2024 Microbiome Symposium Poster Abstracts

1. Parvimonas micra pathogenic potential, host cell interactions, and strain variation among six distinctly sourced isolates in the context of colorectal cancer

Authors: Gwendolyn Freeze (1), Brendan Daisley (1), Sarah Vancuren (1), Emma Allen-Vercoe (1)
Affiliation(s): 1.Department of Molecular and Cellular Biology, University of Guelph
Keywords: Parvimonas micra, colorectal cancer, pathogenesis, strain-variation, tissue culture

Abstract:

The human gut microbiome is composed of trillions of different microorganisms, including bacteria, archaea, viruses, and fungi. A balanced gut microbial community is often associated with overall host health, whereas disruptions to the gut microbial composition can lead to the progression of diseases. In the case of colorectal cancer (CRC), an imbalanced gut microbial community and the enrichment of specific oncomicrobes (i.e., microbes associated with the development of cancer) are often implicated in CRC pathogenesis. One such oncomicrobe, Parvimonas micra, is newly emerging in its striking association with CRC. Recent work has revealed the overabundance of P. micra in CRC patients compared to controls, although few studies have investigated the mechanistic role of P. micra in the disease. As such, the objectives of this study are two-fold: 1) to better characterize the genomic and physiological attributes of P. micra that may be relevant to pathogenesis, and 2) to determine the interactions of P. micra with host cells in vitro. Furthermore, by utilizing six distinctly sourced P. micra isolates, this work also focuses on characterizing strain-variation within the species. Overall, we found that P. micra possesses an abundance of toxin genes and virulence factors, a strong ability to co-aggregate with other known oncomicrobes, a remarkable tolerance to air, as well as a sensitivity to certain gut-related stressors such as pH and short chain fatty acids, with the extent of these effects varying among strains. Furthermore, strains of P. micra were found to differentially stimulate host cell proliferation and inflammatory responses in vitro, as well as adhere to and invade into host cells to varying extents. Overall, this work provides insight into the potential role of P. micra in colorectal tumorigenesis, as well as highlights interesting strain-variation within the species that may be relevant to its pathogenesis in CRC.

2. The impact of human milk oligosaccharides on the gut symbiont, Ruminococcus gnavus, in relation to gastrointestinal and metabolic diseases

Authors: Panuya Athithan (1), Matthew Sorbara (1), Emma Allen-Vercoe (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph
Keywords: Breastmilk, gut microbiome, human milk oligosaccharides, pathobionts, strain-level variation

Abstract:

Breastmilk contains high concentrations of structurally diverse oligosaccharides that constitute the third most abundant component in human milk which are known as human milk oligosaccharides (HMOs). HMOs are best known for their prebiotic effects on the infant gut microbiome and confer several beneficial functions such as aiding in commensal microbe colonization and host immune cell modulation. Due to these benefits, there has been considerable interest in the impact of HMOs on the adult gut microbiome as recent studies have shown that HMOs have the potential to restore health-associated microbial communities in individuals with gut diseases. Dominant species of gut microbes capable of HMO utilization have a growth advantage over those lacking this ability. Recent findings have shown growth advantages employed by a wide diversity of resident gut bacteria isolated from infant fecal samples in the presence of HMOs. However, several strains of bacteria across a variety of species and genera experienced HMO-induced growth inhibition. Due to this novel antimicrobial property, there is considerable interest in investigating the range of HMO-induced inhibition across pathobiont species, including Ruminococcus gnavus (R. gnavus). R. gnavus is widely distributed at low abundance in the gut microbiomes of healthy individuals. However, it is disproportionately overrepresented in gut microbiomes of individuals with chronic metabolic diseases such as inflammatory bowel disease and type 1 diabetes. Thus, the aim of this study is to investigate the impact of HMOs on R.gnavus strains, the nature of HMO inhibition, and discern intra-species variation within R.gnavus. HMO utilization was assessed by treating R.gnavus strains with pooled HMOs(pHMOs) and measuring optical density(OD600). Strain specificity detected amongst R.gnavus through their different growth properties when exposed to pHMOs. While many of the strains demonstrated enhanced growth, some strains experienced a spectrum of pHMO-induced growth inhibition. These findings advance our understanding of HMO interactions with the gut microbiota at the strain level and opens new avenues for exploring the therapeutic potential of HMOs in modulating microbial composition.

3. The effects of high tartrazine exposure on in vitro models of healthy and pre-type 2 diabetic human gut microbiomes

Authors: Hansi Botschner (1), Sarah Vancuren (1), Dr. Joanna Harvey (1), Dr. Emma Allen-Vercoe (1)
Affiliation(s): 1.Department of Molecular and Cellular Biology, University of Guelph; 2. Victoria University of Wellington
Keywords: tartrazine, azo dyes, gut microbiome, diabetes

Abstract:

Azo dyes are the most common synthetic dyes because of their cheap production costs, high stability, and variety of colours. This research focuses on tartrazine, a common additive in food products such as candy and soft drinks. Tartrazine consumption has also been linked to numerous health problems, although the results of some research studies were inconclusive. Importantly, tartrazine has no nutritional value, with their sole purpose being to alter the colour and visual appeal of food products, particularly to children. We previously showed that tartrazine is reduced by several common bacterial members of the human gut microbiota to 4-amine-3-carboxy-5-hydroxy-1-(4-sulfophenyl)pyrazole (SCAP) which spontaneously dimerizes in the presence of oxygen to form a compound we have called purpurazoic acid (PPA), both of which have been found to be cytotoxic in preliminary work. The overall aim of this research was to assess the effects of tartrazine and SCAP on human fecal microbiomes derived from both healthy and unhealthy (obese, pre-type 2 diabetic) donors, modeled in an in vitro bioreactor system which received daily additions of tartrazine. We found that, in both systems, addition of tartrazine resulted in key changes in the community composition that have been previously associated with disease. Additionally, while the concentration of tartrazine increased and decreased along with the additions (as expected), a concomitant buildup of SCAP occurred which correlated with the overall effects on community composition. This suggests that substantial and consistent tartrazine consumption could lead to a similar increase in SCAP in the colon; from our preliminary data, this effect appears to be more marked within the pre T2D -derived ecosystem than the healthy control. Considering that tartrazine has no nutritional value, our findings that it can potentially disturb the colonic microbiome provides weight in favour of banning or restricting its use as a food additive.

4. Creating a genome-wide CRISPR interference functional genomic library in Candida albicans to study fungal gastrointestinal colonization **1st Place in Poster Competition!!!**

Authors: Meea Fogal (1), Lauren Wensing (1), Nicholas C Gervais (1), Rebecca S Shapiro (1), Ilse D Jacobsen (2)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON; 2. Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Jena, Germany
Keywords: CRISPR, Gastrointestinal Colonization, Commensalism, Candida albicans, Fungal Pathogens

Abstract:

Candida albicans is an opportunistic fungal pathogen that also exists as a commensal member of the human microbiota in many healthy individuals. Invasive infections caused by C. albicans can be severe and life-threatening, especially in immunocompromised individuals, with mortality rates of up to 50%. The host gastrointestinal (GI) tract is a reservoir of C. albicans and it is posited that many invasive and systemic infections originate from the gut through translocation of the fungus across the intestinal barrier. Additionally, the use of broad-spectrum antibiotics, a compromised immune system, and an impaired intestinal barrier are all risk factors for systemic infection. Despite C. albicans’ prevalence as a GI commensal, the fundamental molecular mechanisms underpinning colonization and commensalism have been overlooked compared to those that underpin systemic infection. Genetic manipulation techniques can help us understand gastrointestinal colonization. Previous work in the Shapiro lab has validated the use of a transcriptional repression system, known as CRISPR interference (CRISPRi), to study gene function in C. albicans. We propose scaling this technology up to a genome-wide level to identify previously uncharacterized genes with roles in C. albicans GI colonization during antibiotic treatment. We have generated a pooled CRISPRi repression library representing all ~6,100 genes in the C. albicans genome. The pool of mutants will be screened in a murine model of GI colonization in the absence and presence of antibiotics to identify genes that, when repressed, influence colonization under these conditions. Genes of interest from this screen will be subject to follow-up analysis to study their function more comprehensively. This research will help to deepen our understanding of C. albicans’ commensal lifestyle which can also give insight into how infections arise and possible therapeutic strategies to prevent infection.

5. Studying the effects of gut-derived metabolites in brain development to understand its role in the etiology of autism spectrum disorder (ASD)

Authors: J. Capistrano (1), V. Rea (1), P.N.G. Tran (1), T. Ball (1), T. Van Raay (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph
Keywords: Gut-brain axis, metabolites, ASD, neurodevelopment, zebrafish

Abstract:

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders caused by genetic and environmental factors. Due to its complex and often pleiotropic nature, its etiology has been difficult to elucidate—ultimately, hampering the identification of definitive diagnostic markers and development of potential therapies/treatments. Interestingly, most individuals with ASD suffer from gastrointestinal problems, which underscores a potential connection between ASD and the gut. Metabolites produced from the gut have been reported to impact the brain; however, the mechanisms of how these metabolites influence the brain or vice-versa remain poorly understood. Here, we hypothesize that distinct metabolites derived from different diets and/or mental health profiles result in unique phenotypic effects in the developing brain. In the first study, metabolites from naïve fecal samples that were grown in a robogut in the presence of different diets (high fiber Western, low fiber Western, Mediterranean, and Yanomami) were isolated. In the second study, age- and gender-matched fecal samples from neurotypical (NT) and children with ASD were used to isolate metabolites. We then evaluated the effects of these gut-derived metabolites on neurodevelopment by looking into changes in gene and protein expression, sensory organ development, and behavioral responses using germ-free zebrafish as our model. Thus far, our results suggest that the zebrafish model may not be sensitive enough to detect the effects of metabolites derived from different diets. However, we found that zebrafish neurodevelopment seems to be sensitive enough to detect the different effects of ASD and NT metabolites, where we see unique behaviors and distinct alterations in sensory organ development and gene expression profiles. Our goal is to eventually uncover the molecular mechanisms underlying the contributions of gut-derived metabolites on the development of the brain, which has implications for ASD and other relevant diseases and disorders.

6. Bacterial growth stage control NOD2 activation by diverse gut commensal Lachnospiraceae

Authors: Ethel Closa (1), Hannah Wallworth (1), Autumn Sweeney (1), Matthew T. Sorbara (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph
Keywords: Host-microbe crosstalk, NOD2 receptor, growth patterns, peptidoglycan

Abstract:

A central pillar of the homeostatic crosstalk between the mucosal immune system and gut microbiota is the activation of intracellular NOD1/2 pattern recognition receptors (PRRs) that trigger pro-inflammatory signaling. These PRRs recognize conserved fragments of bacterial peptidoglycan that can be taken up by epithelial cells. Notably, a loss-of-function allele in the nod2 gene is strongly associated with Crohn’s Disease (CD), a chronic inflammatory condition of the gastrointestinal tract. This suggests that a tonic level of NOD2 activation is necessary to maintain barrier functions of a healthy gut environment. However, the extent to which anaerobic members of the gut microbiota provide NOD2 signaling is largely uncharacterized. One such taxon is the highly abundant and diverse family of Gram-positive anaerobes, Lachnospiraceae, which produce multiple beneficial metabolites and are often reduced in CD patients. Here, we determine if Lachnospiraceae can activate tonic levels of NOD2 signaling. The ability of diverse Lachnospiraceae to induce NOD2 activation was assessed by stimulating reporter cells expressing human NOD2 receptors with culture supernatants and pellet suspensions. These Lachnospiraceae isolates originated from healthy human donors and were grown in a rich media culture to either 8 or 24 hours prior to the stimulation assay. Bacterial viability was confirmed using plate counts and continuous growth curves were generated for several isolates tested. We demonstrate that members of the Lachnospiraceae family are differentially able to activate NOD2. Some species, including Ruminococcus gnavus and Coprococcus comes, do not activate NOD2 signaling, while others like Anaerostipes hadrus trigger strong NOD2 activation with significant strain level variation. We further demonstrate that changes in NOD2 stimulatory capacity coincide with changes in the cell wall of some Lachnospiraceae upon entry into stationary phase. Our study reveals that the regulation of NOD2 signaling is linked to the species-dependent growth stage in Lachnospiraceae.

7. Prenatal over- or under-consumption of micronutrients contributes to gut microbiota-associated disturbances in the brain serotonergic system in offspring

Authors: Mali Al-Issa (1), Jianzhang Dong (1), Jenny S. Feeney (1), Gia V. Shelp (1), Clara E. Cho (1)
Affiliation(s): 1. Department of Human Health and Nutritional Sciences, University of Guelph
Keywords: Gut microbiota, brain, serotonin, offspring, micronutrients

Abstract:

The gut microbiota can impact host health through the modulation of signaling molecules. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter with diverse biological functions, including food intake regulation via the 5-HT receptor 2C (5-HTR2C) in the hypothalamus. We have previously shown that excess consumption of vitamins including folic acid and under-consumption of the essential micronutrient choline (intake patterns commonly observed in North America) during pregnancy can shape risk of obesity in offspring. Here, we determined central serotonergic system characteristics in offspring exposed to varying amounts of micronutrients in relation to their gut microbiota profiles. Pregnant Wistar rats were fed the AIN-93G diet with one of four micronutrient diets: 1) recommended vitamins (RV); 2) high 10x vitamins (HV); 3) high 10x folic acid with recommended choline (HFolRC); or 4) high 10x folic acid with no choline (HFolNC). Offspring were weaned to a high-fat diet for 12 weeks. Brain 5-HT concentrations, hypothalamic 5-HTR2C expression, and fecal microbiota profiles were determined using enzyme-linked immunosorbent assay, western blotting, and 16S rRNA sequencing. Brain 5-HT concentrations were lower in male and female offspring from HV- or HFolNC-fed dams (P<0.0001) consistent with higher final body weight (P<0.0001). The HFolRC diet resulted in lower 5-HT concentrations in male offspring but not in female offspring. All offspring had lower hypothalamic 5-HTR2C protein expression (P<0.0001), indicating overall disturbances in feeding-related neurocircuitry. Significant beta diversity differences were observed across diet and sex of the offspring. Metabolic integration analysis identified Ruminococcus having the highest association with 5-HT concentrations, which an inverse relationship between Ruminococcus and 5-HT was found (R2=-0.22; P<0.05). In conclusion, excess or imbalanced prenatal consumption of micronutrients leads to gut microbiota-associated alterations in the brain serotonergic system. The significance of our findings is that the microbiota-gut-brain axis modified by early nutrition may be an important target for therapeutic interventions.

8. Self-Reported Food Intolerance in Inflammatory Bowel Disease patients compared to healthy controls

Authors: Rebecca Dang (1), Dominika Boron (1), Jessica Linton (1), Paul Mundra (2), Neeraj Narula (1, 2), Alberto Caminero (1)
Affiliation(s): 1. Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Ontario; 2. McMaster University Medical Centre, Hamilton Health Sciences, Hamilton, Ontario
Keywords: Inflammatory bowel disease, food intolerance, microbiome

Abstract:

Background: Inflammatory bowel disease (IBD) including Crohn’s disease and Ulcerative colitis is classified by chronic intestinal inflammation. IBD patients commonly exhibit food intolerances to various foods. However, the precise dietary triggers and mechanistic pathways responsible for food intolerances are unknown. This study aims to examine self-reported food intolerances and microbiome diversity in IBD participants compared to healthy controls.

Methods: IBD participants with a confirmed UC or CD diagnosis were recruited from the Gastroenterology clinic at McMaster University from August 2021 to September 2023. Healthy controls were recruited through recruitment posters at McMaster University. Participants completed questionnaires pertaining to self-reported food intolerances and demographic information. Also, participants provided a stool sample for 16S rRNA Illumina sequencing to determine microbial composition.

Results: A total of 80 IBD participants (48 CD and 32 UC) and 26 controls completed the questionnaires. We found that IBD participants (88% CD and 90% UC) reported at least one food intolerance compared to controls (30%). The mean number of food intolerances IBD participants reported was 3 (SD=1.78). The most common adverse reactions were reported to dairy (60% CD and 63% UC), wheat (37% CD and 40% UC), peanuts/tree nuts (27% CD and 25% UC), caffeine (27% CD and 25% UC) and fiber (22% CD and 28% UC). The rates of intolerances in all of the food groups were higher than reported by controls. Additionally, IBD participants have a lower fecal alpha diversity compared to healthy controls (p<0.0001).

Conclusion: Our current findings reveal that IBD participants commonly report food intolerances and have a lower alpha diversity measure compared to controls. Common offending foods include dairy, wheat, peanuts/tree nuts and caffeine. Ongoing studies will provide more insights into the relationship between stool bacterial composition and food intolerance symptoms.

9. Antibiotic-induced alteration of the gut and urinary microbiome is implicated in kidney stone disease **Tied for 3rd Place in Poster Competition!!!**

Authors: Riley Fidler (1), Dr. Jeremy Burton (1, 2, 3), Dr. Kait Al (1, 3), and Dr. Jennifer Bjazevic (2)
Affiliation(s): 1. Department of Microbiology & Immunology, Western University; 2. Department of Surgery, Division of Urology, Western University; 3. Canadian Centre for Human Microbiome and Probiotics Research
Lawson Health Research Institute
Keywords: Kidney stone disease, antibiotics, dysbiosis

Abstract:

Background
Kidney stone disease (KSD) is one of the most widespread urologic pathologies. Although previously very rare in children, prevalence has recently risen by 40% per year. Early antibiotic exposure has been proposed as a cause and is corroborated by findings of an altered gut microbiome in adult and child stone formers. To investigate whether antibiotics contribute to KSD and the mechanisms by which it occurs, we investigated stone-inducing properties of cefazolin (CFZ) and ciprofloxacin (CIP). We hypothesized that antibiotics would elevate both the incidence and severity of KSD.

Methods
The effects of CFZ and CIP were quantified in a Drosophila melanogaster (DM) model of KSD by assessing survival, stone burden, and developmental metrics. Changes to the DM microbiota were assessed by bacterial culture. Additionally, the direct crystallization of the antibiotics in urine and adhesion to kidney epithelial cells were assessed.

Results
Exposure to CFZ and CIP rapidly increased stone burden and accelerated health decline and death in adult DM. Conversely, CFZ profoundly increased mass and growth rate in larvae, demonstrating that antibiotic exposure has different consequences at different life stages. Furthermore, CIP increased calcium oxalate (CaOx) crystal adherence to kidney epithelial cells and CFZ increased CaOx crystallization in urine.

Discussion
CFZ and CIP exhibit stone-inducing effects both in vivo and in vitro. Further research is warranted on the mechanistic role of antibiotics and the microbiome in stone disease and how it can be altered to mitigate KSD. By quantifying and comparing the KSD-inducing effects of several antibiotic classes, and further exploring the consequences of antibiotic use during early development, we hope to determine the safest options for children and to center KSD prevention around gut health.

10. Short Chain Fatty Acid profiling in prebiotic treated stool from Fecal Microbiota Transplantation donors in an in-vitro gut microbiome model

Authors: Suyang Jia (1), Esfandiar Shojaei (2), Michael Silverman (1, 2, 3) Gregor Reid (1), Jeremy Burton (1,2), Seema Parvathy(1, 2, 3)
Affiliation(s): 1. Western University, London ; 2. Lawson Health Research Institute; 3. Medicine, Schulich School of Medicine and Dentistry
Keywords: Short chain fatty acids, Fecal Microbiota Transplantation, Human gut, in-vitro model

Abstract:

Background: Gut microbiota derived short chain fatty acids (SCFAs) play a significant role in the regulation of lipid metabolism and inflammation in Metabolic-Associated Fatty Liver Disease (MAFLD). Butyric acid, propionic acid and acetic acid (SCFAs- energy substrates) derived from the intestinal bacteria are known to reduce inflammation, increase insulin sensitivity and promote intestinal barrier integrity, the key factors leading to MAFLD. Selecting Fecal Microbiota Transplantation (FMT) donors with favourable gut microbiome and SCFA production can improve the quality of the FMT and efficacy. Prebiotics are known to improve the growth of favorable gut bacteria and improve SCFA production.

Objectives:

To develop a rapid high-throughput in-vitro model (MiPro) to culture gut bacteria and their metabolites.

Evaluate the in-vitro model for production of SCFAs by gut bacteria using prebiotics (Inulin and XOS) and to potentially predict good donors for future FMT clinical trials in MAFLD.

Methodology: Stool samples were collected from healthy male and female donors (n=9) between the age of 21-48 and with a BMI between 19-29. Diluted frozen stool samples (20% w/v) in PBS with 5% L-cysteine hydrochloride were cultured in a 96 deep well plate experimental model (MiPro- simulates the human gut environment) and treated with 5mg/ml inulin and XOS (prebiotics). Following 24hrs anaerobic incubation in a specialized MiPro media, viable bacterial growth was estimated by serial dilution and plating in Anaerobic agar and SCFA production using nuclear magnetic resonance (NMR- Bruker BioSpin Ltd., Canada). Metabolite spectra were processed and profiled using Chenomx NMR Suite 10.0 Professional software.

Results: MiPro model and specialized media maintained the gut bacterial viability and was successful in evaluating the SCFA production in vitro within 24hrs. The invitro model was conducive for the growth of gut anaerobes (11.2 log10 cfu/ml). Frozen stool storage did not affect the viable count. Among the 9 donors, Donor SH had the highest SCFA levels at baseline in MiPro for acetate (49nM), butyrate (9.4nM) and propionate (23.3nM). Fecal acetate, propionate and butyrate levels increased with addition of XOS and inulin. Concentration of the other SCFAs such as Valerate, Isobutyrate and Isovalerate did not show any significant increase in production in the in vitro model.

11. Genetic Manipulation of Non-Model Blautia luti Reveals a Novel Mechanism of Succinate Production **Tied for 3rd Place in Poster Competition!!**

Authors: Marissa Sim* (1), Bradley Fitzgerald (1), Ailish Moore (1), Matthew Sorbara (1)
Affiliation(s): 1.Department of Molecular and Cellular Biology, University of Guelph
Keywords: Gut Microbiota, Colonization Resistance, Genetic Manipulation, Succinate, Blautia luti

Abstract:

The gut microbiota is a diverse community of microorganisms that perform regulatory processes such as colonization resistance (CR). CR involves safeguarding functions that prevent pathogen expansion. One mechanism is intracellular acidification (IA) which couples an acidic environment with abundant short chain fatty acids (SCFA). This combination lowers the pH inside pathogen cells and inhibits their replication. CR is lost when perturbations disrupt the equilibrium in the gut, increasing host susceptibility to infections. Therefore, strategies to mediate pathogen inhibition and restore CR are critical. Our laboratory investigates the Lachnospiraceae family which are abundant gut anaerobes known for producing SCFA and performing CR. Here, we investigate the genus Blautia for their ability to ferment the metabolite succinate, a mechanism which is currently uncharacterized. Succinate is primarily fermented by the genus Bacteroides; however, we have found that a subset of our isolates produce succinate in equivalent levels. Interestingly, while Bacteroides use succinate intermediately in propionate synthesis, Blautia are seemingly devoid of the terminal components of this pathway. With one pKa value lower than those of abundant SCFA, we hypothesize that succinate aids in establishing an acidic environment that drives IA. Genomic analysis and metabolomic screening allowed us to identify an 11-gene cluster with homology to a flavin based electron bifurcation (FBEB) complex in our succinate producing isolates. We predict this mechanism reduces fumarate to succinate. Traditionally, Blautia have been genetically intractable, however, we implemented a recently established pipeline to knockout a gene in the FBEB complex. To our knowledge, these represent some of the first knockouts in B. luti. Our mutants exhibit succinate elimination, a shift in central metabolism, the inability to lower media pH, and a reduced capacity to inhibit multi-drug resistant Klebsiella pneumoniae. Together, this research will help potentiate the candidacy of succinate producing Blautia as biotherapeutic agents to restore CR.

12. PHB-degrading Flavobacterium sp. and Streptomyces sp. isolated from landfill with predicted novel medium-chain-length PHA depolymerases

Authors: Ariel Tastasa (1), V. Viljakainen (2), L. A. Hug (2)
Affiliation(s): 1. University of Toronto; 2. University of Waterloo
Keywords: Bioplastic degradation; landfill isolates; long-read sequencing; enzyme discovery

Abstract:

Our growing reliance on plastics has significant social benefits but increasingly concerning ecological consequences. Improved resource management and environmental stability will require a sustainable substitute to petroleum-based plastics. Polyhydroxybutyrate (PHB) is a short-chain-length polyhydroxyalkanoate (SCL-PHA) and bioplastic with petroleum-plastic-like characteristics, which is microbially synthesized and degraded. PHB currently costs 10-fold the price of petroleum-based plastics. For PHB to continue to grow in market share, improvements in its production and degradation are required, to increase sustainability and reduce costs. The rate-limiting step of PHB degradation is the initial depolymerization. PHA depolymerases are classified based on their localization and the length of PHA being degraded, with different enzymes localizing intracellularly or extracellularly and depolymerizing short-chain-length (SCL, 3-5C) and medium-chain-length (MCL, 6-14C) PHAs. This study aimed to isolate and identify novel PHB degraders and their extracellular PHB depolymerases. Six PHB-degrading bacteria were isolated from a landfill in Southern Ontario, Canada. Using long-read sequencing, genome sequences were reconstructed for the isolates, and their taxonomic identities and metabolic capacities examined. These bacteria were identified to belong to the genera Flavobacterium and Streptomyces. Members of each genus have been previously shown to degrade PHB. Up to 5 distinct PHB depolymerase genes were predicted for each isolate, all of which grouped with known extracellular MCL-PHA depolymerases. This study indicates that PHA depolymerase activity is not strictly substrate-length dependent and raises questions on the classification of PHA depolymerases based on substrate length specificity. This study contributes novel PHB degraders and MCL-PHA depolymerases that may hold growth and economic potential benefits for the bioplastic industry.

13. Examining carbohydrate utilization within Gram-positive anaerobic bacterium from the Lachnospiraceae family

Authors: Loudon Herold (1), Bradley G. Fitzgerald (1), & Matthew T. Sorbara (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph
Keywords: Carbohydrates, Strain-level, Variability, Nutrient Niche, Operon

Abstract:

The composition of the human gut microbiota is shaped by the host’s diet. A microbe’s ability to replicate in the dense gut environment depends on their success in competing for limited available nutrients compared to their competitors. Understanding how specific microbes access a suite of nutrient niches can provide insight into their contributions to microbial dynamics and the host’s physiological state. Genomic regions have been characterized that express enzymes to transport, cleave, and metabolize carbohydrates. This area has been primarily investigated in the Gram-negative Bacteroidota phylum, but remains relatively unknown in the Gram-positive Bacillota phylum. This project aims to investigate intra- and inter-species variation in the utilization of simple and complex carbohydrates by members of the Lachnospiraceae family and the impacts of different carbon sources on metabolite production.

Strains of Anaerostipes hadrus and Blautia wexlerae were profiled for their carbohydrate utilization, which revealed extensive intra-species variability. Using this dataset, strains of A. hadrus and B. wexlerae can be clustered solely based on carbon source utilization. Tanglegrams were generated from genomic analyses, which revealed a significant correlation between the clustered observed growth phenotypes and whole genome sequence annotation data for all genes and relevant carbohydrate utilization genes. Furthermore, putative utilization operons were identified for carbohydrates that showed strain-dependent growth. Gas-chromatography mass-spectrometry analysis revealed that the SCFAs produced by Lachnospiraceae are both carbohydrate- and species-dependent.

Lachnospiraceae demonstrate intra- and inter-species variability in carbon source use. Genomic analyses demonstrate that these differences can be strongly linked to the strain-level variations in the genomic repertoire. Additionally, the strain and particular carbon source are both determinants in resulting metabolite production. Understanding the impacts of strain-level variation on nutrient niche access and metabolite production will aid in the development of future microbiome-directed therapeutics.

14. Urease-deficient Urinary Bacteria May Use The Arginine Deiminase Pathway to Promote Struvite Kidney Stone Formation

Authors: David Liu (1), Gerrit A. Stuivenberg (2, 3), Jennifer M. Bjazevic (4), Kait F. Al (2, 3), Jeremy P. Burton (2, 3, 4)
Affiliation(s): 1. Department of Physiology and Pharmacology, Western University, London, Ontario, Canada; 2. Lawson Health Research Institute, London, Ontario; 3. Department of Microbiology and Immunology, Western University, London, Ontario, Canada; 4. Department of Surgery, Division of Urology, Western University, London, Ontario, Canada
Keywords: Urinary microbiome, kidney stone disease, struvite, arginine deiminase

Abstract:

Introduction:

Struvite kidney stones are bacterially driven stones that form in alkaline conditions which have been long attributed to urinary tract infection-associated bacteria utilizing urease. However, emerging evidence suggests that urease-deficient bacteria may also alkalize urine using alternative pathways. This study investigates the arginine deiminase (ADI) pathway found in Enterococcus faecalis, which this pathogen uses to neutralize acidic environments. Given the high prevalence of urease-deficient E. faecalis within struvite stones, we hypothesized that the ADI pathway is also used to alkalize urine, potentially contributing to struvite stone formation.

Methods:

E. faecalis ATCC 33186 was grown in artificial urine (AU) supplemented with physiological concentrations of L-arginine, and the final pH was measured after 24 hours. During crystallization experiments, E. faecalis was incubated in AU containing supraphysiological concentrations of arginine to stimulate significant urine alkalization. A co-culture apparatus allowed bacterially driven crystallization to occur in a sterile environment for quantification using optical density. Finally, crystals were isolated from urine and analyzed using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDX).

Results:

Arginine supplementation increased the final pH of AU in a concentration-dependent manner. During crystallization experiments, optical density increased above baseline, indicating that crystallization had occurred in urine. SEM revealed X-shaped crystals that are characteristic of struvite, and the composition of these crystals was confirmed using EDX.

Discussion:

Our strain of E. faecalis could degrade arginine to alkalize urine and form struvite precipitates. Our results contribute to the mounting evidence that urease-deficient bacteria can autonomously form stones using alternative pathways such as ADI. However, a limitation of this study is that our in vitro model may not mimic in vivo conditions. This should be addressed in future studies with the view of eventually testing therapeutic compounds targeting the ADI pathway, expanding much-needed treatment options for struvite stone patients.

15. Distinct Stickland fermentation strategies across Lachnospiraceae alter the depletion of preferred substrates used by Clostridioides difficile

Authors: Ailish Moore (1), Matthew T. Sorbara (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph
Keywords: C. difficile, Lachnospiraceae, Stickland fermentation

Abstract:

Clostridiodes difficile (C. difficile) targets intestinal epithelial cells and causes infectious colitis, resulting in significant mortality, morbidity, and high healthcare costs. As a result, C. difficile is an urgent threat that immediately requires new therapeutics. Several mechanisms of colonization resistance are active against C. difficile, including inhibition by secondary bile acids and high levels of short-chain fatty acids. In addition, resident gut microbiota inhibits the growth of C. difficile by competing for nutrients.

C. difficile utilizes Stickland fermentation, the paired oxidation and reduction of amino acids, including proline, to generate ATP and reducing power. C. difficile also encodes proline dipeptidases, thus it is able to use both free proline and proline dipeptides to produce 5-aminovalerate. Some Lachnospiraceae likewise utilize Stickland fermentation to produce 5-aminovalerate. This suggests that there is competition for proline between healthy gut microbiota, including members of Lachnospiraceae, and C. difficile, but little is known about patterns of Stickland fermentation substrate use in Lachnospiraceae. Therefore, we investigated Lachnospiraceae’s utilization of proline.

Here, we demonstrate that several Lachnospiraceae species encode proline reductase genes and produce 5-aminovalerate. Unexpectedly, we observed that E. intestinalis and C. comes do not deplete free proline, despite producing 5-aminovalerate. Species that do not deplete proline, including E. intestinalis, encode a variety of dipeptidases targeting proline-containing dipeptides and clusters of di- and oligo-peptide transport genes. Finally, growth on minimal medias and defined sources of proline (free or in dipeptides) revealed that some Lachnospiraceae are exclusively able to access proline-containing dipeptides.

Altogether, our results demonstrate that some Lachnospiraceae specialize in the utilization of proline-containing di-/oligo-peptides providing overlap with C. difficile’s metabolic strategy. Therefore, our findings indicate that some, but not all, Stickland-fermenting Lachnospiraceae may be able to compete with C. difficile for colonization within the gastrointestinal tract.

16. The role of acetate compounds and resource-sharing in a human gut microbiota model with a focus on menaquinone production

Authors: Samantha Tapping (1, 2), Omar Abdelraheem (1, 2), John A. Chmiel (1, 2), Kait F. Al (1, 2), Gerrit A. Stuivenberg (1, 2), Jeremy P. Burton (1, 2, 3)
Affiliation(s): 1. Department of Microbiology & Immunology, Western University, London, Ontario, Canada; 2. Lawson Health Research Institute, London, Ontario, Canada; 3. Department of Surgery, Division of Urology, Western University, London, Ontario, Canada
Keywords: Microbiome, Menaquinone, Acetate, Resource-sharing, Symbiosis

Abstract:

A healthy gut microbiota relies on a pool of metabolites produced and shared by the microbes present, known as the pantryome. Disruption of the pantryome, via the loss of taxa and functional pathways, leads to a collapse in the microbiome, termed “dysbiosis,” which has associations with various chronic diseases. Menaquinones are a group of pantryome metabolites of particular interest, since they are important for both microbial cellular respiration chains and human health. Menaquinones have two sources: menaquinone-producing microbes and dietary fermented foods. A loss of menaquinone-producers can lead to microbiota dysbiosis, but our group has shown that abiraterone acetate (AA), an oral prostate cancer medication, promotes the known health-commensal Akkermansia muciniphila and other menaquinone-producers. However, as a hormone synthesis inhibitor, AA cannot be used outside of its therapeutic indication, so we sought to investigate the potential of alternate acetate-containing compounds to beneficially alter the microbiota. As such, we hypothesized that if dietary acetate compounds reached the colon, they would cause a beneficial shift in the microbial communities, like AA, by promoting known menaquinone-producing species. A high-throughput in vitro colonic environment model – MiPro – was used to test human gut microbial communities’ responses to nine acetate compounds at three concentrations. The model was seeded with an optimal liquid medium, fecal slurry from a healthy donor, and food-grade acetate compounds (including AA as a positive control). The most promising candidates are apple cider vinegar and manganese (II) acetate, as they showed the most similar shift in microbial composition to AA. Further, the enrichment of bacterial respiration chains was shown in many of the samples through inferred pathway analysis. These results suggest that, if ingested in adequate amounts, food-grade acetate compounds could be used to modulate the gut microbiota. These results could lead to the development of novel therapies for dysbiosis-associated chronic diseases.

17. The role of urease in gut commensal Lachnospiraceae **2nd Place in Poster Competition!!!**

Authors: Isaac J. Firth (1), Sam Hess (1), Bradley Fitzgerald (1), Matthew Sorbara (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph
Keywords: Urease, Lachnospiraceae, short-chain fatty acids, urea-derived acetate production

Abstract:

The human gut microbiota is a complex group of microbes that are associated with various aspects of host health. Approximately twenty percent of host-derived urea is released into the gut where it is metabolized by gut microbes. Microbial urease cleaves urea into ammonia and carbon dioxide, however the roles of urea utilization in the physiology of commensal gut anaerobes are largely unknown. Health-associated microbiota communities produce high levels of short-chain fatty acids (SCFA) and decrease the pH of the proximal colon triggering inhibition of enteric pathogens. The Lachnospiraceae are a family of anaerobes that are associated with SCFA production, show high levels of genomic diversity, and vary in their capacity to acidify culture media. Here, we investigate the ability of Lachnospiraceae to tolerate pH and SCFA stress, and test the hypothesis that Lachnospiraceae-encoded urease alters SCFA production and acidification. Using a random forest model to predict Lachnospiraceae pH and acetate production based on gene presence/absence identified multiple subunits of urease as important predictors of model outcomes. Here, we demonstrate that urease-encoding Lachnospiraceae show urea-dependent changes in SCFA production, acidification, and growth. Encoding urease increases the tolerance of ureolytic Lachnospiraceae to acid stress and increases SCFA production under acidic conditions. Furthermore, urease-positive Lachnospiraceae directly incorporate the carbon from urea into produced SCFAs particularly under acidic conditions, demonstrating that urease activity directly supports SCFA production. This research provides mechanistic insight into the role of commensal-encoded urease in supporting healthy gut conditions that can inhibit the expansion of pathogens in the gut.

18. Exploring the genetic interactions between host genome and rumen microbiome impacting methane emissions and feed efficiency in crossbred Angus-Simmental cattle

Authors: Fatimatzahra Muhammad (1, 2)
Affiliation(s): 1. Centre for Genetic Improvement of Livestock, Department of Animal Biosciences, University of Guelph, N1G 2W1, Guelph, Ontario, Canada; 2. Faculty of Land and Food Systems, The University of British Columbia, BC, Canada
Keywords: feed efficiency, methane emissions, rumen microbes, genomic selection, beef cattle

Abstract:

Ruminants and their rumen microbes have a symbiotic relationship, crucial for converting fibrous feed into meat and milk for human consumption. The conversion process is assessed by feed efficiency (FE), which is a performance trait that indicates an animal’s ability to transform feed into metabolically available nutrients for meat or milk production. Ruminant livestock are vital to global food security increasing the demand for more economically and environmentally efficient livestock production systems. However, the Canadian animal agriculture sector contributes 5% of all greenhouse gas (GHG) emissions, of which beef production accounts for 71% of the methane outputs, indicating a need to enhance the efficiency and profitability of the cattle industry. Therefore, understanding the genetic relationship between the host and its rumen microbial community, and its impacts on FE and ME, will facilitate the selection of microbial profiles to improve genomic selection for optimized FE and lower ME. The objective of this study is to examine the associations between the host’s genome and rumen microbial metagenome and its impact on ME and FE in low- and high-FE [Residual Feed Intake (RFI; kg/d)] Angus x Simmental beef cattle. Hair and ruminal fluid samples were collected from 82 multiparous cows located at the Ontario Beef Research Center (Elora, ON) for genotyping and functional analysis, respectively. Feed intakes were recorded using Insentec feed bins and enteric emissions were measured every two weeks with a Greenfeed trailer (C-lock Inc.). Body condition scores (BCS), weight, rib fat, and rump fat (ultrasound) were collected every 28 d. Ruminal bacteria and archaea Amplicon sequence variant (ASV) abundance profiles from all cows will be determined using 16s rRNA Amplicon-Sequencing (Amplicon-Seq). Enhanced genetic insights into the interactions of the ruminal microbiome could optimize selection for livestock efficiencies and ME.

19. Exploring Development of the Gut-Brain axis via Maternal Effects in Urban Eastern Grey Squirrels

Authors: Emma Mably (1), Amy Newman (1)
Affiliation(s): 1. Department of Integrative Biology, University of Guelph
Keywords: Development, Urbanization, Maternal Effects, Microbiome, HPA axis

Abstract:

Expansion of the urban environment presents novel stressors which shape the physiology and development of an individual over its lifetime. Utilizing a gradient of urbanized sites we assess microbiome diversity, glucocorticoid concentrations, and Limosilactobacillus reuteri, a metabolite signalling bacteria known to support neurological development in juvenile Eastern Grey Squirrels (Sciurus carolinensis). We aim to address the impact of maternal exposure to anthropogenic sources and environment on offspring physiology. Ultimately, if increased glucocorticoid levels and decreased microbiome diversity are a consistent feature of urban squirrels, this may increase physiological and behavioural costs in urban environments compared to exurban habitats. Aligned with a One Health philosophy, the study contributes insights into the ecophysiological implications of urbanization, offering valuable information for wildlife conservation, urban planning, and sustainability.

20. Dietary supplementation of prebiotics and organic minerals to improve growth performance and gut health of rainbow trout (Oncorhynchus mykiss) subject to low, moderate and high temperatures.

Authors: Samantha Bezner (1), Dr. David Huyben (1)
Affiliation(s): 1. Department of Animal Biosciences, University of Guelph
Keywords: Aquaculture, prebiotics, minerals, yeast, temperature, next-generation sequencing.

Abstract:

As a poikilothermic animal, fish internal temperature and therefore metabolic rates are dependent on the external temperature of the environment. Climate change steadily increases ambient water temperatures in the Great Lakes, resulting in symptoms of heat stress from farmed salmonids: lower feeding rates, gut dysbiosis, higher incidence of disease, and reduced production in net-pens. This project intends to evaluate the efficacy of gut microbiome modulating compounds, such as yeast prebiotics (Saccharomyces cerevisiae) and organic minerals, within the diet by measuring growth parameters, stress and disease biomarkers, and gut microbial composition. Triplicate tanks of fish will be fed graded levels of yeast prebiotic (0.1%, 0.2%, 0.3%), minerals, a mineral-enriched yeast prebiotic (0.1%, 0.2%, 0.3%), or a control diet at a temperature of 8°C. An optimized inclusion level will be chosen from the graded trials based on growth performance and beneficial microbial diversity, which will then be used in triplicate tanks at 15 and 25°C. After each of these 16-week feeding trials, feces will be dissected from the distal intestine. Following DNA extraction from the feces, 16S rRNA gene amplicons were sequences using the Illumina MiSeq platform to determine alpha and beta diversity indices. Blood will also be taken to analyze cortisol and other stress and disease related biomarkers. Using these results, the aquaculture industry will gain a deeper understanding of how temperature and heat stress can be managed through the use of diet, in order to meet the consistently growing demand for high-quality fish protein worldwide.

21. Latent Variable Modeling Reveals Associations between Vaginal Discharge, Stillbirth, and Vaginal Microbiota Dynamics in Gilts

Authors: Maysa Niazy (1, 2, 3)
Affiliation(s): 1. Department of Animal Biosciences, Ontario Agriculture College University of Guelph; 2. Department of Mathematics and Statistics, College of Engineering and Physical Sciences, University of Guelph; 3. Department of Population Medicine, Ontario Veterinary College, University of Guelph
Keywords: Topic modeling, Latent variables, mixed membership

Abstract:

Sow performance is a key component of commercial pig farms’ productivity. The occurrence of stillborn piglets and vaginal discharge can result in reproductive loss. Diverse vaginal microbial communities in females are linked to adverse health outcomes such as premature birth; however, little is known about the associations between sow vaginal microbiota and reproductive performance. This study aimed to evaluate whether vaginal bacterial composition is associated with valvar discharges and subsequent reproductive performance in gilts. Using 16S rRNA gene amplicon sequencing, samples from 50 gilts in their first parity were analyzed two weeks after farrowing. Gilts with no discharge and those with no stillbirth had lower diversity compared to those with severe discharge and a higher stillbirth rate, as indicated by Shannon diversity (P < 0.012, P < 0.022, respectively). Based on the Bray-Curtis dissimilarity index, there were clear shifts in the microbiome of gilts with severe vaginal discharge and a high stillbirth rate compared to gilts with mild or no discharge and a lower stillbirth rate. Firmicutes, Protobacteria, and Bacteroidota were the top three phyla; gilts with high stillbirth rates and severe vaginal discharge had a higher Protobacteria proportion (P < 0.05). We confirmed that the vaginal microbiota was altered in gilts with severe vaginal discharge and stillbirth using the Latent Dirichlet Allocation model (LDA). The LDA uses mixed membership to characterize vaginal communities, addressing the sensitivity and robustness of existing clustering approaches. The LDA model identified seven sub-communities within the study population. Sub-community 1 (Enterococcus, Aerococcus, and Prevotella) and Sub-community 4 (Pseudomonas and Burkholderia) were correlated with gilts with severe discharge, while Sub-community 6 and Sub-community 7 had less diversity and were correlated with gilts with no stillbirth. Our analyses using topic models allow for the exploration of heterogeneous populations and reveal substructures in vaginal ecosystems with potential clinical and biological associations.

22. Characterization of rumen fluid, feces and milk microbiota of Sarda ewes fed with different content of Alfalfa hay (Medicago sativa) assessed by 16S rRNA gene sequencing

Authors: Giannuzzi D. (1), Zardinoni G. (1), Cecchinato A. (1), Correddu F. (1) , Macciotta N. P. P. (3), Atzori A. (3), Carta S. (3), Ledda A. (3), Schiavon S. (1) , Gallo L. (1) , Lam S. (2), Cánovas A. (2), Pegolo S. (1)
Affiliation(s): 1. Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova (Italy); 2. Centre for Genetic Improvement of Livestock, Department of Animal Biosciences, University of Guelph (Canada); 3. Department of Agricultural Sciences, University of Sassari (Italy)
Keywords: sheep, rumen, feces, milk, microbiota

Abstract:

Diet is a major cause of changes in the composition of the rumen and gut microbiota, affecting animal health and productivity. This work aims to characterize rumen fluid, feces, and milk microbiota of 24 Sarda ewes fed with two different diets with equal net energy, protein, and NDF levels but varying alfalfa hay content. Individual rumen fluid, feces, and milk samples were collected at the beginning (day 1, T0) and at the end of the trial (day 22, T1). The sequencing of the V2-4-8 and V3-6, V7-9 hypervariable regions of the 16S rRNA gene performed with Ion GeneStudio S5 allowed to analyze the microbial communities. The raw reads clustering into Amplicon Sequence Variants (ASVs) and the taxonomic assignment were carried out with the QIIME 2 software. The combined effect of diet and time on microbial communities, milk composition and technological traits, and feed efficiency traits were assessed with a linear mixed model by R studio. At the phylum level Proteobacteria and Bacteroidota were the most represented phyla in rumen fluid, while Firmicutes and Bacteroidota in feces, and Proteobacteria and Actinobacteriota in milk, regardless the experimental group. The diet and time interaction seems to influence several low-abundant taxa especially in feces and milk, resulting in a significant difference when comparing the two groups at T1. No effects were observed on the phenotypic traits. Diets did not affect the microbial structure of rumen fluid and milk. However, significant differences in terms of beta-diversity (Bray-curtis and Unweighted Unifrac) were observed in feces at T1. These results suggest that the microbiota is more affected by the nutrient availability rather than variations in dietary ingredients. A study on a larger number of animals may reduce the animal-to-animal variability, allowing for clearer results and a more detailed understanding of the relationship diet-animal health-product quality in sheep.

23. Characterization of the milk somatic cell metagenome bacteria and archaea profiles of high and low feed efficient Holstein dairy cattle

Authors: V. Asselstine (1), S. Lam (1), F. S. Schenkel (1), F. Miglior (1, 2), C. F. Baes (1, 3), P. Stothard (4) and A. Cánovas (1)
Affiliation(s): 1. Centre for Genetic Improvement of Livestock, Department of Animal Biosciences, University of Guelph, Guelph, ON, Canada, N1G 2W1; 2. Lactanet Canada, Guelph, ON, Canada, N1K 1E5; 3. Institute of Genetics, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland; 4. Department of Agricultural, Food and Nutritional Science/Livestock Gentec, University of Alberta, Edmonton, Alberta, Canada, T6G 2R3
Keywords: feed efficiency, milk somatic cell metagenome, 16s rRNA Amplicon Sequencing, Whole Genome Sequencing, RNA-Sequencing

Abstract:

Feed accounts for the largest expense on dairy farms, leading to the need for improvement of breeding strategies for more feed-efficient cattle. The most common trait measured to represent feed efficiency (FE) in cattle is residual feed intake (RFI, kg/d), which accounts for the animals’ actual feed intake and its expected feed requirements for maintenance and production. Characterization of the host genome, transcriptome, and milk somatic cell (SC) microbiome may provide a window into understanding potential host-milk microbial profiles that are different between efficiency statuses in cattle. This study aims to link host genome, transcriptome and milk SC microbiome to determine -OMICs interactions influencing FE in Canadian Holstein dairy cows. Milk SC samples were collected from extreme FE groups [high-RFI (N=6) and low-RFI (N=10)] of cows, selected from a larger population of 200 milking Holstein cows at the Ontario Dairy Research Centre (Elora, Ontario). Samples were analyzed for DNA (host genome and milk SC microbiome) and RNA (host transcriptome). So far, the RNA-Sequencing analysis was completed to identify differentially expressed (DE) genes between RFI groups. Using Whole Genome Sequencing, the DNA sequences were analyzed to identify SNPs significantly associated with RFI. Additionally, milk SC metagenome will be characterized using 16s rRNA amplicon sequencing to determine bacteria and archaea amplicon sequence variants (ASVs) differentially abundant between RFI groups. Preliminary results showed 36 genes DE between Low and High-RFI animals. These DE genes were significantly enriched in 28 metabolic pathways (FDR<0.01), the majority of which were associated with host immune response including the RIG-I-like receptor signaling pathway. Further analysis will involve determining the relationship of the DE genes with differentially abundant bacteria and archaea ASVs and SNPs associated with FE. This knowledge may enhance genetic selection by incorporating host genome, transcriptome and milk SC microbiome interactions to improve FE of Holstein cows.

24. High throughput microbiome characterization of harmful algal blooms in Fairy Lake, Acton ON

Authors: Kathleen Nolan* (1), Koya Basso* (1), Andreas Heyland (1), Robert Hanner (1). *co-presenters
Affiliation(s): 1. Department of Integrative Biology, University of Guelph
Keywords: Harmful algal blooms, freshwater, biodiversity, dysbiosis, amplicon sequencing

Abstract:

Harmful algal blooms (HABs) pose a major threat to the health of freshwaters and those that rely on them. With climate change, freshwater systems are becoming more prone to a state of dysbiosis caused by the overgrowth of microalgae, and the development of tools to assess freshwater microbiomes is crucial for their conservation. Here, we describe a novel approach for high-throughput biodiversity characterization of the freshwater microbiome in a small inland lake (Fairy Lake, Acton ON). This lake is highly accessible with diverse land use within its watershed, and it experiences frequent HABs, making it an excellent model system. In this poster, we describe high-throughput isolation and amplicon sequencing of select microalgal strains from Fairy Lake in order to build a molecular reference library that can be used for environmental sequencing. Additionally, we present methods and preliminary results of a paired molecular and morphological assessment from environmental samples collected during the summer 2024 HAB season. High throughput amplicon sequencing of select ribosomal RNA genes (16S, 18S, and 23S) is coupled with a novel plate-based isolation strategy and a FlowCam 8000 to describe the microalgal community throughout the HAB season. In addition, heterotrophic bacteria, fungi, and zooplankton communities are assessed with high-throughput amplicon sequencing. The goal of this research is to compare molecular and morphological assessment methodologies for microbiome assessment, as well as to parse out the biological contributors to HABs within the microbiome of Fairy Lake. This research will significantly advance the field of aquatic ecosystem health monitoring and create opportunities to better characterize and understand shifts in microbiomes of freshwater ecosystems.

25. Investigating the external and internal pollen microbiome of corn (maize)

Authors: Miraples, A. (1), Khalaf, E. (1), Raizada, M.N. (1)
Affiliation(s): 1. Department of Plant Agriculture, University of Guelph
Keywords: Corn, Microbiome, Pollen, Transmission

Abstract:

Pollen is an essential vector for plant sexual reproduction and microbial transmission contributing to host plant fitness. The microbial communities associated with pollen have been reported to correlate with the fitness of bees and the hive community, as well as having allergenic impacts on humans. Pollen exposure to the atmosphere is believed to be a key mechanism for microbial inoculation onto the pollen surface, suggesting that most of the microbial load resides on the surface of the pollen grain, attached to the exine wall. An endogenous (internal) pollen microbiome has also been alluded to, but it has not yet been explored to the best of our knowledge. An internal pollen microbiome would suggest that pollen inoculation may occur from the host plant itself (i.e. vertical transmission). The proposed research tested the hypothesis that the pollen microbiome of corn is contained both on the surface and inside the pollen grain. To test this hypothesis, pollen from field grown corn of the inbred LH82 was collected and divided into two fractions. One fraction was washed with a 0.1% Tween 80 solution, and the wash solution was kept, while the other fraction was untreated. A comparative taxonomic profiling of the microbiome associated with untreated pollen, washed pollen and the used wash solution was performed using full-length 16S rRNA based PacBio sequencing followed by bioinformatic analysis. The research was replicated across two field seasons. A diversity of bacterial taxa was observed in the pollen wash as well as washed pollen, suggesting that bacteria reside both on the pollen surface and inside the pollen grain or subsurface layers. This research advances our fundamental understanding of the tissue location of the pollen microbiome in maize, with implications for its potential origins, and provides a simple technique to analyze the pollen microbiome of other plant species.

26. Conservation and diversity of the pollen microbiome of Pan-American maize using PacBio and MiSeq

Authors: Eman Khalaf (1), Anuja Shrestha (1), Michelle Reid (1), Benjamin J. McFadyen (1) and Manish N. Raizada (1)
Affiliation(s): 1. Department of Plant Agriculture, University of Guelph
Keywords: pollen, microbiome, maize, landraces, PacBio

Abstract:

Background. Pollen is a vector for diversification, fitness-selection, and transmission of plant genetic material. The extent to which the pollen microbiome may contribute to host diversification is largely unknown, because pollen microbiome diversity within a plant species has not been reported, and studies have been limited to conventional short-read 16S rRNA gene sequencing (e.g., V4-MiSeq) which suffers from poor taxonomic resolution.
Objectives. Here we report the pollen microbiomes of 16 primitive and traditional accessions of maize (corn) selected by indigenous peoples across the Americas, along with the modern U.S. inbred B73. The maize pollen microbiome has not previously been reported.
Methods & Results. The pollen microbiomes were identified using full-length (FL) 16S rRNA gene PacBio SMRT sequencing compared to V4-MiSeq. The Pan-American maize pollen microbiome encompasses 765 taxa spanning 39 genera and 46 species, including known plant growth promoters, insect-obligates, plant pathogens, nitrogen-fixers and biocontrol agents. Eleven genera and 13 species composed the core microbiome. Of 765 taxa, 63% belonged to only four genera: 28% were Pantoea, 15% were Lactococcus, 11% were Pseudomonas, and 10% were Erwinia. Interestingly, of the 215 Pantoea taxa, 180 belonged to a single species, P. ananatis. Surprisingly, the diversity within P. ananatis ranged nearly 10-fold amongst the maize accessions analyzed (those with ≥3 replicates), despite being grown in a common field. The highest diversity within P. ananatis occurred in accessions that originated near the center of diversity of domesticated maize, with reduced diversity associated with the north–south migration of maize. This sub-species diversity was revealed by FL-PacBio but missed by V4-MiSeq. V4-MiSeq also mis-identified some dominant genera captured by FL-PacBio.
Conclusion. The study, though limited to a single season and common field, provides initial evidence that pollen microbiomes reflect evolutionary and migratory relationships of their host plants.

27. sc-MC: Single-cell RNA Profiling for Complex Microbial Community

Authors: Ray Wang (1), Alice Deng (1), Teddy Lee (1), Binh Tran (1), Vanessa Dumeaux (1)
Affiliation(s): 1.University of Western Ontario
Keywords: single-cell RNA sequencing, microbiology, human gut microbiome

Abstract:

Host-adapted microbial communities (or microbiomes) are assemblies of commensal, symbiotic, and pathogenic microorganisms present at specific body sites. The population of bacteria, viruses, archaea, and fungi that comprise the human microbiome depends on functional interactions dictating the community’s composition, stability, and resilience, such as the positive symbiotic and negative antagonistic relationship existing between members of the microbial community. The human gut microbiome, estimated to express 3.3 million genes, plays a critical role in host health. Single-cell RNA profiling has recently been developed to investigate cell-specific gene expression and identify different cell types and states in complex tissues. Our lab and others have developed such methods to profile microbial monocultures, revealing significant heterogeneity even in isogenic cell populations. We extend this method to profile complex microbial communities such as the human gut microbiome, optimizing cell permeabilization for different microbial cell walls and capturing small amounts of microbial RNA, including those from low-abundant microbes. Validation involves real-life stool samples collected from sedentary individuals who participated in an 8-week physical training program. This research presents a unique and effective technique to functionally characterize individual microbes’ heterogeneous behaviour in complex microbial communities. As our understanding of microbial communities evolves, so will the applications to modulate them for a diverse range of biomedical, environmental, food sciences, and synthetic biology applications.

28. crisprHAL: Optimizing sgRNA seletion for bacterial Cas9 genetic engineering

Authors: Tyler Browne (1), Dalton Ham (1), Gregory Gloor (1), David Edgell (1), Tyler Wilson (2), Richard Michael (2)
Affiliation(s): 1. Western University; 2. Tesseraqt Optimization
Keywords: Bacterial, Genetic Engineering, CRISPR-Cas9, Machine Learning

Abstract:

The CRISPR-Cas9 bacterial adaptive immune system is a genetic engineering tool with potential as a next-generation antimicrobial agent. The Cas9 nuclease is targeted to a genomic site by a single guide RNA molecule (sgRNA) where the complex introduces a double-strand DNA break. To ensure the efficacy of a CRISPR system-based antimicrobial, we need to have confidence that the system will be precise in where it cleaves. A still unsolved problem is the impact of nucleotide sequence variations in relation to Cas9 complex on-target cleavage activity in bacteria. Through the proper application of machine learning and accurate data, sequence features impacting sgRNA activity can be extracted, allowing for accurate predictions of bacterial sgRNA-associated Cas9-complex activity. Utilizing a deep learning approach, we developed the bacterial sgRNA/Cas9 activity prediction model crisprHAL. This new model is designed to enhance predictive performance through transfer learning with smaller amounts of high-quality data. Variations of crisprHAL were constructed for the enzymes TevSpCas9 and SpCas9. To test model generalization performance, we used four datasets: one prior, and three of our own datasets. A notable result is the successful performance of crisprHAL on a killing efficiency-based dataset generated in Citrobacter rodentium. Until this point, all bacterial datasets were generated in Escherichia coli, preventing cross-species testing. Unlike eukaryotic models, results indicate that crisprHAL predictions generalize across bacterial species. We present crisprHAL, a sgRNA/Cas9 activity prediction model which recapitulates known sgRNA/Cas9-target DNA interactions and provides a pathway to a generalizable sgRNA/Cas9 activity prediction tool for use in microbial genetic engineering.

29. Harnessing CRISPR-Cas Technology for Targeted Microbiome Editing: A New Approach to Treating Antibiotic-Resistant Infections

Authors: William Layoun (1), Ibrahim Conteh (1)
Affiliation(s): 1. University of Toronto
Keywords: CRISPR-Cas, Microbiome Editing, Antibiotic Resistance, Therapeutic Interventions, Pathogen Control

Abstract:

The escalating challenge of antibiotic resistance demands innovative solutions beyond traditional drug development. This research introduces a pioneering approach using CRISPR-Cas systems to selectively edit the human microbiome, aiming to enhance resistance against pathogenic bacteria without relying on antibiotics.

Utilizing a novel vector delivery system, we designed CRISPR constructs targeting specific genetic sequences in resistant bacterial strains within the gut microbiome. The constructs were engineered to selectively disrupt virulence factors and resistance genes in pathogens like Escherichia coli and Staphylococcus aureus while preserving beneficial bacteria. This precision editing was achieved through careful selection of guide RNAs and Cas proteins, optimized for minimal off-target effects.

In vitro trials demonstrated that our CRISPR vectors could effectively decrease the population of resistant pathogens by up to 90% within mixed microbial communities. Subsequent in vivo studies in murine models confirmed a significant reduction in infection rates and severity without notable impacts on the overall microbiome diversity or health of the host.

This poster will detail the design and optimization of CRISPR vectors, show results from preclinical trials, and discuss the potential implications of microbiome editing as a therapeutic tool. By leveraging CRISPR technology, a new paradigm is proposed in the fight against antibiotic-resistant infections, highlighting a shift towards microbiome-based interventions.

Moreover, our approach sets the stage for developing personalized microbiome therapies tailored to individual genetic and microbial compositions, potentially revolutionizing how we treat a wide array of diseases linked to microbial imbalances.

30. Delineating Compositional and Functional Patterns in Global Gut Microbiomes

Authors: Mohamed Meawad (1), Alice Deng (2), Evelyn Cai (2), Dalwinder Singh (2), Vanessa Dumeaux (1,2,3,4)
Affiliation(s): 1. Department of Anatomy and Cell Biology, Western University, London, Canada; 2. Department of Biochemistry, Western University, London, Canada; 3. Department of Oncology, Western University, London, Canada; 4. Lawson Research Institute, London, Canada
Keywords: Healthy gut microbiome, metagenomics, deep-learning

Abstract:

Background. Host-adapted microbial communities, commonly referred to as microbiomes, consist of diverse microorganisms occupying specific anatomical niches. Studies have found that host-adapted microbiomes are variable across individuals, yet certain microbial configurations (MCs) are observed more frequently across the global population than what we would expect by chance. Multiple factors, ranging from environmental pressures to intrinsic microbe-microbe interactions, could explain the existence of these MCs. In the gut, compositional MCs, also called enterotypes, have been associated with dietary, and various metabolic or immunological markers. While compositional analyses are insightful, several studies have emphasized the need to also analyze the functional potential of gut microbiomes. This research proposes a novel computational framework for defining both compositional and functional MCs found in the global population.

Methods: In this study, we obtained microbial composition and functional pathway profiles for 4,809 healthy adult human gut microbiomes from 47 studies. After determining the statistical distribution of metagenomic data for both compositional and functional data, we used a non-linear archetypal analysis method, to identify dominant microbial configurations (archetypes) which characterize the extreme states of gut microbiome compositional or functional diversity.

Results: For microbial composition, three archetypes were identified, dominated by Prevotella sp002299635, CAG-831 sp900550465, and Akkermansia sp001580195 species respectively. Similarly, three functional archetypes were delineated, each characterized by unique sets of pathways related to central metabolic processes that are reflective of the three macronutrients: fatty acids, proteins, and sugars. Further analyses will enhance our understanding of MCs’ intrinsic structure, stability, resilience, and associations with host attributes.

Conclusions: Overall, our preliminary findings demonstrate the potential of non-linear archetypal analysis to delineate healthy adult human gut microbiomes based on their microbial compositions or functional potential. This deeper knowledge will further elucidate the underlying principles governing complex microbial interactions and their impact on the host or environment.

31. The “BumbleBiome”: Developing a Bioreactor-Based Model for the Bumble Bee Gut Microbiome

Authors: Christine V. Macpherson (1), Brendan A. Daisley (1), Sarah J. Vancuren (1), Dylan J. L. Brettingham (1), and Emma Allen-Vercoe (1)
Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada
Keywords: Bumble bee, microbiome, in-vitro, bioreactor

Abstract:

Bumble bees (Bombus spp.) are highly beneficial pollinator insects who, similar to humans, possess a core gut microbiota consisting of host-adapted symbionts consistently present across spatiotemporal divides. Along with similar microbiota compositions, both bumble bees and humans exhibit similar social structures and encounter the same environmental stressors inhabiting anthropogenic environments, such as antimicrobials and pollutants. However, they notably differ in their species richness, as bumble bees possess a microbiota consisting of 50-100 bacterial species, whereas humans host approximately 1000. Given this magnitude of difference, the bumble bee holobiont can serve as a model of microbe-microbe and microbe-host interactions relevant to health that are less complex to decipher than those of a human. To model these interactions, we are working to establish the “BumbleBiome”, a novel bioreactor-based model which supports the bumble bee gut microbiota. Building upon the framework of the “Robogut”, a similar human colon-based system created in the Allen-Vercoe lab, we adapted a single stage chemostat bioreactor to replicate the conditions of the bumble bee gut. Preliminary results demonstrate promising outcomes, including the establishment of a stable microbial community and the presence of core bumble bee-associated microbes. As well, analysis of 42 small molecule compounds relevant to bumble bee health suggested the presense of pollen-utilizing microbes and confirmed the fermentive capacity of microbes within the BumbleBiome system. The success of our model is promising for the advancement of microbiome research relevant to a wide variety of species. Work is currently underway to optimize a model that will be used for future studies focused on, for example, the impact of xenobiotics and nutrition on the microbiota. Overall, the establishment of the BumbleBiome offers a tractable model with potential to help with the development of novel, microbiome-based treatments to safeguard pollinators, and, therefore, ecological wellbeing and global human food security.

32. Assessing Gut Microbiota Differences in Adolescents at Risk of Type 2 Diabetes Using a Colonic Bioreactor Model

Authors: Connor Gianetto-Hill (1), Alessandra Granato (2), Jill Hamilton (2), Jayne Danska (2), and Emma Allen-Vercoe (1)

Affiliation(s): 1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON Canada; 2. Hospital for Sick Children Research Institute, Toronto, ON Canada

Keywords: Gut microbiome, Type 2 Diabetes, Metagenomics

Abstract

The global incidence of Type 2 Diabetes (T2D) is on the rise, posing an escalating concern particularly among adolescents. Addressing this surge necessitates a deeper understanding of the risk factors contributing to the disease, including the involvement of the gut microbiome.. Existing literature on the gut microbiome’s association with T2D predominantly focuses on populations over 30 already diagnosed with the condition. To bridge this gap, we utilized stool samples obtained from adolescents at risk of developing T2D to inoculate bioreactors designed to model the colonic environment. In parallel, stool from healthy young participants were inoculated in bioreactors to discern differences between the healthy and the at-risk of T2D gut microbiomes. Given the established influence of dietary nutrients on gut microbiome composition, bioreactors were initially supplied with nutrient-rich media, transitioning to a nutrient-poor medium after 28 days to investigate dietary impacts on community composition and function. Shotgun metagenomic sequencing of stool samples revealed some differences in bacterial composition and predicted functions between healthy and T2D risk groups. Furthermore, bacterial community composition in bioreactors was assessed using 16S rRNA marker gene sequencing, while metabolites were identified and quantified using proton nuclear magnetic resonance (1H-NMR), facilitating a comparative analysis between healthy and at-risk adolescents. This approach aims to identify absent bacterial taxa or functions in at-risk adolescent microbiomes, with the ultimate goal of designing potential gut therapeutics to restore these microbial communities.