Development of microbial source tracking markers for detection of human-associated Clostridium perfringens

Microbial source tracking (MST) is a promising approach for identifying human fecal and wastewater contamination in aquatic environments. However, many existing human-associated markers offer limited environmental persistence or restricted applicability. In this study, we developed novel human-associated MST markers based on Clostridium perfringens, which exhibits high environmental persistence and is widely used as a fecal indicator bacterium. A pan-genome-wide association study of 262 fecal-origin C. perfringens genomes, integrating publicly available and newly sequenced data, identified 62 genes with ≥95 % specificity to human sources. Among these, three genes demonstrated specificity of 96.9-100 % and sensitivity of 10.4-36.5 % in an independent dataset of 194 genomes and were designated as novel human-associated markers: HumCp1, HumCp2, and HumCp3. A multiplex PCR assay was developed to detect these markers on a per-colony basis, along with a quantitative PCR (qPCR) assay for quantifying HumCp1 in environmental DNA. Field testing using 49 animal-derived isolates and 85 wastewater treatment plant (WWTP) effluent isolates confirmed their specificity and sensitivity. In environmental C. perfringens strains, marker prevalence increased with contamination level: 0 % in stream water with minimal human fecal impact (n = 28), 30.8 % at river sites with limited WWTP influence (n = 52), and 59.5 % at river sites downstream of multiple WWTPs (n = 37). Quantified HumCp1 marker concentration also reflected the contamination levels across these water bodies. Overall, this study introduces three novel C. perfringens-based MST markers that complement existing MST tools and enhance the ability to trace aged human fecal contamination.