Near on-line detection of enteric bacteria usingluxrecombinant bacteriophage

Abstract

The potential for a revolution in microbiol testing can be perceived with the near on‐line detection of indicator microorganisms. By definition, these are microorganisms present in significant numbers within a food which, while not pathogenic, can be related through increasing count to the increased probability of pathogen contamination. We have used recombinant lux+ bacteriophage to detect enteric indicator bacteria without recovery or enrichment in 50 min, provided that they are present at levels greater than 104 g−1 or cm−2. After a 4‐h enrichment, samples having enteric counts of 10 g−1 or cm−2 can be distinguished from background.

Application of bacteriophages for detection of foodborne pathogens

“…4 The first luciferase reporter phage (LRP) was constructed by Ulitzur and Kuhn in 1987, who inserted the lux operon from Vibrio fischeri (i.e., bacterial luciferase genes) into a lambda-based cloning vector and demonstrated detection of as few as 10 E. coli cells in milk within 1 h. 55 Nowadays, LRPs are available for a multitude of foodborne pathogens, including E. coli, Salmonella, Listeria, Staphylococcus aureus, and Mycobacterium avium. [55][56][57]59,63,66,[68][69][70][71][72][73][74] One of the most prominent examples with relevance for food safety is the A511::luxAB, which is based on the broad host range A511 phage infecting the foodborne pathogen Listeria monocytogenes. 56 This reporter phage features a luxAB fusion gene from Vibrio harveyi immediately downstream of the gene coding for the major capsid protein of A511, resulting in emission of light during the expression of late phage genes inside infected host cells, and allowing detection of very low numbers of Listeria cells in food samples within less than 24 h. 72 This significantly reduced time requirement compared with the standard plating method (4 d) constitutes the major advantage of this assay, while at the same time the high specificity of the phage ensures reliable results even in samples with high levels of background flora.…”

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.…”

Pathogen detection using engineered bacteriophages

“…Using beef samples artificially contaminated with E. coli O157:H7, Willford and Goodridge [60] demonstrated detection limits as low as 100 CFU per 100 cm -2 . The bioluminescent luxAB reporter genes were incorporated into phage λ Charon 30 by Ulitzur and Kuhn [61] for the detection of E. coli in raw milk samples, with a detection limit of ten cells per milliliter in 1.5 h. Kodikara et al [62] used a cocktail of three luxAB phages, including λ Charon 30, to detect enteric bacteria on swine carcasses and on work surfaces along a sausage manufacturing line at 10 4 g -1 or 10 4 cm -2 within 1 h and at 10 g -1 or 10 cm -2 within 5 h. In a different tangent on bioluminescent reporter phages, Ripp et al [63] designed an assay for E. coli using the quorumsensing signaling genes luxI and luxR in combination with a lux-based bacterial bioreporter. 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.…”