CONSTRUCTION OF LUCIFERASE REPORTER BACTERIOPHAGE-A511--LUXAB FOR RAPID AND SENSITIVE DETECTION OF VIABLE LISTERIA CELLS

Specific transfer and expression of bacterial luciferase genes via bac teriophages provides an efficient way to detect and assay viable host cells. Listeria bacteriophage A511 is a genus-specific, virulent myovi rus which infects 95% of Listeria monocytogenes serovar 1/2 and 4 cell s. We constructed recombinant derivative A511:: luxAB, which carries t he gene for a fused Vibrio harveyi LuxAB protein inserted immediately downstream of the major capsid protein gene (cps). Efficient transcrip tion is initiated by the powerful cps promoter at 15 to 20 min postinf ection. Site-specific introduction of the luciferase gene into the pha ge genome was achieved by homologous recombination in infected cells b etween a plasmid carrying A511 DNA flanking luxAB and phage DNA. Recom binants occurred in the lysate at a frequency of 5 x 10(-4) and were r eadily identified by the bioluminescent phenotype conferred on newly i nfected host cells. A511::luxAB can be used to directly detect Listeri a cells. Following infection and a 2-h incubation period, numbers as l ow as 5 x 10(2) to 10(3) cells per mi were detected by using a single- tube luminometer. Extreme sensitivity was achieved by including an enr ichment step prior to the lux phage assay; under these conditions less than 1 cell of L. monocytogenes Scott A per g of artificially contami nated salad was clearly identified. The assay is simple, rapid, inexpe nsive, and easy to perform. Our findings indicate that AS11::luxAB is useful for routine screening of foods and environmental samples for Li steria cells.