Identification of fecal contamination sources of groundwater in rural areas of Vhembe District Municipality, Limpopo Province, South Africa.

Groundwater is a valuable source of drinking water worldwide, recognized as an improved drinking water source. However, on-site sanitation systems may put groundwater at risk of fecal contamination. In the present study, two approaches were used to ascertain the sources of fecal contamination in groundwater used by communities of the Vhembe District Municipality. Overall, 87.5% of boreholes (n = 70) in the wet and 72.5% in the dry season were contaminated with Escherichia coli, and septic tank (n = 18) wastewaters displayed up to 104 cfu/mL E. coli. Host-specific Bacteroidales quantitative polymerase chain reaction (qPCR) assays established the presence of human (BacHum and HF183) and animal (Cytb, BacCan, and Pig-2Bac) genetic markers in groundwater from 15.7% of boreholes (wet) and 10% of boreholes (dry). No strong associations were founded between culturable E. coli counts and the presence/absence of marker genes for all the markers except for Cytb marker, which showed a weak significant correlation (r = 0.217; p = <0.01) between E. coli and the Cytb marker under dry seasonal conditions. Human markers and Cytb were present in the household septic tank wastewater samples. Significant differences in marker genes distribution in wastewater were observed using the Chi-squared test: HF183 (p = <0.001) and BacHum (p = <0.001). Overall, no association was recorded between markers in groundwater and in wastewater for 18 households' septic tanks. A combined culturable E. coli and host-specific Bacteroidales qPCR assays remain an appropriate approach for the identification of fecal contamination of groundwater. Practitioner Points • Households primarily used private boreholes for drinking water, as a primary source • Most households used on-site sanitation systems, including ventilated improved pit latrines and flush toilets connected to septic tanks. • Escherichia coli was detected in groundwater, and the sources of fecal contamination were humans and animals (pigs, dogs, and chickens). • The presence of human and animal markers in groundwater suggests that humans and animals are liable for fecal contamination. • Fecal contamination in drinking water sources poses a significant concern due to pathogenic microorganisms posing potential human health risks.