Avian pathogenic Escherichia coli-targeting phages for biofilm biocontrol in the poultry industry

Avian pathogenic Escherichia coli (APEC) is a principal etiologic agent of avian colibacillosis, responsible for significant economic losses in the poultry industry due to high mortality and disease treatment with antibiotics. APEC and its ability to form biofilms on food and processing surfaces contributes to its persistence within farms. Bacteriophages are promising antibacterial agents for combating APEC. This study focused on characterization of the newly isolated phages UPWr_E1, UPWr_E2, and UPWr_E4 as well as the UPWr_E124 phage cocktail containing these three phages. Methods included efficiency of plating assay, transmission electron microscopy, and characterization of their resistance to different pH values and temperatures. Moreover, phage genomes were sequenced, annotated and analyzed, and were compared with previously sequenced E. coli phages. All three phages are virulent and devoid of undesirable genes for therapy. Phage UPWr_E1 belongs to the genus Krischvirus within the order Straboviridae and both UPWr_E2 and UPWr_E4 belong to the genus Tequatrovirus within the subfamily Tevenvirinae, sharing over 95 % nucleotide identity between them. For their use on poultry farms, UPWr_E phages and the UPWr_E124 phage cocktail were tested for their anti-biofilm activity on two E. coli strains - 158B (APEC) and the strong biofilm producer NCTC 17848 - on two abiotic surfaces: a 96-well microplate, a stainless steel surface, and one biotic surface, represented by lettuce leaves. The reduction of biofilm formed by both strains in the 96-well microplate, on the stainless steel and lettuce leaf surface for bacteriophage treatment was very efficient, reducing biofilms by ranges of 50.2-83.6, 58.2-88.4 and 53-99.4 %, respectively. Therefore, we conclude that UPWr_E phages and the UPWr_E124 phage cocktail are promising candidates for APEC biocontrol.