/c mice received a dietary supplementation with diet supplemented with either 10% w/w cellulose, a non-fermentable fiber, 10% inulin, a fermentable oligosaccharide, 0.6% 5- aminosalicylate (5-ASA), an anti-inflammatory agent, or a control diet lacking fibers. After 2 weeks, mice were subcutaneously injected with CT26 colon carcinoma cells. Tumor development and progression was monitored until 18 days post-injection.
Results: Mice supplemented with inulin displayed a slower tumor progression and smaller tumors than the mice receiving a diet without fibers. Supplementation with cellulose or 5-ASA did not lead to smaller tumors. Quantification of short-chain fatty acids in the gut lumen showed that butyrate, propionate, and acetate were significantly higher in mice supplemented with inulin versus those receiving no fibers. In vitro, butyrate was shown to inhibit both the proliferation of cancer cells and the activation of the pro-inflammatory NF- κB pathway.
Conclusion: Supplementation with inulin, a fermentable oligosaccharide, impedes the growth of extraintestinal CRC in mice. It is associated with an increase in the production of SCFAs, namely butyrate, which has the ability of inhibiting tumor growth and inflammation.
Dr. Vugar Azizov (Poster 10)
Postdoctoral Researcher, Universitätsklinikum Erlangen (Germany)
Alcohol-sourced acetate reduces T cell filamentous actin branching and migration
Vugar Azizov, Michael Frech, Jörg Hofmann, Marketa Kubankova, Dennis Lapuente, Matthias Tenbusch, Jochen Guck, Georg Schett, and Mario M. Zaiss
Alcohol is among the most widely consumed dietary substances. While excessive alcohol consumption damages the liver, heart and brain, clinical observations suggest that alcohol has strong immunoregulatory properties. Mechanisms by which alcohol undermines immune cell functions during autoimmunity and vaccination are currently elusive. Herein, we show that acetate, the main metabolite of alcohol, effectively inhibits the migratory capacity of T cells through acetylation of cortactin, a protein which stabilizes and promotes branching of filamentous actin. Acetate-induced cytoskeletal changes effectively inhibited activation, proliferation, and immune synapse formation in T cells at tissue concentrations reached by alcohol consumption. In summary, our findings show that acetate inhibits T cell-mediated immune responses by modulating the T cell cytoskeletal biomechanics.
Arshpreet Bhatwa (Poster 11)
Graduate Student, McMaster University (Canada)
Obesity and hyperglycemia alter the type of metabolic endotoxemia
Bhatwa A, Anhe GF, Anhe FF, Schertzer JD
Obesity increases the risk of type-2 diabetes and non-alcoholic fatty liver disease. Inflammation associated with metabolic disease can underpin metabolic dysfunction, but the source of this metabolic inflammation is poorly understood. The gut may provide inflammatory cues to the host and an important goal is to move beyond microbial taxonomy and identify bacterial-derived molecules that influence metabolic inflammation. Postbiotics are bacterial components or metabolites derived from living or dead bacteria. Lipopolysaccharide (LPS) is a postbiotic that engages host immune responses and can influence chronic inflammation through activation of toll-like receptor 4 (TLR4) during metabolic endotoxemia, which is a low-level increase of LPS in blood during metabolic disease. Metabolic endotoxemia may be beneficial or detrimental depending on the type of LPS, where hexa-acylated LPS caused dysglycemia, but under-acylated LPS improved blood glucose control in obese mice. It was unclear how under-acylated LPS antagonizes the effects of hexa-acylated LPS. It is also unknown whether certain metabolic disease characteristics alter the type of metabolic endotoxemia. Changes in bacterial composition or gut barrier function are positioned to alter the type and compartmentalization of LPS in the gut lumen and in circulation leading to potential inflammatory and metabolic effects on the host. We used leptin-deficient ob/ob mice of different ages to model hyperglycemia and normoglycemia during obesity and Akita mice with and without insulin delivery to model hyperglycemia and normoglycemia in lean mice. We quantified the TLR4 activation properties of LPS using a cell-reporter assay. Our results show that obesity and hyperglycemia can alter the type and compartmentalization of metabolic endotoxemia. We also found that under-acylated LPS acts as a weak agonist for TLR4 activation and competes with the potent activation of TLR4 by hexa-acylated LPS, which sets up the potential for different types of LPS to be used as a postbiotic to mitigate inflammation.
Dr. Dominique Bihan (Poster 12)
Senior Scientist, University of Calgary (Canada)
Method for absolute quantification of short chain fatty acids via reverse phase chromatography mass spectrometry
Dominique G. Bihan, Thomas Rydzak, Madeleine Wyss, Kier Pittman, Kathy D. McCoy, Ian A. Lewis
Short chain fatty acids (SCFAs; including acetate, propionate, and butyrate) are an important class of biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules in their native form by liquid chromatography mass spectrometry is challenging due to their relatively poor chromatographic properties. Herein, we introduce SQUAD, an isotope-based strategy for absolute quantification of SCFAs in complex biological samples. SQUAD uses aniline derivatization in conjunction with isotope dilution and analysis by reverse-phase liquid chromatography mass spectrometry. We show that SQUAD enables absolute quantification of biologically relevant SCFAs in complex biological samples with a lower limit of detection of 40 nM and a lower limit of quantification ranging from 160 nM to 310 nM. We observed an intra- and inter-day precision under 3% (relative standard deviation) and errors in intra- and inter-day accuracy under 10%. To demonstrate this quantification strategy, we analyzed SCFAs in the caecal contents of germ free versus conventionally raised specific pathogen free (SPF) mice. We showed that acetate was the most abundant SCFA in both types of mice and was present at 200-fold higher concentration in the SPF mice. We also illustrated the use of our quantification strategy in in vitro microbial cultures from five different species of bacteria grown in Mueller Hinton media. This study illustrates the diverse SCFA production rates across microbial taxa with acetate production serving as one of the key differentiating factors across the species. In summary, we introduce an isotope dilution strategy for absolute quantification of aniline-dativized SCFAs and illustrate the utility of this approach for microbiome research.
Kirsty Brown (Poster 13)
PhD Candidate, University of Calgary (Canada)
Effects of microbiome, age and sex on whole body metabolic phenotypes in mice
Kirsty Brown, Carolyn A. Thomson, Ryan Groves, Vina Fan, Ian A. Lewis, Kathy D. McCoy
A growing body of literature indicates that microbial metabolism is a major contributor to the overall metabolic capacity of mammals and perturbations in the microbiome can have a direct impact on the progression of diseases. Despite this, there have yet to be any studies that systematically map the metabolic alterations of diverse host sites in the absence of microbiota and in response to microbial colonization. Herein, we use liquid chromatography high-resolution mass spectrometry (LC-HRMS) to study the metabolomes of germ-free (GF), gnotobiotic (OMM12, 12 species) and conventional (specific pathogen free, SPF) mice. Using this approach, we quantify the impact of commensal bacteria on the metabolic composition of the gastrointestinal tract (GIT), serum, spleen, liver, peritoneal fluid, and urine. We also evaluate the influence of age and sex on metabolite profiles. Using these data, we constructed a computational model describing the relative impact of sampling site, microbiome composition, age, and sex on host metabolism. We show that bacterial colonization has a significant impact on the metabolic composition of all body sites. Not surprisingly, microbiome was the primary driver of variation at sites within in the GIT (R2jejunum=0.265, R2ileum=0.263, R2cecum=0.552 and R2colon=0.655), but also explained the most variation in metabolome of the urine and serum (R2serum=0.156, R2urine=0.383). Significant microbiome-inducted changes were also observed in other systemic sites (Pperitoneal fluid=0.0032, Pliver=0.0001, Pspleen=0.0001), but in these sites age was the primary driver of variance. While significant effects of sex were found in the spleen, urine, and colon, it explained the least amount of variation in all sites. Collectively, these data provide a model describing the interplay between microbiome, age, and sex in the metabolic phenotypes of diverse body sites. This model provides a framework for interpreting complex metabolic phenotypes and will help guide future studies into the role that the microbiome plays in disease.
Diana Changirwa (Poster 14)
MSc Student, University of Calgary (Canada)
Gastrointestinal colonization by Candida albicans modulates systemic host defence in sepsis
Diana Changirwa, Amanda Zucoloto, Katrina Yu, and Braedon McDonald
Pathological dysbiosis of the intestinal microbiome can lead to defects in immune homeostasis and a breakdown in host defense that increases susceptibility to infection. This is particularly relevant in patients with sepsis, a disorder of infection-induced systemic immune dysregulation and multi-organ dysfunction, where dysbiosis of the bacterial microbiome in these patients has been associated with adverse clinical outcomes. However, the impact of the fungal microbiome on sepsis pathogenesis has not been characterized. Analysis of the gut fungal microbiome in critically ill patients with sepsis identified severe fungal dysbiosis driven by marked overgrowth of Candida. Based on these clinical observation, we next sought to investigate the functional impact of intestinal Candida overgrowth on systemic inflammation and host defense using mouse models. We colonized the gastrointestinal tracts of antibiotic-conditioned specific pathogen free (SPF) C57BL/6 mice with different strains of Candida albicans (including both hyphal and yeast morphotypes) for 10 days prior to infection/sepsis with a well-characterized model of S. aureus bloodstream infection. We observed that mice colonized with a yeast-locked strain of C. albicans displayed reduced illness severity, reduced systemic inflammation (lower concentrations of proinflammatory cytokines IL-12p70, IL-1β and IL-6), and enhanced protection against systemic pathogen dissemination compared to uncolonized controls. In contrast, mice colonized with hyphal/filamentous C. albicans displayed similar illness severity, systemic inflammation, and pathogen burden as uncolonized controls. Together, these data demonstrate that fungal dysbiosis with C. albicans overgrowth can modulate the systemic host response to sepsis, and that selective gut colonization by yeast-locked C. albicans may confer beneficial systemic immune modulation that is protective in sepsis.
Dr. Olabisi Coker (Poster 15)
Research Associate, University of Calgary (Canada)
Metagenomic exploration of the gut microbiome in a sibling-controlled pilot study of children with Tourette Syndrome
Olabisi Coker, Hena Ramay, Kevin Muirhead, Tamara M. Pringsheim, Ramon Cortez, Davide Martino and Laura K. Sycuro
Background: Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder characterized by persistent, concurrent motor and vocal tics. Although gut microbes are becoming increasingly important in the etiology of neurodevelopmental disorders, their contributions to TS, and interaction with key factors like host genetics, are poorly understood. This pilot study represents the first sibling-controlled investigation of the gut microbiome in TS.
Methods: Thirty-three patients aged 7–16 years with TS (24 males), with paired unaffected siblings aged 5–18 years (13 males) were recruited from the Tourette and Pediatric Movement Disorders Clinic, Alberta Children’s Hospital, Calgary. Validated neurological and gastrointestinal symptom rating scales were used to assess participants. Shotgun metagenomics was performed on stool specimens, with taxonomic profiles obtained from MetaPhlAn3. Community ecology metrics were evaluated using phyloseq, and differential abundance analysis conducted with DESeq2 (FDR<0.05).
Results: Alpha and beta diversity were not statistically different between patients and siblings. PERMANOVA demonstrated that age (P=0.027), hyperactivity in ADHD (P=0.04) and depression (P=0.015) were associated with bacterial community variation in TS patients. Exploratory Spearman’s correlation studies suggested an inverse relationship between tic severity and Ruminococcus bicirculans and Intestinibacter bartletii, and a positive relationship with Lachnospira pectinoschiza (|ρ| >0.5, P<0.05). Differential abundance analysis adjusted for sex identified 6 enriched and 2 depleted bacterial species in TS children; all were low prevalence taxa (20–33%), but included species previously linked to neuropsychiatric disorders: Clostridium bolteae (associated with autism), Desulfovibrio piger (associated with Parkinson’ disease) and Erysipelatoclostridium ramosum (associated with expressive and receptive language disorder).
Conclusion: This study revealed minimal differences between the gut microbiome of TS patients and their sibling pairs highlighting the benefit of a well-controlled sibling-pair design and the need for a larger sample size. Enrollment is on-going to add power and confirm whether certain low prevalence taxa are linked to Tourette symptoms and neurodevelopmental disorders.
Thibault Cuisiniere (Poster 16)
PhD Candidate, CRCHUM, Université de Montréal Hospital Research Centre (Canada)
Oral iron supplementation shapes the recovery of the gut microbiota and promotes carcinogenesis in the APCMin/+ mouse model
Thibault Cuisiniere, Manon Oliero, Roy Hajjar, Annie Calvé, Gabriela Fragoso, Manuela M. Santos
Introduction: Colorectal cancer (CRC) induces anemia in a large proportion of patients and is usually treated with oral iron supplementation. Surgery, the main treatment for CRC, is routinely accompanied by prophylactic antibiotics to avoid infection. However, the combined effect of antibiotics and luminal iron in the gut on the microbiota and intestinal homeostasis remains unknown.
Materials and Methods: Wild type (WT) mice were subjected to antibiotic treatment followed by oral iron supplementation at different concentrations. The composition of the gut microbiota and its recovery were assessed by 16S rRNA sequencing of the stool. Short-chain fatty acid (SCFA) concentrations were also assessed in the stool. In addition, APCMin/+ mice (a CRC mouse model) received fecal microbiota transplantation (FMT) using samples from anemic CRC patients, followed by oral iron supplementation at different concentrations. Tumor burden and Ki-67-positive colonic cells were quantified, and gut microbiota composition was assessed by 16S rRNA sequencing.
Results: In WT mice, recovery from antibiotics under high luminal iron concentration shifted the gut microbiota toward a Bacteroidetes phylum-dominant composition. Three bacterial species characterized as CRC markers and/or CRC initiators were more abundant under oral iron supplementation and showed a lack of recovery of fecal concentrations of butyrate, an SCFA that inhibits cancer cell proliferation. APCMin/+ mice that received FMT from anemic CRC patient under oral iron supplementation developed more colonic tumors and had a higher proportion of Ki-67-positive cells compared to APCMin/+ fed an iron sufficient diet.
Conclusions: Gut microbiota recovery from antibiotic exposure under oral iron supplementation is frequent in CRC patients, but is also common in the general population. This study identifies possible deleterious effects of the concomitance of these two disruptive agents of the gut microbiota and may lead to modifications in the management of anemia in patients with CRC.
Sonia Czyz (Poster 17)
MSc 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
Our group has previously shown that the mother’s Immunoglobulin G (IgG) can bind metabolites or products derived from the maternal microbiota, which are then transferred to the offspring across the placenta and through the breastmilk. These IgG-bound bacterial metabolites/products drive postnatal immune development. However, the consequence of this transfer on offspring health and the underlying mechanisms remains unclear. The present study aims to investigate whether the transfer of maternal IgG-bound bacterial metabolites/products can promote protection against neonatal sepsis, as well as the requirement of bacterial-specificity and the neonatal Fc receptor (FcRn), a transcytosis receptor located on placental and neonatal gut epithelium, for this transfer.
Germ-free dams were transiently colonized with the auxotrophic Escherichia coli (HA107) through gavages on gestational days E4-E16. Since HA107 cannot survive for more than 48 hours in vivo, the dams were germ-free at delivery. Control dams received PBS. Pups born to HA107-colonized and control dams were challenged with Staphylococcus aureus 7- and 14-days following birth. To determine whether maternal bacterial-induced IgG shows specificity to microbial-derived metabolites/products, germ-free mice were injected systemically with HA107 to induce anti-E. coli IgG. Primed mice were monocolonized with various bacterial species, including the E. coli positive control, to generate metabolites/products that could potentially bind 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. Lastly, intestinal immune cells changes in wild-type and FcRn-knockout pups born to HA107-colonized dams were analyzed to test the requirement for the FcRn to facilitate the transfer of IgG-bound bacterial metabolites/products.
Preliminary data demonstrates no significant difference in offspring health outcomes between 7-day old pups born to HA107-colonized and control dams. However, 14-day-old pups born to HA107-colonized dams showed significantly increased clearance of S. aureus in the liver and kidney when compared to controls.
Dr. Marcela Davoli Ferreira (Poster 18)
Postdoctoral Fellow, University of Calgary (Canada)
Investigating the Role of Human Commensal Bacteria in Promoting Autism Spectrum Disorder
Marcela Davoli-Ferreira, Lukas Mager, Aline Ignacio, Jenine Yee, Thomas Louie, Markus Geuking, Kathy McCoy
Clinical trials using fecal microbiota transplantation (FMT) in ASD children induce beneficial long-term effect, improving ASD behavioral and neurological symptoms, suggesting that the gut microbiota contributes to ASD etiology. In a pilot study using FMT to treat ASD individuals, our group identified several changes in bacterial composition following FMT, with the most predominant effect being the loss of Clostridium innocuum correlating with the improvement of ASD symptoms. Here, we aimed to investigate whether these patient-derived C. innocuum strains contribute to ASD development and/or severity. We used a gnotobiotic ASD genetic model (BTBR T+Itpr3tf/J mice) colonized with C. innocuum strains isolated from ASD or neurotypical individuals and we assessed ASD-like gastrointestinal symptoms and microglial phenotype using flow cytometry and qPCR. The colonization with C. innocuum isolated from ASD donors induced an altered immune phenotype, including increased frequencies of CD11b+CD103– and CD11b+CD103+ dendritic cells, as well as exacerbated Th1 cells in the gut of BTBR mice when compared to mice colonized with neurotypical-derived bacteria. Mice colonized with ASD-derived strains also showed reduced expression of tight junctions in the ileum and increased intestinal permeability, resembling gastrointestinal symptoms often observed in ASD individuals. Notably, some alterations in the central nervous system were also observed under C. innocuum colonization, with microglial cells showing increased levels of CSF1R and reduced CX3CR1expression, suggesting an ‘immature’ microglial phenotype. In conclusion, our preliminary results show that C. innocuum derived from ASD individuals can induce gastrointestinal alterations and microglial phenotype traditionally associated with ASD, suggesting that this bacterial strain could contribute to the etiology of this neurological disorder.
Michelle Dean (Poster 19)
POET Program Manager, University of Calgary (Canada)
A multi-omics approach to examining the microbiome and its potential impact on therapeutic outcomes of de novo metastatic Non-Small Cell Lung Cancer patients at a Canadian tertiary care centre
Dean ML, Ramay H, Mager LF, Groves RA, Lewis IA, McCoy KD, Xu T, Bebb DG, Yip S, Bose P
Introduction and Methods: Gut microbiota have been increasingly associated with therapeutic response to systemic anti-cancer therapy (SACT) across various tumor types. Our objectives are to examine the relationship between the microbiome, metabolome, tumor genomic biomarkers and clinical outcomes. This prospective observational study recruited 12 patients and collected samples prior to, and three months after frontline SACT initiation. Stool and saliva microbial diversity, serum metabolomic profiles, and tumor genomic alterations were evaluated. Clinical and treatment outcome data were abstracted, and therapeutic response was categorized using Disease Control Rate (DCR).
Results: Eight patients experienced disease control as their best response during frontline SACT (DCR=66.7%). Progressive disease was noted in the remaining 4 patients categorized as non-responders (33.3%). Preliminary diversity evaluations show that saliva and stool microbiomes are remarkably different. Decreased alpha diversity was observed in stool samples from patients who received antibiotics during the sampling period, and a possible separation of response may exist based on stool microbiome composition. Beta diversity in saliva samples may also be able to be separated by smoking status. Initial observations of serum metabolomic profiles show some metabolites to be upregulated in responders.
Conclusions and Future Directions: Antibiotics may affect treatment response by decreasing the alpha diversity of gut microbiota, thereby decreasing the presence of potential anti-cancer metabolites. Smoking status in relation to the saliva microbiome is consistent with expectation given the toxicities present in the oral cavities of patients who smoke, and their detrimental effect on the microbial communities residing there. Metabolomic and tumor genomic profile results will be presented, and further outcomes will be reported and discussed. The novel multi-omics approach incorporating investigations of the microbiome and metabolome with tumor genomic profiling demonstrates the unique capabilities of the POET program to use a diverse suite of potential biomarkers in differentiating NSCLC outcomes.
Dr. Vanessa DeClercq (Poster 20)
Research Associate, Dalhousie University (Canada)
Bioinformatic Approaches for Investigating the Role of the Human Microbiome in Chronic Disease
The human microbiome can influence human health in both helpful and harmful ways and has been associated with several chronic diseases. Over the last decade, the level of interest in the role of the microbiome in chronic disease has erupted. The rate at which sequencing data is being generated is faster than current bioinformatic methods can identify robust microbial biomarkers of disease. Thus, there is a great need to improve existing or develop new methods for characterizing the relationship between types of microbes and their function in disease. To date, the most well-studied human microbiome is the gut microbiome, but other microbiomes such as the blood or oral cavity also play a role in human health and disease.
Aim: Develop novel bioinformatic methods to better understand the role of the multiple human microbiomes across various diseases.
Methods: Bioinformatic methods include integrative approaches for combining microbial taxa and function data, new methods for identification of functions, and a novel framework for biomarker discovery and visualization. These bioinformatics approaches will be tested and validated using multiple datasets, including datasets from previously published studies as well as newly generated data from ongoing/future studies. Current datasets include human data from stool and saliva samples of participants with IBD and cancer. Ongoing work includes sequencing and analysis of various biological samples (blood, stool, saliva, tumor, and bronchial fluid) from participants with frailty, mental health conditions, asthma, sepsis, and several types of cancer.
Output: This research will accelerate microbiome research by tackling current limitations in computational analysis and biomarker discovery through the creation of novel bioinformatic tools and approaches for microbiome analysis across diseases.
Dollina Dodani (Poster 21)
Graduate Student, University of British Columbia (Canada)
Translating the vaginal microbiome for improved screening and prevention in endometrial cancers
Dollina D. Dodani, Lauren Tindale, and Aline Talhouk
Endometrial cancer (EC) is the most common gynaecological malignancy in the developed world, with its incidence rising worldwide. If detected early, EC is curable by hysterectomy, but diagnosis at a later stage has poor disease-free and overall survival outcomes. Unlike colorectal, breast, and oral cancers, there are currently no screening programs for EC. While recent screening initiatives for EC have analyzed cell-free DNA (cf-DNA) and vaginal microbiome independently, they alone are unlikely to be discriminatory to cancer risk. However, taken together and in the context of epidemiological risk factors, these tools may provide a feasible approach to screen for malignancy, improving the early detection of cancer. An upcoming study will investigate EC prevention in high-risk asymptomatic women where risk and the need for an endometrial biopsy will be assigned using a lifestyle risk score and the Progesterone Challenge Test. We will then obtain 40 matched self-collected vaginal microbiome swabs and cf-DNA from tampons. We aim to develop a neural network framework to determine a microbiome composition that distinguishes a malignant from a healthy uterine environment and establish the validity of the signature by integrating somatic mutations identified using cf-DNA from matched tampon samples. Our multi-omics approach will provide a proof-of-concept in equipping clinicians with a robust microbiome signature for when menopausal women should have a biopsy to rule out cancer. An improved EC risk prediction substantiated by genomic and epidemiological data will reduce the number of women who need a biopsy, thus making EC prevention initiatives more acceptable.
Sharon Dong (Poster 22)
MSc Student, University of Calgary (Canada)
Genetic Manipulation of Bacteroides for Transient Colonization of Germ-free Mice
Sharon F. Dong, Carolyn A. Thomson, Regula Burkhard, Trevor E. Randall, Joe J. Harrison, Kathy D. McCoy, Markus B. Geuking
Aims: We develop a microbial relationship early in life that impacts our lifelong health. Experimental tools like auxotrophic mutant bacteria that cannot synthesize certain nutrients can be used to temporarily colonize mice. A mutant E. coli strain, HA107, has been used to study effects of gestational colonization of pregnant dams on immune development in the offspring. To expand this toolbox and be able to control the timing and dose of exposure of germ-free mice to relevant Bacteroides species, I aim to generate mutant Bacteroides strains targeting analogous genes disrupted in HA107.
Methods: I use the pExchange recombination system that uses antibiotic resistance genes and a counter-selection gene product, Tdk, that leads to cytotoxicity in the presence of FUdR, to allow for targeted deletion of genes in Bacteroides thetaiotaomicron. This system also allows for sequential deletion of genes without residual antibiotic resistance cassettes. I target genes that synthesize uniquely-bacterial amino acids that are incorporated into peptidoglycan structures of gram-negative bacteria, meso-diaminopimelic acid (mDAP) and the D-isomer of Alanine (D-Ala). The mutants will grow with external supplementation, but not in germ-free mouse guts.
Results: I deleted alr-murF (BT_4101) and asd BT_3636 in B. thetaiotaomicron. The mutants grow when supplemented with D-Ala and mDAP, but not in non-supplemented media. Preliminarily, germ-free mice gavaged with the mutant strain carry lower fecal load of bacteria from 8-24 hours after gavage versus mice gavaged with the wild-type parent strain.
Conclusions: I generated bacterial strains that can transiently colonize germ-free mice by strategic gene deletion. These strains will provide insights into how bacteria communicate with the immune system, like during gestation where maternal intestinal microbes influence fetal immune system development and health outcomes.
Dr. Marija Drikic (Poster 23)
Research Associate, University of Calgary (Canada)
Method for quantifying the metabolic boundary fluxes of cell cultures in large cohorts by high resolution hydrophilic liquid chromatography mass spectrometry
Marija Drikic, Ryan A. Groves, Maryam Mapar, Raied Aburashed, Luis F. Ponce, Stephanie L. Bishop, Thomas Rydzak, Dominique G. Bihan, Hallgrimur Benediktsson, Fiona Clement, Daniel B. Gregson, and Ian A. Lewis
Metabolomics is gaining importance as an investigation tool engaged in the discovery and characterization of novel molecular mechanisms associated with physiological and pathological states of microbiome communities. Microbiome studies are inherently complex and diversified and as such, there is a need for metabolomic methods that are compatible with large sample cohorts that can properly address the many factors at play in each study. The available methods, while performing in smaller cohorts, present limitations when applied to larger cohorts. Many of these limitations come from the inherent time-dependent changes in the liquid chromatography mass spectrometry (LC-MS) system, which affect both the qualitative and quantitative performance of the instrument. In this study, we introduce a new analytical method for addressing these limitations in large-scale microbial studies. Our approach is based on quantifying microbial boundary fluxes using two multiplexed zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns, allowing for a high rate of sample throughput. By applying this method, we show that over 360 common metabolites are detected in 4.5 minutes per sample and those metabolites are quantified with a median coefficient of variation of 0.127 across 1,100 technical replicates. We applied this strategy to analyze 960 strains of Staphylococcus aureus isolated from blood stream infections. These data capture the diversity of metabolic phenotypes observed in bacterial isolates collected from patients and provide an example of how large-scale investigations can benefit from our novel analytical strategy.
Yi (Edward) Fan (Poster 24)
PhD Student, University of Alberta (Canada)
Week-old broiler chickens with high relative Bacteroides abundance have increased cecal short chain fatty acids and reduced expression of inflammatory cytokines
Members from the genus Bacteroides are important commensals in the chicken intestine. Bacteroides are essential complex carbohydrate degraders and short-chain fatty acid (SCFA) producers that are highly adapted to the distal gastrointestinal tract. Previous studies have shown great variation in relative abundance of Bacteroides present in the ceca of young chickens ranging from 2 to 45%. However, limited information is available regarding how these distinct gut microbial communities affect host gut immunity, nutrient utilization, and gut microbial functional genomics. In the current study, we sampled 70 seven-day-old broiler chickens from 14 production flocks to study the cecal microbial structure, functional genomics, SCFA concentration, and host gut immune status. The 16S rRNA gene sequencing result revealed distinct Bacteroides compositions. Based on relative abundance, low-Bacteroides (LB) group and high-Bacteroides (HB) group were identified and further confirmed by Bray-Curtis distance metric and qPCR assay (P<0.01). Spearman pairwise correlation analysis suggested that Lactobacillus composition was negatively correlated to Bacteroides (P<0.05). Shotgun metagenomics sequencing results revealed that the gut microbiota of the LB group had increased abundance of the lactic acid bacteria pyruvate fermentation pathway, whereas the HB group was more abundant in complex polysaccharide degradation and SCFA production pathways (P<0.05, LDA score>2). Moreover, gas chromatography showed that the HB group had increased SCFA concentration in the chicken ceca (P<0.05). The HB group showed decreased gene expression of pro-inflammatory cytokine IL-1β with increased expression of anti-inflammatory cytokine IL-10 and tight-junction protein CLDN1 (P<0.05). In conclusion, this research suggests competitive relationships between Lactobacillus and Bacteroides in the ceca of young chickens. Cecal Bacteroides composition, specifically in the HB microbial community, increased SCFA production which contributed to increased gut integrity and a more immunetolerant gut environment.
Zhi Yi Fang (Poster 25)
MSc Student, Queen’s University (Canada)
Maternal genomics and human milk oligosaccharides are associated with milk microbiota of lactating mothers and risk of asthma among breastfed infants in the CHILD Cohort Study
Zhi Yi Fang, Sara A. Stickely, Amirthagowri Ambalavanan, Yang Zhang, Bianca Robertson, Chloe Yonemitsu, Kelsey Fehr, Shirin Moossavi, Stuart E. Turvey, Piushkumar J. Mandhane, Elinor Simons, Theo J. Moraes, Malcolm R. Sears, Padmaja Subbarao, Lars Bode, Meghan B. Azad, Qingling Duan
The benefits of breastfeeding on asthma have been reported by some earlier studies but others have described no associations. These inconsistencies may be due in part to the consideration of breastfeeding as a homogenous exposure. Yet, human milk is complex and dynamic, including a myriad of components such as human milk oligosaccharides (HMOs) and microbes. In this study, we determine the main and interaction effects of maternal genomics and HMOs on milk microbial composition and lung health of breastfed infants. Milk samples from 1206 mothers of the CHILD Cohort Study, collected between 3-4 months postpartum, were used to quantify the 19 most abundant HMOs by high-performance liquid chromatography. In a subset of mothers’ milk samples (N=885), milk microbial composition was assessed by 16S rRNA sequencing. Genome-wide single nucleotide polymorphisms (SNPs) were genotyped in these mothers using the Illumina HumanCoreExome BeadChip. First, we determined the association between milk microbiota and recurrent wheeze (RW, ages 2-5 years) using a generalized linear model. Our results indicate that breastfed children exposed to increased Pseudomonas in mothers’ milk had lower prevalence of RW (FDR-adjusted P=0.008). Next, we assessed the main and interaction effects of maternal genomics and HMO concentrations on the milk microbes implicated with RW. We identified that interactions between SNPs on chromosome 10 (e.g., rs145643568) and the HMO Disialyl-lacto-N-hexaose (DSLNH) were associated with abundance of Pseudomonas (P=7.35E-9). Thus, our study suggests that maternal genomics and variable abundances of specific HMOs are associated with the milk microbiota and lung health of the breastfed infants. Further investigations, however, are needed to determine a causal link between these human milk components and childhood asthma risk. These results may help facilitate the development of new milk probiotics, which could support lung health of children.
Claire Gerkins (Poster 26)
MSc Student, Université de Montréal (Canada)
The modulation of intestinal inflammation by Parabacteroides goldsteinii in murine models of colitis
Claire Gerkins, Manon Oliero, Roy Hajjar, Hervé Vennin Rendos, Gabriela Fragoso, Annie Calvé, Khoudia Diop, Bertrand Routy, Manuela M. Santos
Inflammatory bowel diseases (IBDs), comprised mainly of Crohn’s disease and ulcerative colitis, are characterized by chronic and relapsing intestinal inflammation. Though the etiology of IBDs is not entirely understood, it is thought that an imbalance of the gut microbiota is one contributing factor, making it a potential target for the treatment of IBDs. This study aims to examine how the potential probiotic Parabacteroides goldsteinii modulates intestinal inflammation in the context of experimental colitis.
Wild type C57BL/6 mice were divided into three groups. One group was given phosphate-buffered saline (PBS) via oral gavage without any further treatment. The second group was treated with 1% dextran sodium sulfate (DSS) for twelve days induce colitis and given PBS every three days. Finally, the third group was also treated with 1% DSS for twelve days and given P. goldsteinii via oral gavage every three days. Weight loss, stool consistency, and intestinal bleeding were monitored and were used to calculate the Disease Activity Index (DAI) daily. Postmortem, colon length was measured, and inflammation score was evaluated on hematoxylin and eosin-stained samples of the colon and spleen. Finally, interleukin (IL)-6 levels were measured in the colon tissue and the serum by ELISA.
Colonization was confirmed at day three after gavage by real-time PCR. Preliminary results show that DSS-treated mice lost significantly more weight by day twelve than mice colonized with P. goldsteinii. Additionally, on days eight and twelve, mice colonized with P. goldsteinii had significantly lower DAI scores than mice that received only PBS. Finally, mice colonized with P. goldsteinii had significantly longer colons than those that received only PBS. The preliminary data indicates that mice colonized with P. goldsteinii had less severe symptoms of colitis than mice only given PBS. This suggests that P. goldsteinii may attenuate inflammation in mice treated with DSS.
Chanel Ghesquiere (Poster 27)
Graduate Student, University of British Columbia/British Columbia Children’s Hospital Research Institute (Canada)
Assessing the ability of genetically engineered Live Biotherapeutic Products to reduce intestinal inflammation in a SHIP-deficient mouse model of Crohn’s disease
Chanel Ghesquiere, Susan Menzies, Maggie Ma, Deanna Gibson, Laura Sly
Inflammatory bowel disease (IBD) is a major health burden in Canada, and can be classified into two primary clinical subtypes, Crohn’s disease, and ulcerative colitis. IBD cannot be prevented, and current therapies do not work for everyone, highlighting the urgent need for new treatments. Live biotherapeutic products (LBPs) involves ingestion of non-pathogenic microorganisms to exert a positive effect. LBPs are a potential novel therapy for IBD; however, the variable ability of bacteria to colonize and persist in the inflamed gut limits clinical applicability. Two genetically engineered LBPs have been modified to enhance colonization or persistence in the IBD gut (Gibson: PCT/CA2018/050188). These LBPs were developed from the parent strains of two different probiotic human gut microbes. To assess the ability of these LBPs to reduce inflammation in the IBD gut, we tested the LBPs in the SHIP deficient mouse model of Crohn’s disease-like ileal inflammation. We administered the LBP or parent bacteria to our SHIP mouse model and healthy control mice by oral gavage. Following a four-week monitoring period, intestinal tissues and contents were collected, full-thickness ileal homogenates were used to analyze inflammatory cytokine levels, and histology of ileal cross-sections was performed to analyze histological damage scores. LBP and total bacterial load were examined in ileal, colon, and cecum contents to assess relative abundance along the GI tract. One LBP demonstrated a modest ability to reduce ileitis severity, with moderately reduced IL-1β levels in ileal homogenates and histological damage scores compared to controls. In contrast, the other LBPdid not show any evidence of reduced ileal inflammation. The ability of these LBPs to reduce the severity of ileitis likely depends primarily on their localization to, and persistence in, the site of inflammation.
Dr. Thaís Glatthardt (Poster 28)
Postdoctoral Fellow, University of Calgary (Canada)
Malassezia restricta gut colonization impacts lung immune responses to Respiratory Syncytial Virus
Thaís Glatthardt, Erik van Tilburg Bernardes, Mackenzie Gutierrez, Leanne Bilawchuk, David Marchant, Eugenia Corrales Aguilar, Marie-Claire Arrieta
Respiratory syncytial virus (RSV) is a major cause of infant bronchiolitis and pneumonia worldwide. Besides the risk of hospitalization and severe disease, early life RSV infection is associated with increased asthma risk. While the mechanisms involved in this connection are incompletely understood, the gut-lung axis is hypothesized to be involved given the roles of the gut microbiome in immune development, asthma risk, and lung immune responses to other respiratory viruses. While most microbiome studies are focused on gut bacteria, we and others have demonstrated that colonization with gut fungi potentiates mucosal and systemic immunity. Further, we recently showed that infant antibiotic use leads to an overgrowth of the fungi Malassezia restricta, and that colonization with this fungus increased susceptibility to allergic airway inflammation in mice. Thus, we hypothesized that colonization of M. restricta may also exacerbate inflammation after RSV infection and increase susceptibility to allergic airway inflammation post-infection. Eighteen day-old gnotobiotic BALB/C mice colonized with bacterial consortium Oligo-MM12 alone or in combination with M. restricta were infected intranasally with RSV (A2 strain). Lungs were collected for immune cell profiling via FACS on the fifth day post-infection. Despite poor viral particle recovery from infected lungs, we detected an increase in monocytes (CD45+Ly6C+IRF5+) and macrophages (CD45+CD64+CX3CR1+IRF5+) and Th1 T cells (CD45+TCR+CD4+Tbet+) in RSV infected mice colonized with M. restricta compared to mice colonized only with Oligo-MM12. This suggest that colonization with M. restricta exacerbates inflammation to control RSV infection. Further experiments are necessary to evaluate the impact of M. restricta on susceptibility to airway inflammation post RSV infection. Results from this work will help elucidate the mechanistic underpinnings of the gut-lung axis during early life, how it contributes to the risk of asthma and potential therapeutic strategies.
Dr. Alessandra Granato (Poster 29)
Research Associate, The Hospital for Sick Children (Canada)
Transfer of gut microbes isolated from infants at risk for type 1 diabetes into germ free NOD mice regulate spontaneous diabetes
Alessandra Granato, Steve Mortin-Toth, Simone Renwick, Christina Ohland, Christopher Yau, Tiffany Kong, Michelle Daigneault, Kathy D. McCoy, Emma Alen-Vercoe, Jayne Danska
Studies in humans and rodent models of spontaneous type-1 diabetes (T1D) link gut microbiota to disease pathogenesis. In the non-obese diabetic (NOD) mouse model, greater vivarium hygiene or treatment with antibiotics elevate diabetes frequency. Previously we showed that manipulation of the NOD mouse gut microbiota promotes alterations in host metabolism and confers robust protection from islet autoimmunity. In humans, prospective studies of infants and children with high-risk HLA haplotypes have identified early life risk factors, including multiple anti-islet autoantibodies (IAB) predictive of disease progression. DIABIMMUNE is a longitudinal birth cohort study of T1D high-risk infants from northern Europe. A lower complexity in gut bacterial composition was identified in DIABIMMUNE participants who progressed to T1D compared to nested controls who did not, but a causal relationship between specific early life gut microbes and progression to diabetes has not been established. Here, we investigate the functional impact of DIABIMMUNE participant’s gut bacteria by transfer into germ-free NOD mice. To generate reproducible, well-defined bacterial consortia, we first cultured DIABIMMUNE infant fecal samples in a host-free bioreactor system that models the distal human colon and propagates bacterial communities representative of the source sample. Hundreds of bacterial strains were isolated, sequenced and selected based on their prevalence in samples originated from infants who did or did not progress to IAB+ seroconversion or a T1D diagnosis. Following colonization of young germ-free NOD mice, defined consortia of bacterial strains isolated from IAB- infants had far lower T1D incidence compared to mice colonized with bacterial strain consortia isolated from IAB+ high-risk infants. These results show that gut microbes isolated from IAB- compared to IAB+ infants differentially regulate spontaneous autoimmune diabetes and suggest that early life intestinal microbes exert a causal impact on the disease.
Mackenzie Gutierrez (Poster 30)
PhD Student, University of Calgary (Canada)
Early-life Fungal Colonization Mediates Host Metabolism and White Adipose Tissue Inflammation in Mice
Mackenzie W. Gutierrez, Erik van Tilburg Bernardes, Kristen Kalbfleisch, Faye Chleilat, Marie-Claire Arrieta
The gut microbiome plays an important role in host energy metabolism and metabolic inflammation. However, the role of the mycobiome in this context has been understudied. We explored the contribution of early-life fungal colonization with Candida albicans and Rhodotorula mucilaginosa in host metabolism and metabolic inflammation under normal and obesogenic diet conditions. Gnotobiotic mice were colonized from birth with a community of 12 mouse-derived bacteria (Oligo-MM12) alone or in combination with either fungal species. C. albicans colonization reduced body weight in mice fed a control diet, and induced resistance to weight gain and increased adiposity in mice fed an obesogenic diet. In contrast, R. mucilaginosa colonization was associated with increased adiposity in mice fed control diet, and elevated glycemia and LDL-cholesterol in mice fed the obesogenic diet. Fungal colonization had a broad impact on immune cells in gonadal white adipose tissue. C. albicans colonization was associated with increased adipose tissue inflammation with elevated neutrophils, cDC1s, 𝛾𝛿T cells and ILC1s independently of diet. Additionally, vascular associated macrophages, CX3CR1+ macrophages and DCs, ILC3s and Th17 were elevated in mice fed control diet, while mice fed the obesogenic diet displayed elevated B cells, CD8+ T cells, NK cells and eosinophils. In contrast, R. mucilaginosa colonized mice displayed decreased adipose tissue B cells and Tregs when fed control diet, and increased ILC3s when fed the obesogenic diet. Elevated adipose tissue inflammation in C. albicans colonized mice suggests dysfunction of energy storage and may explain their decreased body weight and resistance to diet-induced obesity, while the immune changes in R. mucilaginosa colonized mice may contribute to alterations in glucose metabolism. This work revealed that two common fungal colonizers have distinct and striking influences on host metabolism and metabolic inflammation and prompts for the inclusion of fungi in microbiome studies on host metabolism.
Diana Haider (Poster 31)
PhD Candidate, Dalhousie University (Canada)
Mock microbial community meta-analysis using different quality thresholds
Diana Haider, Michael Hall, Julie Laroche, Robert Beiko
The abundance of new high-throughput sequencing datasets is increasing rapidly with researchers studying microbes from every conceivable niche. Comparability and reproducibility across studies from the same discipline are still a challenging task: while software pipelines such as QIIME2 are available, the correct workflow and analysis parameters are not clear, and biases can be introduced at any step from library preparation to ecological interpretation. In this study, we systematically evaluate the impact of varying the trimming length of Illumina 2x300bp reads generated from 16S and 18S rRNA mock microbial communities of known composition. Read trimming discards low-quality nucleotides from reads to avoid false discoveries, and maintains all reads at the same length for clustering into Amplicon Sequence Variants (ASVs). Trimming lengths were varied from zero (i.e., retention of the entire read) to the entire read length (i.e., elimination of the entire read) for both forward and reverse reads. We compared taxonomic trees between observed and expected communities trimmed at different combinations of trim lengths, and observed that there is a trade off between the type of false positives: groups taxonomically distant from the expected community at low frequencies, or groups closely related to expected at higher frequencies. Keeping forward reads longer than 230 nt lead to better taxonomic classifications down to the species level, and keeping reverse reads longer than 110 nt induced erroneous discoveries due to the poor quality of reverse reads. Regardless of the trim length selected, there are groups that are constitutively underrepresented due to mismatches with the primer. To summarize, small variations in the filtering of amplicon reads changes the final composition of microbial communities, supporting the need for an effort to evaluate the discrepancies generated from a range of different parameters throughout the analysis of sequencing data to find suitable parameters to optimize filtering and denoising.
Dr. Alya Heirali (Poster 32)
Postdoctoral Fellow, University of Toronto (Canada)
Peripheral biomarkers of the gut microbiome and response to microbiome-targeting therapies in individuals with solid tumors
Alya Heirali, Daniel V. Araujo, Tira J. Tan, Marc Oliva, Pierre H.H. Schneeberger, Bo Chen, Matthew K. Wong, Lee-Anne Stayner, Aaron Richard Hansen, Samuel D. Saibil, Ben X. Wang, Sawako Elston, Kyla Cochrane, Keith Sherriff, Emma Allen-Vercoe, Wei Xu, Lillian L. Siu, Anna Spreafico, Bryan Coburn
Background: The gut microbiome has been causally implicated in response to immune checkpoint inhibitors (ICI) for solid tumors. Therapeutic augmentation of the microbiome to improve patient outcomes is a research priority. Fecal microbiome transplantation (FMT) with stool from ICI responders overcame resistance in 30-40% of patients with ICI-refractory melanoma in two recent trials. Microbial ecosystem therapeutics-4 (MET4) is a consortium of 30 bacterial species designed as an alternative to FMT. Microbial-derived metabolites including bile acids (BA) and short chain fatty acids (SCFA) can be detected in blood. However, our understanding of blood as a biomarker of gut microbiome composition, function, and response to microbiome-targeting therapies is limited. We sought to assess these as biomarkers in patients with solid tumors receiving ICI +/-MET4.
Methods: Plasma samples were profiled for panels of BA and SCFA. Patients were defined as ecological responders (EcoR) to MET4 if they had at least 5 MET4 taxa increase by at least log10 post MET4. Differences in metabolites were measured longitudinally, between MET4 recipients and controls and compared between EcoR and ecological non-responders (EcoNR).
Results: Forty patients were enrolled in a trial assessing the safety of MET4 in combination with ICI. We assessed a subset of these patients whom plasma samples were available at week 3-4 pre-ICI/MET4 (n=25 samples), week 3-4 post ICI (n=25 samples) and week 3-4 post MET4 (n=18 samples). No significant differences were observed in SCFA temporally, between MET4 recipients and controls, nor between EcoR and EcoNR. However, we observed a decrease in taurochenodeoxycholic acid (median log 2 fold -2.47, p=0.041) and glycocholic acid (-0.71, p=0.021) in EcoR post MET4 but not in EcoNR.
Conclusions: Plasma BA and SCFA were stable pre/post ICI +/-MET4 initiation. However, MET4 EcoR had a significant decrease in BA, validating the pursuit of these biomarkers as correlates of ecological response.
Dr. Aline Ignacio Silva (Poster 33)
Postdoctoral Fellow, University of Calgary (Canada)
Eosinophils: the forgotten players of intestinal homeostasis
A. Ignacio, K. Shah, J. Bernier-Latmani, Y. Köller, G. Coakley, M. Moyat, R. Hamelin, F. Armand, N. C. Wong, H. Ramay, C.A. Thomson, R. Burkhard, Hazhoe Wang, A. Dufour, Markus. B. Geuking, B. McDonald, T.V. Petrova, N.L. Harris, K. D. McCoy
Intestinal homeostasis following postnatal microbial colonization requires the coordination of multiple processes including the activation of immune cells, cell-cell communication, the controlled deposition of extracellular matrix, epithelial cell turnover and differentiation. The intestine harbors the largest number of resident eosinophils of all homeostatic organs, yet the functional significance of eosinophil recruitment to this organ has long remained enigmatic. Using the eosinophil-deficient dbl.GATA1 mouse strain this study investigated the impact of eosinophils deficiency to small intestine (SI) homeostasis. We hypothesized that commensal microbes are central regulators of eosinophil activation and function. To address whether the intestinal microbiota influences eosinophil-mediated functions, Δdbl.GATA1 mice were re-derived to germ-free status followed by colonization in adulthood for a period of 4 weeks. Balb/c mouse strain was used as control. To elucidate the direct role of loss of eosinophils in intestinal homeostasis, bone marrow-derived eosinophil were generated in vitro and transferred into colonized Δdbl.GATA1 mice. Remarkable, we found the microbiota to be a critical regulator of eosinophil activation and function on the small intestine. We observed that eosinophils play a role in regulating villous architecture where eosinophil deficiency resulted in impairment of epithelial barrier integrity, increased intestinal motility, and decreased number of Ly6C-MHCII+ terminally differentiated macrophages in the SI. Eosinophil transfer rescued microbial-induced villous atrophy, transit time and increased the number of terminally differentiated macrophages in the SI. Overall, our work provides rationale for the early life recruitment of eosinophils in the small intestine, implicating these cells as critical facilitators in the establishment of proper mutualistic interaction between host and microbiota.
Baofeng (Justin) Jia (Poster 34)
PhD Candidate, Simon Fraser University (Canada)
Metagenome-assembled genome binning methods with short reads disproportionately fail for plasmids and genomic islands
Baofeng Jia, Finlay Maguire, Kristen L. Gray, Wing Yin Venus Lau, Robert G. Beiko, and Fiona S. L. Brinkman
Metagenomic methods enable the simultaneous characterization of microbial communities without time-consuming and bias-inducing culturing. Metagenome-assembled genome (MAG) binning methods aim to reassemble individual genomes from this data. However, the recovery of mobile genetic elements (MGEs), such as plasmids and genomic islands (GIs), by binning has not been well characterized. Given the association of antimicrobial resistance (AMR) genes and virulence factor (VF) genes with MGEs, studying their transmission is a public-health priority. The variable copy number and sequence composition of MGEs makes them potentially problematic for MAG binning methods. To systematically investigate this issue, we simulated a low-complexity metagenome comprising 30 GI-rich and plasmid-containing bacterial genomes. MAGs were then recovered using 12 current prediction pipelines and evaluated. While 82–94 % of chromosomes could be correctly recovered and binned, only 38–44 % of GIs and 1–29 % of plasmid sequences were found. Strikingly, no plasmid-borne VF nor AMR genes were recovered, and only 0–45 % of AMR or VF genes within GIs. We conclude that short-read MAG approaches, without further optimization, are largely ineffective for the analysis of mobile genes, including those of public-health importance, such as AMR and VF genes. We propose that researchers should explore developing methods that optimize for this issue and consider also using unassembled short reads and/or long-read approaches to more fully characterize metagenomic data. Considering this bias and limitation of MAGs, we further developed a readbased approach to detecting AMR genes in metagenomics data, named AMRTime, that will recover AMR genes with high accuracy, including those located on MGEs.
Dr. Tingting Ju (Poster 35)
Postdoctoral Fellow, University of Alberta (Canada)
The presence of gut commensal Escherichia coli aggravated high-fat diet-induced obesity and insulin resistance
Tingting Ju, Benjamin C. T. Bourrie, Andrew J. Forgie, Deanna M. Pepin, Stephanie Tollenaar, Consolato M. Sergi, and Benjamin P. Willing
The enrichment of the family Enterobacteriaceae, largely represented by Escherichia coli, induced by a high-fat diet (HFD) feeding has been correlated with impaired glucose homeostasis; however, whether the enrichment of Enterobacteriaceae in a complex gut microbial community in response to a HFD contributes to metabolic disease has not been established. To investigate whether the expansion of Enterobacteriaceae amplifies HFD-induced metabolic disease, a tractable mouse model with the presence or absence of a commensal E. coli strain was established. With a HFD treatment, but not a standard chow diet, the presence of E. coli significantly increased body weight and adiposity and induced impaired glucose tolerance. In addition, E. coli colonization led to increased inflammation in liver, adipose and intestinal tissue under a HFD regimen. Although the presence of commensal E. coli had a modest effect on microbial composition, it resulted significant changes in the predicted functional potential of microbial communities. The results demonstrated the role of commensal E. coli in glucose homeostasis and energy metabolism responding to a HFD treatment, indicating contributions of commensal bacteria to the pathogenesis of obesity and type 2 diabetes. This is the first study to show the addition of a single bacterial species to an animal already colonized with a complex microbial community can increase severity of metabolic outcomes. The findings of this research identified a targetable subset of the microbiota in the treatment of people with metabolic inflammation.
Dr. Larisa Kovtonyuk (Poster 36)
Postdoctoral Fellow, University of Calgary (Canada)
Inflammageing of Hematopoietic Stem Cells is driven by microbiome via IL-1
Larisa V. Kovtonyuk, Francisco Caiado, Santiago Garcia-Martin, Eva-Maria Manz, Patrick Helbling, Hitoshi Takizawa, Steffen Boettcher, Fatima Al-Shahrour, Cesar Nombela Arrieta, Emma Slack, and Markus G. Manz
Lifelong continuous blood production is sustained through a stepwise differentiation program by a very limited number of self-renewing Hematopoietic Stem Cells (HSCs) in the bone marrow (BM). Hematopoietic cell development is tightly controlled by both cell intrinsic and extrinsic factors and its dysregulation can lead to aplasia or neoplasia. During ageing, hematopoietic stem cells (HSCs) increase in number, reduce self renewal capacity, skew towards myeloid differentiation, and show mitigated bone marrow (BM)–homing ability. We here evaluated how and to what extend HSC-extrinsic factors determine HSC behaviour during aging. Firstly, we observed that aged specific pathogen free (SPF) wild-type mice, in contrast to young SPF mice, produce more IL-1a/b in bone marrow (BM), with most of IL-1a/b being derived from myeloid BM cells. Secondly, blood of aged WT SPF mice contains higher levels of microbe associated molecular patterns (MAMPs), specifically TLR4 and TLR8 ligands. Thirdly, upon LPS stimulation, BM myeloid cells from aged mice produce more IL-1 in vitro, and aged mice show higher and more durable IL-1a/b responses in vivo. Given these observations together, we hypothesized that HSC ageing is driven via microbiome/IL-1 axis, to test this we evaluated HSCs from IL-1R1KO (SPF) and WT germ free (GF) mice. Indeed, aged HSCs from IL-1R1KO (SPF) and WT (GF) mice show significantly mitigated ageing-associated inflammatory signatures and maintain unbiased lympho-myeloid hematopoietic differentiation upon transplantation, thus resembling the functionality of young WT (SPF) HSCs. Finally, reducing the inflammatory burden by in vivo antibiotic suppression of microbiota or pharmacologic blockade of IL-1 signalling in aged WT mice was similarly sufficient to reverse myeloid biased output of their HSC populations. Our data demonstrate that ageing associated phenotype and myeloid-biased differentiation of HSCs is a result of signals derived from the microbiome, that act through increased IL-1 signalling, locally in BM.
Of note: the work presented in this abstract was done at the University Hospital Zurich and the University of Zurich (Switzerland)
Hannah Lantz (Poster 37)
MSc Student, University of Alberta (Canada)
Exploring Early Life Escherichia coli and Amoxicillin Interventions on Subsequent Salmonella Resistance in a Swine-derived Bacterial Gnotobiotic Mouse Model
Hannah Lantz, Tingting Ju, Stephanie Tollenaar, Camila Marcolla, Janelle Fouhse, Benjamin Willing
Many associations between the gut microbiota, host health, and disease have been revealed, however, mechanistic research to determine causality is necessary. Preliminary results suggesting that Escherichia coli and amoxicillin administration in early life can enhance piglets’ immune responsiveness to intraperitoneal heat-killed Salmonella enterica serovar Typhimurium (S. Tm) were tested in our swine-derived bacterial defined community (DC) mouse model. Female C57BL/6J mice colonized with the swine DC with or without E. coli were bred, and during the first two weeks of their pups’ lives dams were either administered sterile amoxicillin drinking water (25mg/kg/day) or sterile drinking water. The 4 pup treatment groups (E. coli + Amoxicillin (EA), n=5; E. coli, n=8; Amoxicillin, n=8; and Control, n=8) were then challenged with live S. Tm SL1344 at 6-weeks-of-age, after normalization of the gut microbiota on post-natal day 21. The S. Tm load in both the ileum and liver significantly differed by treatment, with the lowest loads observed in the EA group, suggesting that the combined neonatal exposure to E. coli and amoxicillin does facilitate local and systemic pathogen burden later in life (P<0.05). Sex differences in S. Tm load and cytokine production were observed without accompanying microbiota differences, but no treatment cytokine differences were observed. Males suffered from significantly greater S. Tm loads in the ileum, cecum, and liver with significantly increased IL-6 and IL-10 (P<0.05). While largely underexplored in S. Tm infection, sexual dimorphism was consistent with other enteric infections and may be due to a greater innate and cell-mediated immune response without excessive proinflammatory cytokine production. While these results show that early life microbial and antibiotic interventions have long lasting impacts on S. Tm resistance, further research is needed to unravel the mechanisms behind early life E. coli and amoxicillin exposure on immune system development and subsequent disease resistance.
Sukyoung (Suzie) Lee (Poster 38)
MSc Student, University of Calgary (Canada)
Of Sex, Gut and Brain: Functional studies in a mouse model of Amyotrophic Lateral Sclerosis-Frontotemporal Dementia
Sukyoung Lee, Yulan Jiang, Matthew Stephens, Keith Sharkey, Gerald Pfeffer and Minh Dang Nguyen
Amytrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) is a spectrum of neurodegenerative diseases characterized by the progressive loss of the motoneurons (typical of ALS), and cognitive dysfunction (characteristic of FTD). Previous studies have identified biological sex as an important determinant in the presentation and risk of ALS-FTD, but the mechanisms underlying these sex differences are poorly understood. Emerging studies suggest that the gut microbiota plays a key role in ALS disease initiation and progression. In humans, the microbiota composition varies significantly between males and females, suggesting that such variation may account for the male susceptibility in disease. Using 16S sequencing, we analyzed the gut microbiome composition in mice expressesing a mutant form of the TAR DNA binding protein 43 (TDP43) linked to ALS-FTD. We performed the analysis based on genotype, age and sex, and related our findings to the sexually dimorphic motoneuron disease and lifepsan of the animals. We went on to functionally test the role of a bacterial product derived from a pathogenic species identified in the gut of TDP43 mice. Details of our results will be presented at the meeting.
Dr. Karen Lithgow (Poster 39)
Postdoctoral Fellow, University of Calgary (Canada)
Resolving glycogen and related enzymes reveals correlates of Lactobacillus crispatus dominance in a cohort of young African women
Karen V. Lithgow, Athena Cochinamogulos, Kevin Muirhead, Shaelen Konschuh, Lynda Oluoch, Nelly R. Mugo, Alison C. Roxby, and Laura K. Sycuro
Background: A healthy vaginal microbiome is dominated by Lactobacillus species that produce lactic acid, lowering vaginal pH and limiting pathogen colonization. Lactobacillus dominance (LD) is established during puberty, but many women, especially those of Black race, lose LD during their reproductive years. Loss of LD increases the risk for acquiring sexually transmitted infections (STIs), including HIV. Glycogen is thought to be a key host nutrient that supports vaginal lactobacilli and their fermentative lactic acid production, but mechanisms of vaginal glycogen utilization are incompletely understood.
Methods & Objective: We established new methods that biochemically segregate glycogen and glycogen-derived maltodextrin, as well as the activities of glycogen-degrading enzymes in clinical samples. We coupled these with metagenomic profiling of the vaginal microbiome to determine how glycogen catabolism relates to Lactobacillus dominance in a pilot study of young African women (N=17) with limited sexual experience.
Results: A majority (88%) of girls exhibited LD and 65% were dominated by L. crispatus. Glycogen and pullulanase activity, but not maltodextrin and amylase activity correlated with lactic acid. Pullulanase activity was 4-fold higher in L. crispatus-dominated samples, but 33% of dominant L. crispatus strains in our cohort lacked a functional pullulanase (pulA) gene, a 3-fold higher frequency of gene inactivation compared to other published cohorts.
Conclusions: Our results indicate that although amylase and pullulanase (amylopullulanase) activities jointly drive the accumulation of glycogen catabolites in vaginal fluid, pullulanase appears to specifically contribute to maximal D-lactic acid production by L. crispatus. However, this is only possible when a functional pulA gene is present, which was not the case in a substantial proportion of young African women with dominant L. crispatus. Scaling this analysis to a larger cohort will address whether genomic and enzymatic indicators of L. crispatus pullulanase activity are predictive of sustained LD, vaginal health and protection against STIs.
Sijie (Nicole) Liu (Poster 40)
Graduate Student, University of British Columbia (Canada)
Characterization of Probiotic Strains and Their Ability to Survive in Perturbed pH and Osmolality
Sijie Liu, Sagar Pannu, Katharine M. Ng, Carolina Tropini
Probiotics have been identified as potential therapeutic vessels for numerous intestinal conditions, but we do not yet understand their ability to colonize a host, particularly one whose gut environment has been affected by disease. For example, patients with severe inflammatory bowel disease can have significantly decreased colonic pH and increased gut osmolality due to malabsorption. Physical factors such as pH and osmolality are key in determining the ability of bacteria to survive and colonize within the gut and need to be investigated in the context of probiotic therapy. Lactic acid bacteria (LAB) and Bifidobacterium are common probiotic microorganisms that have been used in the management and towards treatment of inflammatory conditions, allergy, and cancer. This project aims to investigate the strain-specific adaptability of commercially available probiotics strains to pH and osmolality growth conditions identified in disease. Different strains of LAB and Bifidobacterium were isolated from ten commercially available probiotics and assessed for their growth in various pH and osmolality conditions. We quantified their ability to grow in limiting conditions and to impact their surrounding abiotic environment. We found that probiotics of the same species exhibit differential growth responses to high osmolality and extreme pH conditions, which we hypothesize are caused by the variations in bacterial genomes. Biotherapeutics such as probiotics are a promising treatment alternative to conventional drugs, but their effectiveness in disease management remains controversial. This study will help identify characteristics that are important for probiotic strain colonization and therapeutic efficacy and will ultimately facilitate the development of microbiota-based therapies.
Dr. Pekka Määttänen (Poster 41)
Associate Professor, Burman University (Canada)
Lifestyle Medicine and the Microbiome: Do Lifestyle and Diet Immersion Programs with Significant Health Benefits Shift the Microbiome?
Naomi Akanmori, Richard Gonzalez, Ethan Burgess, Boss Lee, Tiina Keronen, Aki T. Pirskanen, Cami Gotshall, Neil Nedley, Francisco E Ramirez, Devin Holman and Pekka Määttänen
Background: The human microbiota affects physical and mental health, but the extent to which changes in the microbiota are necessary or sufficient for healthy outcomes in humans remains unclear. The Karpalo program is a 5-day live-in with 6-week follow-up program that focuses on cardiometabolic health. The Nedley Depression and Anxiety Recovery Program (NDARP) is a 10- day intensive residential program focused on mental health. Both programs serve strictly plantbased diets and emphasize exercise, fresh air, sunlight, adequate hydration, abstinence from harmful substances, rest, and trusting relationships. Both programs consistently result in significant improvements in both mental and physical health in their participants.
Methods: Cardiometabolic health (blood pressure, cholesterol, weight, BMI, fasting glucose, triglycerides, pulse rate, hsCRP) and mental health (BDI, PHQ-9) were measured in participants in the Karpalo program (n=12-23), while mental health scores were collected from 26 participants in the DARP. Stool samples were collected from all participants before and after enrollment, DNA was extracted, and 16S rRNA gene sequencing was used to analyze the microbiota. Functional profiling of the microbiomes was also carried out using shotgun metagenomic sequencing, and these results were compared with participant food questionnaires and other metadata to determine potential functional links to changes in health status.
Results: Despite significant weight loss, decreased BMI, lower systolic and diastolic blood pressure, lower cholesterol, decreased fasting serum glucose, and lower depression scores, no overall significant differences in microbial diversity or taxonomy were detected in the gut microbiota of patients after participation in each program, although certain individuals did show dramatic shifts in their microbiomes. Linkage of these differences, for example, to major dietary changes, did not reveal obvious patterns for the shifts.
Conclusion: Lifestyle immersion programs that significantly impact the health of individuals do not necessarily cause dramatic changes in gut microbial taxonomy.
Emily Mercer (Poster 42)
PhD Candidate, University of Calgary (Canada)
Interkingdom dynamics and nutrition are associated with divergent maturational patterns of the infant bacterial and fungal gut microbiome in the first year of life
Emily M. Mercer, Allan B. Becker, Elinor Simons, Piushkumar J. Mandhane, Stuart E. Turvey, Theo J. Moraes, Malcolm R. Sears, Padmaja Subbarao, Meghan B. Azad, and Marie-Claire Arrieta
The infant gut microbiome undergoes ecological shifts characterized by increasing bacterial and decreasing fungal alpha diversity in early life. Research has shown that not all infants follow these maturational trends, but our understandings of the ecological and environmental factors linked to atypical microbiome maturation patterns are limited. We assessed patterns of gut microbiome maturation in the first year of life in 100 infants from the CHILD Cohort Study and identified factors associated with atypical maturational trajectories. 16S and ITS2 analyses of the microbiome at 3 and 12 months revealed 24% and 20% of infants displayed atypical alpha diversity trajectories for bacteria and fungi, respectively. Atypical maturation patterns were associated with reduced relative abundance of Bacteroides spp. and increased Candida spp. at 3 months. Functional analysis by untargeted urine metabolomics revealed that an atypical bacterial trend was associated with increased trimethylamine n-oxide, indole acetic acid, creatine, and 2-furoylglycine, whereas an atypical fungal trend was associated with increased lactate. Using random forest analyses, the strongest predictors of atypical trajectories for both bacteria and fungi were interkingdom dynamics, breastfeeding duration, and maternal diet during pregnancy. Logistic regression revealed that an atypical bacterial alpha diversity trend was positively associated with delivery via cesarean section and infant fut2 secretor genotype and inversely associated with exclusive breastfeeding at 3 months and maternal fut2 secretor genotype, whereas an atypical fungal trend was positively associated with gestational consumption of artificially sweetened beverages and inversely associated with prenatal antibiotics exposure. Together, this highlights the importance of multi-kingdom analyses at the individual level to generate more nuanced understandings of microbiome maturation in early life. While known microbiome-modifying factors proved to be important determinants of maturational trajectories, future analyses will evaluate the role of interkingdom dynamics in mediating these relationships and the impact of atypical maturational patterns on infant development.
Dr. Chunlong Mu (Poster 43)
Postdoctoral Associate, University of Calgary (Canada)
Targeted gut microbiota manipulation attenuates seizures in a model of infantile spasms syndrome
Chunlong Mu, Naghmeh Nikpoor, Thomas A. Tompkins, Anamika Choudhary, Melinda Wang, Wendie N. Marks, Jong M. Rho, Morris H. Scantlebury, Jane Shearer
Infantile spasms syndrome (IS) is a devastating early-onset epileptic encephalopathy associated with poor neurodevelopmental outcomes. When first-line treatment options including adrenocorticotropic hormone and vigabatrin are ineffective, the ketogenic diet (KD) is often employed to control seizures. As the therapeutic impact of the KD is influenced by the gut microbiota, we examined whether targeted microbiota manipulation, mimicking changes induced by the KD would be valuable in mitigating seizures. Employing a rodent model of symptomatic IS, we show the both the KD and antibiotic administration to reduce spasm frequency and to be associated with improved developmental outcomes. Spasm reductions were accompanied by specific gut microbial alterations including increases in Streptococcus thermophilus and Lactococcus lactis. Mimicking the fecal microbial alterations in a targeted probiotic, we administered these species in a 5:1 ratio. Probiotic administration reduced seizures and improved locomotor activities in control diet-fed animals, similar to KD-fed animals while a negative control (Ligilactobacillus salivarius) had no impact. Probiotic administration also increased antioxidant status and decreased pro-inflammatory cytokines. Results suggest that a targeted probiotic reduces seizure frequency and improves locomotor activity in a rodent model of IS and provides new insights into microbiota manipulation as a therapeutic avenue for pediatric epileptic encephalopathy.
Dr. Van Ortega (Poster 44)
Postdoctoral Fellow, University of Calgary (Canada)
The influence of early-life gut microbiome disruption on microglial development, HPA stress axis dysregulation and allergic asthma susceptability
Van A. Ortega, Colin Mackenzie, Fernando A. Vicentini, Muhammad Hafeez, Shirin Moossavi, Erik van Tilburg Bernardes, Emily Mercer, Kristen Kalbfleisch, Jumana Samara, Veronika Kuchařová Pettersen, Keith A. Sharkey, Marie-Claire Arrieta
Asthma is a major inflammatory disease of the bronchial airways that affects 1 in 8 Canadian children and is the leading cause of school absenteeism and pediatric hospitalizations. While the underlying causes for asthma development in children are not fully understood, chronic exposure to stress during prenatal or post-natal periods may be a key pathway for asthma. We explored the influence of early life microbiome disruption on gut-to-brain signaling pathways critical for the programming of the hypothalamic-pituitary-adrenal (HPA) stress axis, which regulates physiological responses to stress, including inflammation. Microglial cells in the brain are speculated to mediate cross-talk between the microbiome and HPA and may contribute to increased stress activation and asthma susceptibility when their development is disrupted. We evaluated susceptibility to ovalbumin-induced airway inflammation after altering the gut microbiome of newborn mice pups using two antibiotic cocktails (augmentin-clavulanate or broad spectrum), while also inducing maternal separation stress until weaning age. We observed significant microbial alterations from the combined or individual effects of antibiotics and chronic stress, including increased alpha-diversity and relative abundances of Staphylococcus, Streptococcus, Klebsiella and Escherichia in stressed mice treated with broad-spectrum abx, or from stress alone. Early-life stress and/or abx exposure also resulted in disrupted glucocorticoid levels (hormonal end-product of HPA activation), significantly higher proportions of activated microglia at weaning with reduced immune responses to lipopolysaccharide, and altered dendritic and macrophage splenocyte populations. Early-life exposure to stress and/or augmentin-clavulanate also increased susceptibility of adult mice to allergic asthma. Following our observation that the microbiome is a major regulating factor for neuroendocrine stress axis development, we designed experiments that inducibly knocked-out microglia in mice during early-life developmental windows to demonstrate their direct role in mediating microbiome influence on neuroendocrine stress, and on resulting inflammatory disorders like asthma.
Stuti Patel (Poster 45)
Undergraduate Student, University of Calgary (Canada)
Associations Between Prenatal Depression, Prenatal Maternal Microbiome, and Infant Temperament
Stuti Patel, Marcel van de Wouw, Lianne Tomfohr-Madsen, Catherine Lebel, Marie-Claire Arrieta & Gerald Giesbrecht
Background: 17-25% of pregnant individuals experience clinically elevated depression symptoms. This has the potential to adversely affect infant temperament. Microorganisms within the gastrointestinal tract (i.e., the gut microbiome) are linked to both depressive symptoms and temperament. Notably, lower alpha diversity and reduced Prevocella, Firmicutes, and Bacteroides families are linked to depressive symptoms. However, the association between prenatal maternal microbiome and infant temperament has not been studied.
Aim: This research aimed to understand if maternal gut microbial bacteria are associated with prenatal depression symptoms and infant temperament, and if these microbes mediate the relationship between prenatal depression symptoms and infant temperament.
Methods: Data were obtained from the Pregnancy During the Pandemic cohort (n=249 mothers and n=200 infants). Maternal stool samples were collected during the third trimester of pregnancy and analyzed using shotgun metagenomics sequencing. The Edinburgh Postpartum Depression Scale was used to assess prenatal depression symptoms and the Infant Behaviour Questionnaire-Revised was used to assess infant temperament at six months of age. Data were analyzed using Spearman correlations followed by the Benjamini-Hochberg correction.
Results: Maternal depression symptoms were not significantly associated with the relative abundance and alpha diversity of bacteria. However, infant temperament was associated with bacterial abundance- negative affectivity with Adlercreutzia equolifaciens, Prevotella copri, Parabacteroides merdae and Ruminococcus callidus ( r= .247, p = .000421; r = -.221, p = .002; r = .221, p = .002; r = -.209, p = .003), and orienting/regulation with Rothia mucilaginosa (r = .254, p = 0.000267).
Conclusion: These findings suggest that the maternal microbiome is associated with the infant’s negative affectivity and orienting/regulatory ability.
Roxanne Plante (Poster 46)
MSc Student, Université Laval (Canada)
Duodenal microbiota as a new target for the study of type II diabetes development
Plante, R., Lebeuf, M., Nguyen, G., Gill, M.E., Pilon, G., Couture, P., Rancourt Bouchard, M., Lemelin, V., Di Marzo V., Raymond, F., Marette, A.
Introduction: The prevalence of type 2 diabetes (T2D) has taken on epidemic proportions over the past decades. The discovery of the metabolic impacts of the composition of the intestinal microbiota has changed our perception of the pathogenetic causes of this disease. It has been shown that a drastic change in the microbiota in people with obesity and T2D can lead to low-grade inflammation, glucose intolerance and insulin resistance. The focus on the colon microbiota has left out other intestinal segments such as the small intestines which is also of interest based on its important role in the absorption of nutrients and glucose.
The objective of this project is to identify bacteria from the duodenum of patients with or without T2D and to assess the impact of the duodenal microbiota on inflammation and glucose metabolism.
Methods: The subjects (n = 40) men or women chosen for the study are aged between 18 and 60 years old and are overweight with or without T2D. The collected duodenal biopsies are homogenized and seeded on various culture media promoting bacterial diversity. The isolated strains are identified by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of flight) and stored at -80°C for subsequent analyses.
Results: The 95 bacteria isolated so far represent 17 different bacterial genera and belong to three main phyla: Actinobacteria, Firmicutes and Proteobacteria.
Conclusion: Presently, our laboratory is screening these bacteria in vitro to determine their pro and anti-inflammatory effects in macrophages and their potential to modulate the incretin GLP-1, involved in the control of insulin, with the GLUTag cellular model. Ultimately, it will be possible to validate their effects in vivo with animal models such as the C. elegans worm and the zebrafish, which will make it possible to investigate the impact of these different strains on intestinal permeability and glucose metabolism.
Tausha Prisnee (Poster 47)
Graduate Student, University of Alberta (Canada)
Maternal mycobiome but not antibiotics alter fungal community structure in neonatal pigs
Tausha L. Prisnee, Janelle M. Fouhse, Natalie E. Diether, Tingting Ju, Hannah L. Lantz, Benjamin P. Willing
Antibiotic exposure during early life has been associated with diverse long-term adverse health outcomes. Despite being immunomodulatory, the impact of antibiotics on the fungal community (mycobiome) has received little attention. The objectives of this study were to determine the impact of commonly prescribed infant antibiotic treatments on fungal load and community structure in conjunction with the impact of antibiotics on bacterial load and community structure. A total of 32 piglets were divided into four treatment groups: amoxicillin (A), amoxicillin + clavulanic acid (AC), gentamicin + ampicillin (GA) and flavored placebo (P), and were treated orally with antibiotics starting on post-natal day (PND) 1 until PND 8, except for GA which was given on PND 5 and 6 intramuscularly. Fecal swabs were collected from piglets on PND 3 and 8 and sow feces was collected 1 day post-farrowing. Impacts of antibiotics on bacterial and fungal communities were assessed by sequencing of the 16s rRNA and ITS2 rRNA genes, and quantitative polymerase chain reaction (qPCR) was performed to determine total bacterial and fungal load. Antibiotics did not alter fungal 𝛼-diversity (Shannon Index, P = 0.834) or 𝛽-diversity (PERMANOVA, P = 0.565) on PND 8. Treatment with AC increased the ratio of total fungi: total bacteria on PND 8 (GLM, P = 0.035). There was strong clustering of pigs by litter on PND 8 (PERMANOVA, P < 0.001), which corresponded to differences in the sow mycobiome (P < 0.05). We observed a strong litter effect and show that the maternal mycobiome is essential for the shaping of the piglet mycobiome in early life.
Rajibur Rahman (Poster 48)
Graduate Student, University of Alberta (Canada)
Transplantation of wild boar derived live microbiota into conventional piglets is associated with increased fecal microbiota diversity and immune activation
Rajibur Rahman1, Janelle M. Fouhse1, Ryan Brook2, Roman Nosach2, John Harding2, Benjamin P. Willing1*
Recent work has identified that wild microbiomes make lab mice more resistant to disease. We hypothesized that the strict biosecurity and antibiotic use in pig production has resulted in the loss of co-evolved species, making them less resistant to disease. The objective of this study was to culture and reintroduce wild missing microbes into conventional piglets to understand their colonization dynamics and effect on host physiology. On post-natal day (PND 23), 48 piglets selected from 6 litters were exposed to either a cultured mixed microbial community (MMC) derived from 1) Wild boar (WB); 2) Conventional sow (Sow); 3) Wild boar + Sow (Combined); or 4) 1X PBS (Control) on alternate days until PND 28. There were no differences in pre-transplantation fecal microbial β- and α-diversity among the treatment groups, whereas post-transplantation (PND 48) WB microbial community structure was different from the Control and Sow groups and had higher species richness and diversity compared to the Combined group. DESeq2 analysis revealed that genera Asteroleplasma, Dialister, Actinobacillus, Succinivibrionaceae and Megasphaera were differentially abundant bacteria in the WB group compared to the Control. Cecal metabolite profile analysis revealed that the WB group had significantly enriched histamine and acetyl-ornithine as compared to Control and Sow piglets. Interestingly, this higher level of histamine in WB pigs was associated with an increase in Lactobacillus (a key histamine producer) abundance in the cecum. Ongoing metagenomic analysis will reveal whether unique species of Lactobacillus are found in the wild boar inoculum. Plasma cytokine analysis indicated that IL-1α and TNFα were significantly higher in WB pigs as compared to the Combined pigs. Importantly, no differences were observed among the treatments for weight gain and diarrheal incidents, indicating no adverse effects of inoculation. These findings indicate the potential ability of a WB derived MMC to colonize the gut of conventional piglets and to produce immune modulating metabolites, and impact immune activation.
Dr. Hena Ramay (Poster 49)
Senior Bioinformatician, University of Calgary (Canada)
FemMicro16S: Advancing the science of women’s health through open data sharing
Hena R. Ramay, Sahar Bagheri, Kevin Muirhead and Laura K. Sycuro
The vaginal microbiome impacts a woman’s health throughout her lifetime, contributing to her likelihood of acquiring sexually transmitted infections (STI), experiencing preterm birth, or suffering cervical cancer. The microorganisms inhabiting this important body site remain poorly understood, however, due to limited research funding and a lack of cooperative, open source research platforms. As sequencing projects expand to more cohorts from around the globe, it is increasingly important to examine these data in aggregate and encourage validation studies. To promote data sharing and build a more inclusive research community, we collected more than 20 publicly-available Illumina 16S rRNA gene amplicon datasets (2500+ female genital tract samples collected from 6 continents and covering a wide range of conditions such as preterm birth, bacterial vaginosis and STIs), and packaged it as a bioconductor R package called FemMicro16s. For each dataset, we removed primers, performed quality trimming (cutAdapt), and processed reads through DADA2 to generate amplicon sequence variant (ASV) tables. For assigning taxonomy to individual ASVs, we used RDP and IDTaxa classifiers with four different databases: SILVA and RDP for being well curated, and GTDB and URE (a custom database) for including full-length 16S rRNA gene sequences from uncultured species. To increase species assignment at ASV level, we further cluster ASVs at 98.6% identity using Cdhit. Within each cluster, if one or more ASVs have species assignment, those species are assigned to all the ASVs in that cluster. Key metadata have been curated, including geography, race/ethnicity, age, pregnancy status, and presence of bacterial vaginosis. This toolset simplifies access to curated taxon tables, with flexible assessment of taxa using different databases and similarity thresholds. This effort thus provides a foundational open data resource that we hope to expand through engagement with the global women’s health research community.
Laura Rojas (Poster 50)
MSc Student, University of Calgary (Canada)
Investigating the Association Between Prenatal Stress and Neurodevelopment: The Contribution of the Gut Microbiota
Laura Rojas, Marcel van de Wouw, Yanan Wang, Deborah Dewey, Raylene A. Reimer, Nicole Letourneau, Tavis Campbell, Gerald Giesbrecht
Prenatal stress impacts 1 in 3 pregnant persons worldwide and is associated with adverse neurodevelopmental outcomes in children. Emerging evidence suggests that the child’s gut microorganisms (e.g., gut microbiota) may mediate the relationship between prenatal stress and neurodevelopmental outcomes.
This study aims to investigate the relationship between (1) prenatal stress and Full-Scale Intelligence Quotient (FSIQ), and (2) features of the child’s gut microbiota and FSIQ in children aged 3-4 years from the Alberta Pregnancy Outcomes Nutrition study.
Both objective and subjective measures of stress were used to operationalize prenatal stress during each trimester. Maternal salivary cortisol levels were used for the objective measures. For the subjective measures, a psychological distress score was generated using the average score of the Edinburgh Postnatal Depression Scale and the Anxiety scale of the Symptom Checklist-90-Revised. Child stool samples collected at 3-4 years of age were assessed for the gut microbiota using 16s rRNA sequencing and metabolomics. The Wechsler Preschool and Primary Scale of Intelligence-IV (WPPSI-IV) was used for the neurocognitive assessments.
There was a significant negative association between psychological distress in trimester 3 and FSIQ (r = -.17). However, when controlling for relevant covariates (e.g., socioeconomic status), the multivariate model was not significant F(6,147) = 1.638, p =.141. Finally, greater Shannon alpha diversity (i.e., different bacteria types) was associated with a lower score on the Verbal Comprehension Index (r = -.14) but not with FSIQ.
Overall, these findings suggest that the child’s gut microbiota does not mediate the relationship between prenatal stress and FSIQ at 3-4 years of age. Although there is strong evidence connecting the brain and gut microbiota, the current findings do not link them together.
Laura Rojas (Poster 51)
MSc Student, University of Calgary (Canada)
Investigating the Impact of Prenatal Stress on the Child Gut Microbiota at Preschool-Age
Laura Rojas, Marcel van de Wouw, Yanan Wang, Deborah Dewey, Raylene A. Reimer, Nicole Letourneau, Tavis Campbell, Gerald Giesbrecht
Prenatal stress impacts 1 in 3 pregnant persons worldwide and is associated with adverse neurodevelopmental outcomes in children. Emerging evidence suggests that prenatal stress may influence the composition of the child’s gut microorganisms (e.g., gut microbiota) which has been linked to neurodevelopment.
This study investigates the relationship between prenatal stress and features of the child’s gut microbiota in children aged 3-4 years from the Alberta Pregnancy Outcomes Nutrition study.
Both objective and subjective measures of stress were used to operationalize prenatal stress for each trimester. Maternal salivary cortisol levels were used for the objective measures. For the subjective measures, a psychological distress score was generated using the average score of the Edinburgh Postnatal Depression Scale and the Anxiety scale of the Symptom Checklist-90-Revised. Child stool samples collected at 3-4 years of age were assessed for the gut microbiota using 16s rRNA sequencing and metabolomics.
Maternal cortisol levels were positively associated with the child’s gut Faith PD alpha diversity (i.e., different bacteria types) in trimester 1 (r = .32) and negatively with Shannon alpha diversity in trimester 2 (r = -.27). Psychological distress was positively associated with Chao1 alpha diversity (r = .24) in trimester 2. In addition, maternal cortisol levels in trimester 2 were associated with ten distinct bacterial taxa. Finally, maternal cortisol levels in trimesters 1 and 3 were associated with 12 and 3 different metabolic pathways, respectively.
Prenatal stress affects the composition and function of the prenatal child’s gut microbiota in a trimester-dependent manner. This may be important as the child’s gut microbiota has been associated with health outcomes. As such, supporting mental health during pregnancy may positively support child development, possibly through the gut microbiota.
Isla Skalosky (Poster 52)
MSc Student, University of Calgary (Canada)
The impact of intestinal microbiota complexity and antigen mimicry of a viral epitope on anti-viral T cell responses
Isla Skalosky and Markus Geuking
It is well known that the intestinal microbiota impacts the development and regulation of the immune system in the context of immune-mediated diseases such as allergies and autoimmunities. The role of the intestinal microbiota and its metabolites in anti-viral immune responses or vaccine efficacy is less clear and is an important question in the current pandemic environment. It has been further demonstrated that microbial mimicry of self-antigens can be either beneficial or detrimental to autoimmune or autoinflammatory diseases, depending on the context. The consequences of intestinal microbiota antigen mimicry of a viral epitope on anti- viral T cell responses has not been studied.
The proposed research will use a genetically modified intestinal commensal Escherichia coli strain expressing a lymphocytic choriomeningitis virus (LCMV)-derived epitope mimic in combination with gnotobiotic mouse models of precisely defined and controlled intestinal microbiota composition. The LCMV infection model is an extremely well characterized viral model to study T cell responses and has already been successfully established in the lab with corresponding readouts to measure epitope mimic-specific T cells.
Germ-free or gnotobiotic mice harboring a defined 12-species intestinal microbiota will be colonized with either a wil-type commensal E. coli or a genetically modified E. coli expressing the LCMV epitope mimic. What impact the presence or absence of the LCMV epitope mimic has on anti-viral T cell responses will be tested by challenging these mice eith either an acute or chronic LCMV infection, followed by characterization of LCMV epitope mimic-specific T cell responses by tetramer staining and intracellular staining.
Erin Strachan (Poster 53)
MSc Student, University of Alberta (Canada)
IgA-mediated Immunity in Type I Diabetes
Erin Strachan, Jessica Groat, Masoud Akbari, Sue Tsai
Type 1 Diabetes (T1D) is an autoimmune disease that involves the destruction of insulin-producing cells in the pancreas by autoreactive lymphocytes. Research has shown that abnormalities in the gut play a significant role in disease development. Two key features of a healthy gut include the presence of a community of microorganisms called the microbiota as well as secretions of Immunoglobulin A (IgA) into the gut. During infancy when the immune system is immature, breast milk provides the primary source of immunity largely in the form of IgA delivered directly to the infant’s gut. In infants and adults, the microbiota and IgA secretions work together to preserve the integrity of the gut barrier and regulate immune responses both locally and systemically. Interestingly, patients with T1D exhibit signs of subclinical gut inflammation, altered gut microbiota, loss of gut integrity and have an increased incidence of IgA deficiency. We hypothesized that IgA in the gut plays a protective role in the development of T1D and a deficiency of IgA would enhance disease. We are addressing this using a non-obese diabetic mouse model (NOD), comparing disease progression in IgA-deficient mice vs IgA-sufficient littermates. Unexpectedly, we observed a dominant disease-modifying effect of maternal IgA availability. Specifically, IgA-sufficient offspring of IgA-deficient dams show significantly lower disease incidence compared to offspring of IgA-sufficient dams. Furthermore, diabetes protection afforded by maternal IgA deficiency is lost in IgA-deficient offspring. Analysis of lymphocyte populations in these mice has shown differences in proportion, activation state and cytokine production in the bowels and mesenteric lymph nodes. We are endeavoring to further characterize these population differences and determine their significance in disease. Overall, our current data suggest that maternal influences promote disease susceptibility in offspring. In the absence of maternal IgA, these effects are ameliorated and protection is maintained by endogenous IgA. Whether these maternal influences are exerted during gestation, early postnatal period or both is actively being investigated.
Dr. Marcel van de Wouw (Poster 54)
Postdoctoral Fellow, University of Calgary (Canada)
Associations Between the Gut Microbiota and Sleep in Preschool-Aged Children
Introduction: Sleep plays a crucial role in the development of mental and physical health of children. The relationship between sleep and the gut microbiota is becoming increasingly clear in animals and human adults, even though this relationship remains unexplored during preschool – a key developmental period. This study aims to investigate the link between the gut microbiota and sleep in preschool-aged children
Methods: The stool microbiota and metabolite levels were assessed from a community sample of typically developing children (4.37 ±0.48 years, n=143). Sleep measures included total nighttime sleep (TST), sleep efficiency (SE), and wake-time after sleep onset (WASO) assessed using actigraphy.
Results: Differences in beta-diversity between children with low and high TST (p =0.048) suggest gut microbiota community alterations. Specifically, the relative abundance of Bifidobacterium was higher in the high TST group, while Bacteroides was higher in children who had higher SE and low WASO (LDA score >2). In contrast, some Lachnospiraceae members (e.g., Blautia and Coprococcus 1) were associated with shorter night-time sleep duration and less efficiency, respectively. We also found a group of stool metabolites, including specific neuroactive and immunomodulating metabolites, that were associated with greater sleep efficiency and less time awake at night. Notably, tryptophan and its metabolizing products were higher in children who had higher SE or lower WASO (LDA score >2); concentration of propionate was higher in children with lower WASO (p =0.036).
Conclusions: These data provide novel insights into the relationship between the gut microbiota and sleep during preschool. Longer night-time sleep and greater sleep efficiency correlated with selective commensal bacteria that may regulate sleep through modulating the metabolism of neuroactives.
Dr. Marcel van de Wouw (Poster 55)
Postdoctoral Fellow, University of Calgary (Canada)
The Relationship Between Internalizing Behaviors and the Gut Microbiota in Preschool Children
Introduction: The connection between the brain and the gut microbiota, including its metabolites like short-chain fatty acids, is becoming increasingly clear. However, the relationship between the gut microbiota and mental health problems (e.g., internalizing behaviors) in preschool-aged children remains largely unknown. This study investigates the associations between the gut microbiota and internalizing and externalizing behaviors in preschool children.
Methods: The stool microbiota and SCFA levels were assessed from a community sample of typically developing children (3-5 years of age, n=248). The parent-reported Child Behavior Checklist was used to assess child internalizing and externalizing behaviors. Spearman correlations followed by an adjustment for multiple testing were used to investigate associations between the gut microbiota and child behaviors, with subanalysis conducted in children clinically “at risk” for behavioral problems compared to those who were not.
Results: There was an association between Shannon alpha diversity with internalizing behaviors (rs = -0.134, p = 0.035) and its subscale somatic complaints (rs = -0.144, p = 0.023). Children clinically “at risk” for internalizing problems had decreased alpha diversity (U = 551, p = 0.017). Internalizing behaviors correlated with valerate and isobutyrate (rs = -0.147, p = 0.021; rs = -0.140, p = 0.028, respectively). In addition, the somatic complaints subscale also correlated with acetate and butyrate (rs = -0.219, p = 0.001; rs = -0.241, p < 0.001, respectively). These findings were additionally present in children “at risk” for internalizing problems (U = 569, p = 0.026; U = 571, p = 0.028) and somatic complaints (U = 164, p = 0.004; U = 145, p = 0.001).
Conclusions: These results provide novel insights into the relationship between internalizing behaviors and the gut microbiota in preschool children. Furthermore, the relationship between somatic complaints and acetate and butyrate warrants future investigation into interventions that increase SCFA production.
Erik van Tilburg Bernardes (Poster 56)
PhD Candidate, University of Calgary (Canada)
Antibiotic-induced Malassezia spp. expansion in infants elicits immune dysregulation and airway inflammation in gnotobiotic mice
Erik van Tilburg Bernardes, Mackenzie W. Gutierrez, Carolyn A. Thomson, Kathy D. McCoy, Stephen B. Freedman, Marie-Claire Arrieta
The neonatal immune system undergoes important developmental changes that are dependent on microbial colonization post-birth. Early-life antibiotic use deleteriously impact the gut microbiome, leading to immune dysregulation and increased risk of childhood asthma. However, it is currently unknown if the fungal microbiome (mycobiome) contributes to antibiotic-induced dysbiosis and to immune dysregulation conducive to allergic asthma. We aimed to evaluate the effect of antibiotic-induced mycobiome changes on neonatal immune development and experimental airway inflammation. We ran an observational, prospective clinical study of 47 infants (<6 months) receiving antibiotics. We compared the bacterial and fungal microbiome in fecal samples collected before and after antibiotics via shallow shotgun and ITS2 sequencing, as well as qPCR for fungal and bacterial DNA. Antibiotic use decreased bacterial and increased fecal fungal DNA and induced expansion of Malassezia spp. in infants. To evaluate the effect of Malassezia spp. colonization on host immune development, and to differentiate it from the effect of other common fungal colonizers, we compared immune development in gnotobiotic mice colonized with consortia of 12 mouse-derived bacteria (Oligo-MM12), or bacteria with Candida albicans, Saccharomyces cerevisiae and/or Malassezia restricta. M. restricta colonization increased Th1 and Th2 cells, eosinophils, and delayed macrophage maturation in the colonic lamina propria. M. restricta also increased migratory dendritic cells, eosinophils, Th2, Th17 and ILC3 cells in mesenteric lymph nodes, suggesting elevated immune responses deemed critical in atopy development. M. restricta colonization also increased house dust mite-induced airway inflammation, with elevated cellularity and marked eosinophilia in the bronchoalveolar lavage. This translational work shows that fungal overgrowth and expansion of Malassezia spp. are previously overlooked collateral effects of infant antibiotic use, which causally contribute to immune dysregulation and increased susceptibility to allergic airway inflammation in mice.
Dr. Yan Wang (Poster 57)
Postdoctoral Fellow, University of California Los Angeles School of Medicine (Canada)
Overview of the relationship between oral microbiome and oral health disparities among children
Yan Wang, Janni Kinsler, Francisco Ramos-Gomez
Background: Oral health disparities refers to “the existence of differences in the incidence, prevalence, mortality, and burden of oral diseases and other adverse health conditions, as well as the use of health care services, among specific population groups in the United States”. The oral microbiome plays an essential role in oral and systemic health. It is defined as the collective genome of microorganisms that reside in the oral cavity. The latest high-throughput sequencing techniques, such as targeted sequencing for the bacterial 16S ribosomal RNA (rRNA) gene, has completely changed clinical microbiology. We reviewed the current literature about the connection between oral microbiome and oral health disparities among children.
Methods: A PubMed search was conducted in April 2012 using the key words “oral”, “microbiome” and “disparities” to identify all papers that were in this field. Only papers focused on children or adolescents with oral microbiomes biomarkers were included in the detailed review.
Results: Thirteen papers were found in search results. Three papers in 2016 (review), 2020 (supragingival), and 2021 (saliva) studied the oral microbiome and oral health disparities among children or adolescents. The 2020 and 2021 papers identified a significant difference at taxa-level in terms of both bacterial diversity and composition among ethnic groups. The findings suggest a strong association between oral microbial profiles and race/ethnicity. Seven ethnic group-specific bacterial taxa were identified after adjusting for oral diseases and hygiene habits.
Conclusions: Oral microbiome may change the way oral health disparities are measured. The potential biomarkers that may be used for risk prediction and early detection of oral diseases need further investigation, and reinforces the need for a rigorous design of longitudinal microbiome data at population level to study the oral health disparities.
Nadia Yorke (Poster 58)
Undergraduate Student, University of Calgary (Canada)
The effect of intestinal colonization status on T cell development in the thymus
Nadia Yorke, Kirsten Wilson, Roopa Hebbandi Nanjundappa, Markus Geuking
The microbiota, particularly the commensal bacteria that colonize the gut, influence the immune system and changes in the microbiota are associated with immune-related diseases. This project aimed to investigate the influence of the intestinal microbiota composition on T cell development in the thymus, focusing on negative selection and natural regulatory T cell (nTreg) generation, which is still poorly understood. Both negative selection and nTreg generation are vital in ensuring the development of an immune system that is not self-reactive and defects within these processes may have implications on the development of autoimmune or autoinflammatory disorders. First, an organotypic thymic culture system was set up with three different T cell receptor (TCR) transgenic mouse models including SMARTA, OTII and TCR-M, to identify the model most suitable to study nTreg generation in vitro. Peptide titration experiments revealed that, amongst the three models, SMARTA thymocytes were the best model to study antigen-specific negative selection and nTreg generation in vitro. The influence of the gut microbiota was determined using co-cultures of wild type thymi from different hygiene states together with immature SMARTA thymocytes. Three hygiene states were compared, Germ-free, sDMDMm2 (12 species) and SPF (complex microbiota) thymi were used, as they have varying levels of colonization complexity and different levels of microbial metabolites which could influence the thymic microenvironment. Analysis of transgenic thymocyte populations by flow cytometry indicated that thymic microenvironment is impacted by microbial colonization and mice with higher colonization diversity had better nTreg generation and CD4+CD8+ double positive survival than germ-free animals. These findings indicate that there is a gut-thymus axis that promotes immune regulation already at the level of T cell development. As a next step, we will investigate this in more detail in vivo.
Kassandra Zachos (Poster 59)
PhD Researcher, University of Toronto (Canada)
The Microbiome-Mitochondria Crosstalk: Assessing the Microbiome Composition of Patients with Mitochondrial Disease
Kassandra A. Zachos, Neil Sondheimer & Ana C. Andreazza
Mitochondria are highly unique double-membraned organelles that are critical for the production of energy. Dysfunctional mitochondria have the ability to cause a wide range of complex physiological consequences. In the context of primary mitochondrial disease (PMD), the phenotype-genotype discrepancy makes PMDs challenging to diagnose, target and treat. Similarly, an individual’s microbiota is highly unique and composed of trillions of bacterial species critical in the maintenance of human health. It is known that these gut microbes interact with the mitochondria to maintain biochemical pathways, however this relationship, coined the microbiome-mitochondria crosstalk, warrants further investigation. The microbiome composition in patients with mitochondrial disease has yet to be evaluated but has proven to be effective in understanding the underlying biochemical modulations in other diseases. Furthermore, there is no effective treatments for individuals suffering from mitochondrial diseases. Evaluating the microbiome composition, and thus their metabolite production acts as a potential mechanism to be targeted for therapeutics at a patient-specific, personalized level. The objective of this pilot study is to determine the role of the microbiome in mitochondrial diseases. Specifically, we aim to investigate any potential mitochondrial disease-specific trends in the microbiome composition, sex-specific differences, and the role of microbial metabolites in the underlying biochemical mechanisms. We hypothesize that the microbiome composition of patients with mitochondrial disease will have a reduced relative diversity and a microbial signature correlated to their mitochondrial mutation. This prospective pilot study will include 10 patients with various mitochondrial diseases. Fecal samples will be collected, and two analyses will be conducted: (1) an exploratory analysis using 16s rRNA sequencing to assess the composition and (2) an untargeted metabolomic assessment will be conducted to further support the understanding of the ongoing mechanisms in the mitochondria-microbiome crosstalk. This study is currently ongoing, and results are pending.
