Characterization of Listeria phages for application to biosanitation

Listeria monocytogenes poses a public health risk due to its ability to cause severe illness and to persist in conditions relied upon in food processing environments (FPEs) to inhibit the growth of pathogenic bacteria. When present in FPEs, L. monocytogenes typically resides in biofilms. Bacteriophages are a promising approach for control of pathogenic foodborne bacteria. Here we present phenotypic characterization of a group of related Listeria phages, members of the genus Pecentumvirus, with regard to their host range, efficiency of degrading mono-species biofilms of L. monocytogenes and killing efficiency in liquid culture. The nine Listeria phages (eight newly isolated plus Listex P100™) efficiently infected (efficiency of plating ≥ 0.1) from 16/25 to 21/25 of the tested Lsiteria strains. They completely inhibited growth in liquid culture from 9/21 to 16/21 Listeria strains tested over a 40h period when initial concentrations of 107 CFU/ml and 107 PFU/ml were used. Phage CKA15 was selected to further assess its suitability for application to biosanitation. Using confocal laser scanning microscopy with a live-dead stain, the phage was shown to significantly reduce the viability of five day old L. monocytogenes biofilms at an initial titre of log 5 PFU/ml and a contact time of 16 hours. The phage rapidly adsorbs to bacteria in planktonic as well as sessile physiological states, and to planktonic bacteria grown at 15 ̊C in the presence of milk macromolecules. The respective adsorption constants for phage CKA15 to L. monocytogenes ATCC19111 under the above conditions were 1.64±0.11 × 10-9, 1.83±0.28 × 10-9, 1.76±0.36 × 10-9 PFU/ml/min. These results demonstrate that phage CKA15 has potential utility as a biosanitation agent in dairy facilities, where the average temperature in drains is 15 ̊C, where milk residue is expected to be present, and where Listeria spp. are expected to be in sessile physiological states.