Carolina Tropini

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. The specificity of phage-bacterial interactions has generated renewed interest in phage research as a potential alternative strategy to counter the increasing threat of antimicrobial resistant bacteria. While there has been some success in using phage therapy to combat bacterial septic infections, we still do not have the foundational understanding of phage-bacteria-host dynamics within our gut ecosystems that is needed for their safe development.Recent studies demonstrating that phages adhere to intestinal mucus through specific capsid proteins (Hoc) have suggested that phages may protect the underlying epithelium from bacterial invasion, providing a host-extrinsic mechanism to maintain intestinal homeostasis. Here, I build upon these findings to investigate the kinetics between Escherichia bacteriophage T4 (containing a Hoc domain) and its target bacterium, Escherichia coli, within the intestinal tract of a gnotobiotic mouse model. I determined that T4 phage and E. coli can stably coexist within the murine gastrointestinal tract in the absence of other microbes, despite continual phage predation. However, I was unable to conclude that T4 phage retention within the murine gut requires Hoc protein-mediated mucus adhesion. Further, my data suggest that gut-colonising T4 phage elicit a type 1 immune response in the gut-draining lymph nodes, without causing inflammatory disease. Together, these results suggest that T4 phage is well tolerated in the gastrointestinal tract of gnotobiotic mice by the bacterial and metazoan hosts and may contribute to immune system priming.

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Bowel preparation (prep) is a common practice that precedes colonoscopy procedures in the clinic to clear out luminal contents of the gastrointestinal tract (GIT). Most commonly, osmotic-based laxatives are used for bowel prep based on ease of administration and are considered harmless to patients. One of the most common osmotic laxatives used in the clinic is polyethylene glycol (PEG), which functions by increasing osmolyte concentration in the gut lumen and promoting gut motility. The human gut microbiota, a diverse group of microorganisms in the GIT, plays a crucial role in health. Recent studies indicate that osmotic stress alters the gut environment, selecting for osmotically tolerant bacteria in the microbiota. This study aimed to investigate the effects of bowel prep on the gut microenvironment and microbiota.Using a murine model, we observed changes in the GIT essential for colonization resistance. To determine if bowel prep impairs this resistance, we examined pathogen infection and expansion in the gut microbiota. We tested Salmonella enterica, an organism known for its osmotic resistance and role in host-pathogen interactions. Our findings showed that bowel prep disrupts the gut's physical environment, reducing colonization resistance and significantly increasing Salmonella colonization and translocation to extra-intestinal organs. GIT tissue imaging showed changes to the mucus layer and epithelial damage due to Salmonella post-bowel prep.Building on these findings, we explored how bowel prep affects microbiota with existing pathogenic bacteria, focusing on inflammatory bowel disease (IBD), a chronic GIT disorder. Although the cause of IBD is unknown, research highlights the prevalence of pathobionts, bacteria that become pathogenic under certain conditions. Some IBD patients experience inflammatory episodes post-bowel prep, leading to emergency room visits and reluctance to undergo colonoscopies. We transplanted an IBD patient’s microbiota into a mouse model and found that post-bowel prep, IBD pathobiont bacterial translocation persisted for two weeks.These findings offer insights into pathogen infection mechanisms following bowel prep and reveal how osmotic perturbations can exacerbate pathogen and pathobiont activity in the gut. This research investigates potential mechanisms behind pathogen infection post-gut disruption and highlights the systemic effects of bowel prep on the host.

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Crohn’s disease and ulcerative colitis, collectively termed inflammatory bowel disease (IBD), are chronic and incurable diseases of the gastrointestinal tract. Beyond the gastrointestinal tract, IBD patients exhibit high rates of hormonally driven sexual, reproductive, and psychiatric disorders. For instance, delayed puberty is reported in up to 85% of pediatric IBD patients, and sexual dysfunction is reported in up to 60% of adult female patients and up to 94% of adult male patients. Further, psychiatric illness is two to three times as prevalent in IBD patients compared to the general population.Previously, systemic sex steroid levels were thought to be exclusively controlled by the hypothalamic-pituitary-gonadal axis. However, recent research has identified gut microbes capable of degrading androgens and reactivating estrogens in a clinically significant manner. Based on these findings, we proposed that the gut microbiota could play a role in driving the high prevalence of neuroendocrine comorbidities observed in IBD. Specifically, we hypothesized that IBD-like alterations to the murine gut would induce sexual, reproductive, and psychiatric effects like those seen in IBD patients. To test this hypothesis, we used the well-known dextran sodium sulfate (DSS) model of IBD to disrupt the murine gut microbiota and induce inflammation at specific developmental time points, and measured the effect on the gut, systemic sex steroid levels, reproductive development, brain cell morphology and behavior. Confirming our hypothesis, we found that DSS induced inflammation reshapes the composition of the gut microbiota, compromises the integrity of the gut epithelium, impedes the development of the seminal vesicles, and causes changes in sex-specific behavior. In contrast, DSS did not appear to disrupt systemic sex steroid levels, affect the timing of pubertal onset, cause damage to the reproductive organs or alter mating behavior. Taken together, these results suggest that DSS inflammation selectively impacts certain aspects of the gut-endocrine-brain axis in IBD while leaving others unaffected. This points to the need for further research that can take a multi-system approach to investigating the complex and nuanced interplay between the gut microbiota, immune system, endocrine system, reproductive organs, brain and behavior in IBD.

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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. Physical factors 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. This project aims to characterize the strain-specific adaptability of commercially available probiotic strains to disease-relevant physical parameters – pH and osmolality. Different strains of lactic acid producing bacteria and Bifidobacterium spp. were isolated from ten commercially available probiotics and assessed for their growth in various pH and osmolality conditions and for their ability to impact their surrounding abiotic environment. We found that probiotic strains from the same phyla exhibit differential growth responses to high osmolality and low pH conditions, and we performed comparative genomic analysis to identify candidate genes involved in probiotic stress response. This study highlights the impact of biophysical parameters on commensal bacteria survival and helps to inform characteristics that are important for probiotic strains to establish sufficient viability in the dynamic gastrointestinal environment and will ultimately facilitate the development of other microbiota-based therapies.

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