Circulation of enterotoxigenic Escherichia coli (ETEC) isolates expressing CS23 from the environment to clinical settings

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infant diarrhea in low- and middle-income countries (LMICs). Diarrheal pathogens are transmitted through environmental reservoirs; however, the bacterial clones that spread across the human-environment interface remain unexplored. We aimed to determine the relationship and clonal dissemination of ETEC between children with diarrhea and polluted water samples from a local river in La Paz, Bolivia. By using WGS and the PhenePlates phenotypic system to analyze ETEC strains, we showed that ST218 and ST410 LT+STh ETEC expressing the colonization factor (CF) CS23 were found with high frequency in both samples. The CS23 ETEC isolates were found within several STs, E. coli phylogroups, and across ETEC lineages. Comparative genomic evaluation and PhenePlate screening of globally distributed clinical ETEC strains suggest that the CS23 gene is likely carried on plasmids acquired independently of the bacterial chromosomal background. Clinical strains were more often multidrug-resistant (MDR) than environmental isolates and harbored the class 1 integron-integrase gene intI1 next to the MDR cassettes. Retrospective analysis of antibiotic resistance in ETEC revealed a high frequency of MDR in clinical isolates. The LT+STh CS23 environmental ETEC isolates, showed an increased biofilm ability at environmental temperature, equal cytotoxicity, and significantly lower adherence to human epithelial cells compared to ETEC expressing other CFs. Together, we suggest that CS23 is more prevalent in ETEC than previously estimated, and the Choqueyapu River is a reservoir for LT+STh CS23 ETEC containing strains capable of causing diarrheal cases in children. IMPORTANCE The importance of clean water cannot be overstated. It is a vital resource for maintaining health and well-being. Unfortunately, water sources contaminated with fecal discharges from animal and human origin due to a lack of wastewater management pose a significant risk to communities, as they can become a means of transmission of pathogenic bacteria like enterotoxigenic E. coli (ETEC). ETEC is frequently found in polluted water in countries with a high prevalence of diarrheal diseases, such as Bolivia. This study provides novel insights into the circulation of ETEC between diarrheal cases and polluted water sources in areas with high rates of diarrheal disease. These findings highlight the Choqueyapu River as a potential reservoir for emerging pathogens carrying antibiotic-resistance genes, making it a crucial area for monitoring and intervention. Furthermore, the results demonstrate the feasibility of a low-cost, high-throughput method for tracking bacterial pathogens in low- and middle-income countries, making it a valuable tool for One Health monitoring efforts.