Clostridium difficile: A Study on its Potential as a Food-borne Pathogen and Strategies for Controlling its Transmission

Clostridium difficile is a gram-positive, spore-forming, anaerobic, nosocomial pathogen affecting hospitalized patients taking long-term antibiotics. The bacterium is responsible for over 500,000 cases and 29,000 deaths annually (Lessa et al., 2015), with ~ $3 billion in health-care costs (O\u27Brien et al., 2007). There has been the emergence of a new hypervirulent strain of C. difficile known as North American Pulsotype 1 (NAP1), which is responsible for severe infections. Further, an increase in the incidence of C. difficile infections outside of hospital environments (Hensgens et al., 2012; McDonald et al., 2006; Rupnik et al., 2009) is observed. It is reported that 32% of all C. difficile illnesses in the United States are community associated C. difficile infections, especially in individuals with no recent hospitalization (Lessa, 2013). C. difficile has been isolated widely from the environment in many countries, especially from soil and water. The persistence of C. difficile in the environment and isolation of hypervirulent C. difficile from food animals and foods suggest a potential role for foods as a source of C. difficile infection in the community. In this dissertation, the prevalence of C. difficile in ready-to-eat lettuce collected from retail stores in Connecticut was investigated. The results showed a prevalence rate of 23% in ready-to-eat lettuce, with the isolates being toxigenic and resistant to multiple antibiotics. The survival of C. difficile in ready-to-eat lettuce bags packaged with modified atmosphere packaging (MAP) conditions was also studied. C. difficile spores were found to remain viable under MAP, but did not increase in number over 10 days of refrigerated storage. As the second objective, the viability of C. difficile spores in ground beef subjected to chilling, freezing and cooking was investigated. Results revealed that chilling for a week or freezing for 12 weeks did not affect the survival of C. difficile spores (p \u3e 0.05). Moreover, cooking ground beef to the USDA recommended internal temperature (71.1°C) decreased spore counts only by 1 log CFU/g compared to uncooked controls. Since no previous research investigated the viability of C. difficile spores in any fermented meat product, the effect of acidity (fermentation) and cooking on the viability of C. difficile spores in ready-to-eat, fermented pork summer sausage over a period of 60 days at 4oC was determined. Results indicated that C. difficile spores can survive the acidity and cooking in fermented pork summer sausage, underscoring the need for effective intervention strategies to control C. difficile contamination of pork carcasses. C. difficile spores can persist on hospital surfaces and equipment for prolonged periods of time, thereby serving as a potential source of infection. Commonly contaminated hospital surfaces and equipment include, floors, toilets, windowsills, bedrails, bedside-tables, thermometers, blood-pressure cuffs, and intravenous catheters (Dubberke et al., 2008; Gerding et al., 2008; Guerrero et al., 2012). C. difficile spores are resistant to most of the commonly used physical and chemical disinfectants, including household dilutions of sodium hypochlorite (Edwards et al., 2016; Fawley et al., 2007; Gerding et al., 2008). Moreover, sodium hypochlorite is not suitable for sanitizing some medical surfaces and devices. Therefore, as the fourth objective, the efficacy of a novel disinfectant, namely octenidine hydrochloride (OH), for reducing C. difficile spores on stainless steel surfaces was evaluated. Application of OH (5%) directly on stainless steel surfaces or wiping with OH decreased populations of C. difficile spores significantly compared to controls. In infected humans, sporulation of C. difficile vegetative cells occurs in the colon, and newly formed spores shed in the feces can potentially contaminate hospitals and healthcare facilities, leading to new infections through the fecal-oral route (Barbut, Menuet et al. 2009). Therefore, it is critical to control C. difficile sporulation in the human gastrointestinal tract for controlling the infection and its transmission. Aspirin (acetylsalicylic acid) is a non-steroidal anti-inflammatory drug widely used in medicine (Soni 2005). In an observational study of adults diagnosed with C. difficile infection, aspirin was identified as a factor that reduced mortality (Saliba, Barnett-Griness et al. 2014), although the mechanism behind this is not known. Therefore, as the final objective, the effect of aspirin on C. difficile toxin production, spore production, spore germination and spore outgrowth was investigated. Although aspirin had no effect on C. difficile toxin production, it reduced spore production and spore outgrowth significantly in C. difficile (p \u3c 0.05). Results justify follow-up studies in an appropriate animal model to validate the anti-sporulation effect of aspirin against C. difficile