Substrate-independent luminescent phage-based biosensor to specifically detect enteric bacteria such as E. coli

Water quality is a major safety consideration in environments that are impacted by human activity. The key challenge of the COMBITOX project is to develop a unique instrument that can accommodate several biodetector systems (see the accompanying COMBITOX papers) able to detect different pollutants such as bacteria, toxins, and heavy metals. The output signal chosen by our consortium is based on luminescence detection. Our group recently developed phage-based biosensors using gfp as a reporter gene to detect enteric bacteria in complex environments such as sea water, and the main challenge we faced was to adapt our biodetector to a luminescent signal that could fit the COMBITOX project requirements. Another key point was to use a substrate-independent reporter system in order to avoid substrate addition in the detection prototype. This paper describes the development of a phage-based biodetector using a luminescent and substrate-independent output to detect some enteric bacteria, such as Escherichia coli , in water samples. We have successfully engineered various prototypes using the HK620 and HK97 bacteriophages that use different packaging systems, and both proved functional for the integration of the full luxCDABE operon controlled by two different bacterial promoters. We show that the luxCDABE operon controlled by the P rplU bacterial promoter is the most efficient in terms of signal emission. The emission of luminescence is specific and allows the detection of 10 4 bacteria per milliliter in 1.5 h post-infection with neither a concentration nor enrichment step.