Addressing Underestimation of Waterborne Disease Risks Due to Fecal Indicator Bacteria Bound in Aggregates

Microbiological water quality monitoring is critical for managing waterborne disease risk; currently, regulations rely on quantifying culturable fecal indicator bacteria using traditional culture-based methods. These approaches cannot distinguish between planktonic forms and aggregates harboring higher loads of bacteria and associated pathogens, potentially underestimating exposure risks. By using size fractionation and ALERT, an automated rapid method for comprehensive quantification of culturable bacteria, we reveal widespread and substantial presence of aggregate-bound indicator bacteria across a variety of water matrices and geographies. We observe comprehensive bacteria counts exceeding traditional method counts by significant multiples (e.g., 3.4x on average at the Seine River 2024 Olympic venue, occasionally 100x in irrigation canals and wastewater plant effluent). The results, corroborated by microscopic and molecular analyses, highlight a major systematic bias in global water safety regulatory frameworks. Automated comprehensive culture-based rapid quantification methods could provide higher-accuracy risk assessments, enabling effective monitoring, including in remote and resource-limited settings.