Characterization of a Multi-Receptor Phage Cocktail and Understanding the Cost of Phage Resistance Emergence in Salmonella enterica

Salmonella is one of the most prevalent food-borne bacterial pathogens around the world causing gastroenteritis in humans. Several salmonellosis outbreaks have been associated with the consumption of contaminated poultry products. This has been linked to contamination during processing and urges improvement in the prevention and control programs. The use of broad-host range lytic bacteriophages (phages) as biocontrol agents has recently emerged as a novel approach to enhance food safety. However, emergence of phage resistance remains one of the major challenges of this technology. Here, a multi-receptor phage cocktail was fully characterized. This cocktail targets four different receptors: O-antigen, BtuB, OmpC, and rough Salmonella strains. The host range, morphotype, receptor, infection kinetics, genome sequence, temperature and pH stability of all phages comprising the cocktail was characterized. The phage cocktail showed an outstanding host range, significantly inhibiting the growth of a total of 66 Salmonella strains (spanning 22 serovars). Moreover, the efficacy to control the growth of Salmonella in chicken skin was assessed. Cocktail treatment of 7 log10 PFU/mL showed a 3.5 log10 CFU/cm2 reduction after 48 hours with treatments of at 25 and 15°C, and 2.5 log10 CFU/cm2 at 4°C, compared to the control. Lastly, the emergence of bacteriophage resistance mutants (BIMs) against the phage cocktail was studied in Salmonella Enteritidis. The average BIM frequency against the cocktail was calculated to be 6.22 x 10-6, significantly lower compared to single phage (P=0.0095). A single BIM strain showing cross-resistance to all phages in the cocktail was isolated to study the impact of phage resistance development. A genome sequence analysis revealed mutations in multiple genes encoding for tRNA ligase (thrS), vitamin B12 receptor (btuB), and O-antigen biosynthesis (rfbK and rfbP). More experiments will be performed to understand the cost of phage resistance, including antibiotic susceptibility assays, phenotypic microarrays, and transcriptomics. Together, these experiments will shed light on the understanding of phage resistance mechanisms and will contribute to the development of better antimicrobials involving phage for food safety and therapy.