Development of a bacteriophage cocktail with high specificity against high-risk avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is a widespread pathogen that poses a significant threat to the poultry industry globally. A recent typing scheme has proposed several APEC pathotypes, including non-APEC, High Risk non-APEC (HR non-APEC), APEC, and High Risk APEC (HR APEC), based on the presence of the ompT and hlyF genes, sequence types (ST) ST131, ST23, ST428, ST355, and the O78 antigen. In Brazilian broiler farms, a higher prevalence of lesions in carcasses has been linked to the presence of HR APEC pathotypes. Due to the growing concern over antimicrobial resistance, bacteriophage-based formulations are emerging as a promising alternative for controlling APEC outbreaks. In this study, we isolated and sequenced 142 strains from Brazilian commercial broiler farms, classifying them as APEC (70), HR APEC (12), HR non-APEC (3) and non-APEC (57). Notably, 38 % of the isolates were classified as multi-drug resistant (MDR), with serotypes H10 and ST155 being the most frequently identified. Additionally, we isolated, sequenced, and classified 66 bacteriophages that exhibited lytic activity against these bacterial strains in both qualitative and quantitative assays. Eight of the bacteriophages demonstrated complementary host ranges against the bacterial collection. Bacteriophage cocktails were assembled, tested in quantitative assays, and shown to be effective against APEC. A cocktail consisting of four bacteriophages (AC-01) displayed a broad lytic spectrum in vitro, inhibiting the growth of 56.3 % (n = 80/142) of the isolates with a mean inhibition of 32.9 %. Remarkably, the in vitro lytic activity of the cocktail was significantly more effective against HR APEC isolates (12/12, 65.9 % mean inhibition) and HR non-APEC isolates (3/3, 58.0 % mean inhibition). Our results emphasize the importance of genetically characterizing target bacteria when developing an effective and specific bacteriophage cocktail against APEC.