Characterization of Campylobacter, Salmonella, and Diarrheagenic Escherichia Coli from Food, Food Waste and Water in the Chobe Region of Botswana

Introduction and Justification: Diarrheal disease is a leading cause of death in children in low- and moderate-income countries. Food, food waste, and water are all vehicles that can promote the spread of diarrheal disease-causing bacteria like Campylobacter, Salmonella enterica, and E. coli. Resistance to commonly used antibiotics is on the rise, making them difficult to manage. This study aimed to determine prevalence and antibiotic resistance profiles of Campylobacter, S. enterica, and E. coli isolated from food, food waste, and water samples obtained from the Chobe Region of Botswana. In addition, the survival of two common pathogens, E. coli and C. jejuni, on kale, a type of leafy green commonly consumed raw, was determined. Methods: Samples were collected from the Chobe region of Botswana in 2022 including water from the local river, food (produce, beef, pork, and poultry) from local vendors, and food scraps from the landfill. Food samples were enriched in the appropriate selective media: Brilliant Green Bile Broth for E. coli, Bolton Broth for Campylobacter, and Rappaport Vassiliadis Broth for S. enterica. Water samples were collected using modified USEPA methods1103.1 and 1604, E.coli isolation was performed by plating on RAPID E.coli2 agar and incubation at 37°C for 2h and 44°C for 16-22h. Campylobacter, S. enterica, and E. coli were isolated from meat, poultry, and water samples before being sent to Virginia Tech, while enriched bacterial pellets from the produce were shipped for screening and isolation at Virginia Tech. E.coli were confirmed by PCR detecting the phoA gene (all E. coli), and classified as pathogenic through screening for the eae (present in enterohemorrhagic and enteropathogenic E.coli), stx1 and stx2 (present in enterohemorrhagic E. coli) and est1b ( present in Enterotoxigenic E.coli) genes. Campylobacter isolates were confirmed using a genera-specific PCR while S. enterica isolates were confirmed using invA primers. These enrichment and primer sets were tested as part of a study to determine the survival of E. coli O157:H7 and C. jejuni on kale during a 21-day shelf life. E. coli and S. enterica isolates were subjected to antibiotic resistance testing using the Kirby-Bauer Disk Diffusion method. Results: Methods for detection of inoculated E. coli O157:H7 on kale indicated survival for the majority of the shelf-life (up to 19 d), in comparison, C. jejuni was undetectable by day 13 using enrichment and PCR or plating. From the Botswanan samples, E. coli was isolated from 20% of produce, 49% of meat, and 84.7% of water. Salmonella was only isolated from produce samples (2.4%, 7/294). Resistance was uncommon among the Salmonella isolates with only one isolate being resistant to chloramphenicol. No Campylobacter were isolated from the screened produce, meat, or food waste. E. coli resistant to 3 or more classes of antibiotics (MCR) were identified in 15.5% of produce, and 22.2% of meat isolates. Isolation of E. coli or Salmonella from meat was not associated with a particular food type. In contrast, isolation of E. coli was more common from certain types of vegetables and fruits. Antibiotic-resistant E. coli were isolated more commonly from beef, poultry, and pork than from produce. Multi-class resistant E. coli were isolated from fruits, greens, soil associated, and above ground associated vegetables, beef, and poultry. Water samples were collected from the same time period as the food samples. E. coli isolation, especially pathogens (based on eae presence) was more frequent from environmental water samples collected during the wet season compared to the dry season. Water samples collected during periods of increased rainfall were more likely to contain E. coli isolates, especially pathogens. S. enterica and Diarrheagenic E. coli isolates, especially MCR isolates, pose a significant risk of illness to consumers. Strategies to reduce the circulation of these pathogens in foods and water sources are needed. Master of Science in Life Sciences People can get sick with diarrheal diseases after consuming contaminated food and water. These illnesses are difficult to treat and control when the bacteria causing them are resistant to antibiotics. Campylobacter, Salmonella, and diarrheagenic E. coli are three types of bacteria that can cause illness from food and water. These illnesses disproportionately affect people, especially children, in low-and moderate-income countries like Botswana. Little is known about the prevalence of Campylobacter, Salmonella, and diarrheagenic Escherichia coli in Botswana. This study aimed to determine the prevalence of these microorganisms as well as how resistant they are to different types of antibiotics. Samples from produce, beef, poultry, pork, and recreational water sources were collected in the Chobe region of Botswana over the course of 2022. Food samples were collected from different vendors and food scraps were obtained from the landfill. E. coli and Salmonella were isolated out of meat and water samples in Botswana while produce samples were shipped as mixed cultures to Virginia Tech where E. coli, Salmonella, and Campylobacter isolation or confirmation was done. Once the target bacteria were isolated, their resistance to certain antibiotics was tested. Salmonella was only found in produce from samples collected during October-December. No Campylobacter was found from produce, meat, or food waste. More E. coli was isolated from fruit or vegetable food waste collected from the landfill than from produce bought at local vendors. E. coli was obtained from meat purchased from local vendors more often than samples from the landfill. E. coli was found more often in water when there was more rainfall. E. coli that causes illness was also more likely to be obtained during the wet seasons. Resistant E. coli that could not be killed by the screened antibiotics, were classified as multi-drug resistant when it was resistant to more than three antibiotics. Rainfall, season, and the food source influenced if E. coli isolates were likely to be multi-drug resistant. While more research is needed to determine how these bacteria are moving in the environment and gaining resistance to antibiotics, the findings of this study show they are present in the environment and require further research.