Survival and adaptative strategies of Enterotoxigenic E. coli (ETEC) to the freshwater environment

Waterborne pathogenic enterobacteria are adapted for infection of human hosts but can also survive for long periods in water environments. To understand how the human pathogen enterotoxigenic Escherichia coli (ETEC) adapts to acute and long-term hypo-osmotic stress and oligotrophic water conditions, this study aimed to explore the effects of short- and long-term freshwater exposure on ETEC isolates by examining transcriptional responses, survival mechanisms, and antibiotic resistance development. RNA sequencing revealed that over 1,700 genes were differentially expressed, with significant transcriptional reprogramming occurring early within the first two hours of water exposure. Early responses included activation of catabolic pathways for nitrogen and carbon assimilation and downregulation of energy metabolism and anabolic processes to mitigate osmotic stress. Notably, the arnBCADTEF operon was upregulated, facilitating lipid A modification and membrane enforcement which also confers colistin tolerance. ETEC carries virulence genes on large plasmids which cause diarrheal disease in humans. Plasmid gene analysis indicated repression of virulence genes and upregulation of mobilization and toxin-antitoxin systems during the first 48 hours in water, suggesting a shift towards genetic adaptability. Prolonged exposure over weeks enhanced biofilm formation capacity and adherence to human epithelial cells, and ETEC isolates evolved towards increased colistin resistance. These findings stress the significant influence of freshwater on ETEC adaptive strategies, suggesting a role of waterborne transmission for human pathogens in development of persistence, biofilm formation capability and the emergence of antibiotic tolerance. Importance Environmental conditions play a vital role in shaping the behavior of pathogenic bacteria, influencing their survival, virulence, and resistance to treatments. This study reveals how freshwater environments act as crucial reservoirs for enterotoxigenic Escherichia coli (ETEC), one of the most common causes of diarrhea in children, by driving genetic adaptations that enhance biofilm formation and antibiotic resistance. These adaptive changes increase resilience and ability to cause disease, posing significant public health risks by facilitating persistent waterborne infections. Understanding the environmental factors that influence pathogenic bacterial behavior is essential for developing effective strategies to prevent waterborne outbreaks and manage antibiotic-resistant infections, ultimately protecting vulnerable populations from severe diarrheal diseases.