Background: C. difficile is one of the primary infectious causes of morbidity and mortality in Canada. Colonized patients can pose a risk to others as a factor in the transmission and development of hospital-associated C. difficile infections. Despite immense efforts and resources invested in the reduction in C. difficile transmission within Canada and Hamilton Health Sciences – further reduction in these rates are unlikely, and novel screening strategies are imperative in this field of study. Methods: This project was a retrospective cohort study of adult in-patients admitted to either The Juravinski, Hamilton General, or St. Joseph’s Healthcare Hamilton Hospitals from January to April 2018 and September 2018 to August 2019. MSRA/VRE swabs were collected during admission or through universal point prevalence screening and subsequently tested for colonization. Results: From the 1056 patients in the data sample, 72 were colonized with asymptomatic C. difficile resulting in a prevalence rate of 6.81%. In-patient point prevalence screening strategies identified more carriers than admission swabs alone (p < 0.001). Risk factors for colonization on admission were being female (OR 2.66, 95% CI 1.02-8.33) and previous CDI (OR 4.76, 95% CI 1.49 – 13.86). During hospitalization, risk factors for colonization were previous CDI (OR 4.75 95% CI 2.14-9.94) and recent hospitalization within the last 12 months (OR 2.35, 95% CI 1.30-4.42). The multi-level Cox PH model identified those with a recent hospitalization (OR 2.21, 95% CI 1.32 – 3.73) and those with previous CDI (OR 2.40, 1.34 – 4.30) were twice as likely to develop asymptomatic C. difficile colonization throughout hospitalization. Conclusion: The addition of universal point prevalence screening in addition to admission screening helped identify more than double the amount of carriers in the population. Moreover, a previous hospitalization, previous CDI, and being female may indicate patients at the highest risk of colonization. / Thesis / Master of Science (MSc) / C. difficile infection (CDI) is a severe infectious disease. Patients with asymptomatic C. difficile present a risk to others as they can contribute to the spread and development of hospital-associated CDI. We are currently unsure of the proportion of adult in-patients colonized with asymptomatic C. difficile. Identifying these carriers early on in their hospital stay is imperative to reduce CDI rates in health care settings. Our study objectives were to determine the best screening strategies to identify asymptomatic carriers, identify risk factors for carriage, and understand the transition from asymptomatic C. difficile to symptomatic CDI. We demonstrated that being female, being recently hospitalized or previously having CDI may increase a patient's risk of being an asymptomatic carrier. Also, timely screening throughout a hospital stay in addition to admission screening helped identify more colonized in-patients. Lastly, we determined that 1 in 5 carriers would go on to develop symptomatic CDI infection.

Clostridioides difficile (C. difficile) and human rotavirus (HRV) are leading causes of bacterial and viral gastroenteritis worldwide. Treatment and vaccination options for both pathogens have significant limitations. C. difficile infections are treated with antibiotics, which is paradoxical as C. difficile itself is associated with antibiotic usage. In the United States, two live oral attenuated vaccines (Rotarix and RotaTeq) are licensed for protection against HRV. Since receiving approval from the World Health Organization (WHO), Rotarix and RotaTeq have been widely implemented into global national childhood immunization schedules, with one report finding 59 countries using Rotarix and 25 using RotaTeq. However, these vaccines have much lower efficacy rates in low- and middle-income countries. Because of these caveats, there is an urgent need to generate novel prophylaxes and treatments for C. difficile and HRV. In order to address this need, animal models that replicate the nuances of each infection are imperative. We have developed gnotobiotic (Gn) pig models for each pathogen. Gn pigs infected with spores of the hypervirulent UK1 strain of C. difficile develop classical signs of infection, including watery diarrhea and weight loss. Gross necropsy reveals colonic distention and discoloration, and histopathological evaluation shows volcano lesions, pseudo membrane formation, and epithelial cell erosion. Gn pigs infected with a G4P[6] strain of HRV also display pathogen specific signs of infection, including diarrhea, fecal rotavirus shedding, and damaged intestinal villi. A dose response study of the G4P[6] strain revealed diarrhea and virus shedding occurred at all tested doses, however the most severe diarrhea and virus shedding, measured by cumulative diarrhea score, area under the curve (AUC) of diarrhea, peak virus titer, and AUC of virus shedding, were all detected in the highest dose group. Based on the presentation of clinical signs of infection, 105 fluorescent focus units was selected as the optimal challenge dose for future studies. These models enable us to test candidate therapeutics, but also elucidate unique replicative features of the pathogens. For example, we found that HRV can replicate in the salivary glands and nasal cavity of Gn pigs in addition to the small intestine. HRV infection primed immune responses in the ileum, tonsils, and facial lymph nodes; infection also induced high levels of systemic and mucosal rotavirus specific antibody responses. Moving forward, we hope to expand upon this replication study to identify what cell types within the glands are infected as well as look at local cellular immune responses to HRV infection. Additional future directions include determining the protective efficacy of next generation HRV vaccines and evaluating effectiveness of an engineered probiotic yeast in reducing severity of C. difficile infection and disease. The Gn pig models of C. difficile and G4P[6] HRV are clinically relevant, and they will continue to serve as useful tools to better our understanding of pathogenesis, infection, and prevention of these pathogens. / Doctor of Philosophy / Clostridioides difficile (C. difficile) and human rotavirus (HRV) both cause gastrointestinal related symptoms when they infect humans. Treatments available for C. difficile and HRV all have significant drawbacks. This represents a gap in knowledge which we aimed to fill by developing germ-free (gnotobiotic [Gn]) pig models of C. difficile and HRV infection and disease. Animal models that mimic the outcomes of disease seen in humans are essential for evaluating protectiveness of new therapeutics. The more similar the disease presentation, the more likely the treatment results will be translational to humans. We began with C. difficile; pigs were orally fed C. difficile and monitored for a week post infection for development of signs of infection. Inoculated pigs lost weight and developed diarrhea. Bacterial cells and toxins were isolated from fecal samples collected on various days post infection. Multiple changes were observed in infected pigs’ large intestinal tissues, including severe bleeding, tissue distension, and fluid buildup. Infected pigs also had significant upregulation of pro-inflammatory cytokines, indicating activation of the immune response. We performed a similar procedure for the establishment of the HRV model. Gn pigs were orally challenged with differing doses of G4P[6] HRV and followed for several days post infection. Consistent with HRV infection in children, the pigs developed watery diarrhea that lasted for multiple days. Small intestinal tissues collected at necropsy had several signs of damage, including blunted villi, fluid buildup, and immune cell invasion. These lesions were also consistent with HRV infection in humans. Taken all together, these results indicated successful establishment of both C. difficile and HRV models. While the primary goal of generating these models was to evaluate new treatments, a secondary goal was to use them to better our understanding of pathogen replication dynamics. For example, the small intestine was thought to be the primary site of HRV infection. Using a pig model of HRV, we expanded on this knowledge to show that HRV can replicate in the nose and salivary glands as well. Additionally, we found HRV infection to induce immune responses near the sites of infection, including the intestine, the tonsils, and the facial lymph nodes. Overall, these studies demonstrate the utility of germ-free pig models and are an important first step in generating more effective treatments for bacterial and viral infections.

Bacterial biofilms are 1,000 times harder to kill than planktonic bacteria. Spores are also harder to kill compared to vegetative cells. We developed a rapid model for establishing dry surface bacterial biofilms for disinfectant efficacy testing and challenged them with seven EPA-registered disinfectants. We also demonstrated that during the disinfection of surfaces contaminated with bacterial spores, disinfectant wipes may transfer spores from contaminated to uncontaminated surfaces.

Clostridioides difficile is a bacterial pathogen that causes pseudomembranous colitis and the majority of antibiotic-associated diarrheal cases. Broad-spectrum antibiotic usage disrupts the normal gut microbiota and thereby compromises colonization resistance, the main defense against C. difficile infection. Treatment options are limited to vancomycin, fidaxomicin, and the fecal microbiota transplant. Addressing the scarcity of these therapeutics, we documented two explorations in C. difficile drug discovery: (i) evaluation of antibacterial and toxin-suppressing activity of (+)-puupehenone and similar derivatives, and (ii) clarification of a discrepancy in the hypothesized mechanism of auranofin against C. difficile. A better understanding of how C. difficile colonizes and thrives in the gut can greatly benefit therapeutic development. Interestingly, C. difficile can scavenge nutrients such as amino acids and possibly even purines during infection. Amino acids including proline and glycine act as substrates for Stickland metabolism, a bioenergetics scheme that partially relies on enzymes containing selenium in the form of selenocysteine (e.g., D-proline reductase and glycine reductase). Purines such as xanthine and uric acid can be degraded by bacterial molybdenum hydroxylases harboring an uncharacterized form of selenium, though the role of these enzymes in C. difficile physiology is poorly understood. Selenium likely plays a key role in the scavenging of these nutrients during C. difficile infection. Our investigation of these selenium-dependent enzymes revealed two new findings in C. difficile biology: (i) a link between proline-dependent growth and D-proline reductase, characterized as an energy "addiction," and (ii) a previously uncharacterized selenium-dependent pathway involved in the catabolism of xanthine and uric acid. Overall, these physiological analyses of C. difficile provide promising candidates for therapeutics and key information regarding the organism's nutrient preferences.

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Investigating novel treatment approaches to combat Clostridioides difficile Rusha Pal ABSTRACT Clostridioides difficile is the leading cause of antibiotic-induced diarrhea and colitis in hospitals and communities worldwide. The enteric pathogen, classified to be an "urgent threat" by the United States Center for Disease Control and Prevention (CDC), capitalizes on disrupted intestinal microbiome to establish infection with disease symptoms ranging from mild diarrhea to potentially fatal conditions. Disruption of the intestinal microbiome, caused mostly by antibiotic use, enables C. difficile to colonize and proliferate within the host. Paradoxically, antibiotics are used to treat C. difficile infection. These antibiotics decimate the gut microbial community further, thus priming the gastrointestinal tract to become more prone to recurrence of infection. To tackle this clinical setback, we utilized a combination of traditional and non-traditional drug discovery approaches and identified chemical entities and targeted treatment options effective against this toxin-producing intestinal pathogen. Herein, we exploited the strategy of high-throughput screening to identify leads that harbor anticlostridial activity. Our primary phenotypic screen of FDA-approved drugs and natural product libraries led to the identification of novel molecules that were further characterized for their anticlostridial efficacy both in vitro and in vivo. The most potent scaffolds identified were those of mitomycin C, mithramycin A, aureomycin, NP-003875, NAT13-338148, NAT18-355531, and NAT18-355768. Of these, mithramycin A, aureomycin, and NP-003875 were also found to harbor anti-virulence properties as they inhibited toxin production by the pathogen. Furthermore, natural product NP-003875 could confer protection to 100% of the infected mice from clinical manifestations of the disease in a primary infection model of C. difficile. Our final approach has been to develop targeted therapeutics called peptide nucleic acids (PNAs). PNAs are antisense agents capable of inhibiting gene expression in bacteria. In this study, antisense inhibition of the RNA polymerase  subunit gene (rpoA) of C. difficile was found to be bactericidal for the pathogen and could also inhibit the expression of its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene was found to be non-deleterious for the tested commensal microflora strains. Given their intriguing anticlostridial properties, it can be concluded that our research opened exciting possibilities that can be further evaluated to uncover new treatments for CDI. / Doctor of Philosophy / Investigating novel treatment approaches to combat Clostridioides difficile Rusha Pal LAYMAN LANGUAGE ABSTRACT Clostridioides difficile is a prominent human pathogen that can colonize the gut and cause fatal infections. C. difficile is the most common cause of microbial healthcare-associated infection and results in substantial morbidity and mortality. The "most urgent worldwide public health threat" label has been assigned to C. difficile by the United States Centers for Disease Control and Prevention (CDC). There is a pressing need to develop new classes of antibiotics with improved efficacy to treat C. difficile infections (CDI). To address the need for novel strategies to combat the growing problem of CDI, we screened FDA-approved drugs and natural products library in search of novel drugs that possess potent and specific anticlostridial activity. Several promising hits were identified and evaluated successfully both in vitro and in vivo. The most potent and novel hits that displayed exceptional activity were mitomycin C, mithramycin A, aureomycin, NP-003875, NAT13-338148, NAT18-355531, and NAT18-355768. Furthermore, a murine model of C. difficile infection revealed that compound NP-003875 conferred 100% protection to the infected mice from clinical manifestations of the disease. Interestingly, these compounds were non-toxic to the gut microflora and human cells. Our final approach has been to develop non-traditional therapeutics to target specific genes in C. difficile. These novel therapeutics are called peptide nucleic acids (PNA). Herein, we designed a PNA targeting RNA polymerase  subunit gene (rpoA) of C. difficile. The designed PNA could successfully inhibit the growth of the pathogen and expression of its virulence factors. In conclusion, our research opened exciting possibilities that can be further evaluated to uncover new treatments for CDI.

Irritable Bowel Syndrome (IBS) is the most common gastrointestinal disorder which affects approximately 4% of the population worldwide, according to the Rome IV criteria. It is characterized by abdominal pain and altered bowel movements in the absence of relevant structural abnormalities. The diagnosis of IBS is based on symptom profiles as no biomarkers exist to guide diagnosis or treatment stratification. Accumulating data suggests that altered gut microbiota and chronic low-grade inflammation play important roles in genesis of IBS. However, the mechanisms are unclear. My thesis first addresses the hypothesis that changes in fecal β-defensin secretion reflect compositional changes in the intestinal microbiota. This was driven by the understanding that compositional changes in the microbiota (“dysbiosis”) may play a role in the expression of IBS, and that a marker of these will identify those patients who might benefit from microbiota-directed therapies. I used a murine model in which I disrupted the microbiota using interventions relevant to the natural history of IBS i.e., antibiotics, stress, or dietary changes. I showed that experimentally induced compositional changes in the microbiota, with the exception of stress, altered the secretion of fecal β-defensin. My study indicates that monitoring fecal β-defensin over time identifies compositional changes in microbiota. I next investigated mechanisms and treatment options for a recently recognized variant of post-infectious IBS (PI-IBS) developed following antibiotic treatment in patients recovering from Clostridioides difficile infection (CDI). I refer to this variant as post-CDI gut dysfunction. I used a humanized mouse model in which germ-free mice were colonized with fecal microbiota from patients with post-CDI gut dysfunction, or age and sex matched healthy controls. I found that mice colonized with microbiota from a patient with severe slow transit constipation post-CDI reproduced the donor phenotype. Mice developed slow colonic transit due to macrophage mediated damage to the interstitial cells of Cajal (ICC) that maintain normal motility. These changes were reversed after fecal microbiota transplantation (FMT) from healthy donor mice thus confirming that the post-CDI gut dysfunction is microbiota driven. Similar results were obtained in a patient with slow transit constipation without a history of infection. My findings prompted me to next evaluate the therapeutic potential of microbiota-directed dietary therapies. I chose psyllium, the flavonoid quercetin, and pectin based on their demonstrated ability to alter microbiota composition. Psyllium and pectin each normalized colonic transit, and this was accompanied by an alteration in macrophages morphology, restoration of the disrupted ICC network and an increase in short chain fatty acids production. My results demonstrate the importance of a dysbiotic microbiota in this post infectious- IBS (PI-IBS) variant and, identify two potentially useful dietary based therapeutic approaches to improve gut dysfunction in these and similar patients. If findings from my thesis are confirmed in humans, it could offer novel approaches for identifying those IBS patients who might benefit from microbiota-directed therapeutics. / Thesis / Candidate in Philosophy

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Clostrioides difficile is a common cause of nosocomial (hospital-acquired) infections. Patients receiving antibiotic treatment experience dysbiosis of gut microbiota, and C. difficile, normally held in check by the various other organisms, takes this opportunity to propagate. Symptoms of infection generally include diarrhea, colitis, dehydration, and fever. Understanding that C. difficile generally only causes illness when it is the dominant bacterium (i.e. when growth is relatively unchecked by other microbes), it is appropriate to investigate potential competitive organisms that may be introduced after antibiotic courses or during active C. difficile infection to effectively displace it. Fecal samples from the University of Central Florida Lift fecal collection station were aseptically plated onto modified cycloserine cefoxitin fructose agar (CCFA). Visually remarkable colonies (certain colonies that looked unique in comparison to others) were restreaked on new plates of the same media to verify growth, then transferred to brain heart infusion-supplemented (BHIS) plates for propagation. Colonies were inoculated in glycerol stocks for storage, then grown in BHIS liquid media to prepare for identification. Genomic extraction was performed on each sample, and spectrophotometric quantification and gel electrophoresis were executed to confirm successful extraction. Genomic samples will be sent to an external laboratory for identification via polymerase chain reaction and Sanger sequencing. We hypothesize that at least one bacterial strain from the fecal collection station will potentially inhibit C. difficile infection. Should such an organism be identified, it follows that the efficacy of its application in conventional hospital settings may be examined. Current regulation of fecal microbiota transplants, an effective therapeutic practice, is cumbersome, and changing the classification of fecal transplants may improve timeliness and effectiveness of treatment.