Exploratory Characterization of the Water Microbiome, Resistome, and Fecal Contamination Sources in Two Chadian Waterbodies

Background/Objectives: Fecal contamination in surface waters can introduce pathogens and antibiotic resistance genes (ARGs), creating potential risks for human and animal health, especially in low- and middle-income countries. Yet microbial water quality in Chad remains understudied. This exploratory study characterized the fecal contamination sources, resistome and microbial communities of two waterbodies in Chad. Methods: Surface water samples were collected from two waterbodies in Ali Garga, Chad, and concentrated by membrane filtration, and DNA was extracted (n = 4). Droplet digital PCR (ddPCR) assays were performed for microbial source tracking (MST) and ARGs. Shotgun metagenomic sequencing was used to characterize bacterial and microeukaryotic communities and the environmental resistome. Results: Sulfonamide and tetracycline resistance genes were abundant. The sul1 gene was detected at 4.84 and 3.74 log GC/100 mL at Sites 1 and 2, respectively. At each site, more than half of the ARGs identified through metagenomic analysis were annotated as able to confer resistance to glycopeptide antibiotics, including vancomycin. MST results revealed substantial avian and canine fecal contributions at both sites. At both sites, the Pseudomonadota and Actinomycetota bacterial phyla were the most abundant. Chlorophyta and Ciliophora co-dominated the eukaryotic community at Site 1, and Chlorophyta dominated at Site 2. Conclusions: While samples for this study come from two waterbodies and are not generalizable, the exploratory findings provide initial evidence of fecal contamination and diverse ARGs in two surface waters in Chad. They also demonstrate the value of combining MST, targeted ARG quantification, and metagenomics to investigate water quality from a One Health perspective. This approach can inform future studies of environmental and animal-associated exposures, including those relevant to Guinea worm disease.