Addressing underestimation of waterborne disease risks due to fecal indicator bacteria bound in aggregates

AIMS: This study aims to identify and address significant limitations in current culture-based regulatory methods used for monitoring microbiological water quality. Specifically, these methods' inability to distinguish between planktonic forms and aggregates containing higher bacterial loads and associated pathogens may lead to a severe underestimation of exposure risks, with critical public health implications. METHODS AND RESULTS: We employed a novel methodology combining size fractionation with ALERT (Automatic Lab-in-a-vial E.coli Remote Tracking), an automated rapid method for comprehensive quantification of culturable fecal indicator bacteria (FIB). Our findings reveal a substantial and widespread presence of aggregate-bound indicator bacteria across various water matrices and geographical locations. Comprehensive bacterial counts consistently exceeded those obtained by traditional methods by significant multiples, such as an average of 3.4× at the Seine River 2024 Olympic venue, and occasionally up to 100× in irrigation canals and wastewater plant effluent. These results, supported by microscopic and molecular analyses, underscore a systematic bias in global water safety regulatory frameworks. CONCLUSIONS: Our research demonstrates the inadequacy of traditional culture-based techniques in assessing microbiological risks posed by aggregate-bound FIB and associated pathogens, particularly in water matrices affected by FIB-rich fecal particles from recent sewer overflows or sediment, which can carry higher infectious risks. Incorporating comprehensive FIB analysis techniques, including molecular methods and rapid culture-based approaches as shown in this study, offers a promising and effective solution to these risk assessment limitations.