MSc (Microbiology) / Department of Microbiology / Background: Escherichia coli is a facultative anaerobic bacterium that forms part of the gut microbiota. It is used as an indicator that confirms recent faecal contamination. E. coli have been identified amongst the pathogens that are mostly responsible for moderate to severe diarrheal outbreaks in the low and middle-income countries. With South Africa facing an issue in water scarcity, issues concern poor sanitation and hygiene practices results in serious public health problems and allows E. coli to be transmitted from infected human or animal faeces to a new susceptible host using environmental reservoirs such as soil, water, hands as the transmission pathway. Objective: The primary objective of the study was to characterize E. coli strains from rural communities of Vhembe district, Limpopo, South Africa. Methodology: Households of 7 villages in the Vhembe district were randomly selected. A total of 81 households (HHs) were part of the study. In each household, a structured questionnaire was used to background information on WASH practices. Samples taken from each HH included toilet seat swabs, floor swabs, child and mother handwash samples, stored water samples and running tap water samples. A total of 399 samples were analysed using Colilert® Quanti-trays®/2000 method to detect the presence of Escherichia coli. Positive E. coli samples were further identified using multiplex polymerase chain reaction (m-PCR) to determine the pathogenic strains of E. coli. Transmission pathways were established using identified strains. Results: Data from the structured questionnaires showed common problems of availability of running tap water; lack of provision of sanitation; open practice on defaecation and very little hand hygiene practices. A total of 91 (22.81%) samples tested positive for E. coli with the Colilert® Quanti-trays®/2000 method. The mothers’ handwash samples had the most E. coli prevalence followed by stored water samples. The most prevalent E. coli pathotype was EPEC with the virulence gene eae. Atypical EPEC (60.44%) outnumbered the typical EPEC (5.49%). The pathotype ETEC was detected in 41.76% samples and EHEC in 9.89% samples. Transmission pathway was observed from the different households; with eae gene (aEPEC) being the most detected from samples looking at the LT gene (ETEC). v | P a g e Discussion: All 7 villages are facing common issues such as lacking running water, poor sanitation and improper hand hygiene practices. The mothers were the most contaminated and it was observed that its due to the daily activities that they perform around the house. It is of importance for them to practice proper hand hygiene to prevent transmission of pathogenic E. coli to the children via direct or indirect transmission route. The pathogenic E. coli was detected from all different samples collected from the households including the floor and toilet seat samples. EPEC was detected the most, and studies have shown that this strain is known to cause diarrheal infections in young children from developing countries. Conclusion: The members of the study village households were aware of the WASH services and its importance. However, proper implementation into their day-to-day life is lacking due to the high number of TC and E. coli detected from handwash samples and stored water samples from the households. Recommendation: Appropriate WASH strategies should be established to ensure good health at the village households. Further studies should be done to check possible transmission pathways from more village households. / NRF