Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

Abstract

Aim: To incorporate into the lambda phage genome, a luxI‐based acyl‐homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux‐based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. Methods and Results: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage‐based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL‐specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target‐specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 × 108 to 1 CFU ml−1 within 1·5–10·3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 × 108 to 130 CFU ml−1 was achieved within 2·6–22·4 h, respectively. Conclusions: The initial feasibility of binary phage‐based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. Significance and Impact of Study: With further modification, binary phage‐based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

Recent advances in recognition elements of food and environmental biosensors: A review

“…The target cells that contain these bioluminescence genes can then be detected with a luminometer. As bioluminescence genes, the complete lux operon can be used, but also a part of this lux operon, like luxI (Birmele et al, 2008;Ripp et al, 2006Ripp et al, , 2008 or luxAB can be used (Chen and Griffiths, 1996;Kodikara et al, 1991;Loessner et al, 1997). With bioluminescence phage biosensors, it is possible to determine rapidly low concentration of food pathogens compared to standard plating methods, taking approximately four days.…”

Bacteriophage-Based Pathogen Detection

“…Ripp et al [14] used lux in a uniquely different manner in phage reporter assays designed to detect E. coli . Conventional luxAB phage reporters require the addition of n -decanal before bioluminescence can be generated.…”

Pathogen detection using engineered bacteriophages

“…The luxI gene product synthesizes acyl homoserine lactone (AHL) autoinducer molecules that diffuse out of cells and interact with neighboring cells, where they bind with the LuxR protein to stimulate transcription of luxCDABE and luxI. The luxI gene was integrated into phages λ and PP01 for the general detection of E. coli and the specific detection of E. coli O157:H7, respectively [64,65]. When these luxI-carrying phages infect their E. coli hosts, the luxI gene is inserted and expressed and the E. coli cells begin emitting AHL molecules.…”