Bacteriophage-Based Bioluminescent Bioreporter for the Detection of Escherichia coli O157:H7

Abstract

The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli O157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli O157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP01-luxI to detect several strains of E. coli O157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PP01-luxI and E. coli O157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within 1 h when exposed to 104 CFU/ml of E. coli O157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 104 CFU/ml of E. coli O157:H7 in 2 h after a 6-h preincubation.