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 3.4x at the Seine River 2024 Olympic venue, and occasionally up to 100x 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.