Abstract
Swine wastewater, characterized by a high bacterial density and significant pressure on antibiotic selection, serves as a distinct reservoir for various bacteriophages. In this study, we report the simultaneous isolation and identification of two novel bacteriophages, vB_EcoM_BYEP01 and vB_SalS_SP14, from a single pig wastewater sample. These phages specifically target original host strain Escherichia coli BYEC01 and Salmonella enterica ATCC 14028, respectively. Both phages exhibit activity within the temperature range of 30-50 °C and remain stable over a pH range of 6-10. Transmission electron microscopy revealed that vB_EcoM_BYEP01 belongs to the Myoviridae family and vB_SalS_SP14 to Microviridae. Whole-genome sequencing and comparative analyses further confirmed these phylogenetic differences. Notably, despite these significant structural and genomic differences, both bacteriophages demonstrated efficient lytic life cycles. They lack genes associated with toxins or antibiotic resistance, while demonstrating biological safety. They share key functional characteristics, including rapid adsorption rates, short incubation periods, and large outbreak sizes, highlighting their efficacy as potent bacterial killers. Both bacteriophages can be combined with antibiotics to enhance antibacterial activity and demonstrate protective effects in food (such as milk and pig skin) and mouse infection models. The coexistence of these unrelated yet highly effective bacteriophages within a single microenvironment highlights the inherent functional redundancy and evolutionary adaptability of bacteriophages. This study provides valuable insights for developing targeted phage cocktail therapies against multiple bacterial pathogens.