Rapid, sensitive, and low-cost detection of Escherichia coli bacteria in contaminated water samples using a phage-based assay

Inadequate drinking water quality is among the major causes of preventable mortality, predominantly in young children. Identifying contaminated water sources remains a significant challenge, especially where resources are limited. The current methods for measuring Escherichia coli (E. coli), the WHO preferred indicator for measuring fecal contamination of water, involve overnight incubation and require specialized training. In 2016, UNICEF released a Target Product Profile (TPP) to incentivize product innovations to detect low levels of viable E. coli in water samples in the field in less than 6 h. Driven by this challenge, we developed a phage-based assay to detect and semi-quantify E. coli. We formulated a phage cocktail containing a total of 8 phages selected against an extensive bacterial strain library and recombined with the sensitive NanoLuc luciferase reporter. The assay was optimized to be processed in a microfluidic chip designed in-house and was tested against locally sourced sewage samples and on drinking water sources in Nairobi, Kenya. With this assay, combined with the microfluidic chip platform, we propose a complete automated solution to detect and semi-quantify E. coli at less than 10 MPN/100 mL in 5.5 h by minimally trained personnel.