Smart design of a phage cocktail to mitigate Salmonella contamination risk during poultry processing

Citation

Carlos Martinez, Michael McClelland, Cezar Khursigara, and Hany Anany*. Smart design of a phage cocktail to mitigate Salmonella contamination risk during poultry processing. Program book of the Oxford Bacteriophage Virtual Conference – Phages 2021.

Plain language summary

Salmonella is one of the most prevalent food-borne bacterial pathogen around the world causing gastroenteritis in humans. Poultry meat and by-products are considered to be major vehicles of Salmonella pathogenesis to humans. The use of broad-host range bacterial viruses as biocontrol agents has recently emerged as a novel approach as it offers numerous advantages compared to traditional methods. Here, we isolated and assessed the host range of 33 Salmonella phages against 22 Salmonella Enterica serovars using a high throughput turbidimetric assay. Application of the phage cocktail in liquid culture in vitro at virus to bacteria ratio of 1000 completely inhibited the growth of the top ten Salmonella serovars circulating in Canada, while delaying the growth of another six serovars for more than five hours. Furthermore, Salmonella Enteritidis was used to study the emergence of bacteriophage insensitive mutants (BIMs) and the growth inhibition at 25°C and 15°C for 48 and 96 hours, respectively. The phage cocktail exhibited a low BIM frequency of 6.16x10-4±1.9x10-4. BIMs isolated after cocktail treatment only developed resistance to one phage, while remaining susceptible to the rest of the phages suggesting no cross-resistance to all phages in S. Enteritidis. Bacteria challenge experiment using difference cocktail concentrations showed complete growth inhibition at 25°C and 15°C for 48 and 96 hours, respectively. These results suggest that using phage candidates that bind to different receptors to formulate the phage cocktail is a promising strategy to ensure mitigating Salmonella contamination risk during poultry processing.

Abstract

Salmonella is one of the most prevalent food-borne bacterial pathogen around the world causing gastroenteritis in humans. Poultry meat and by-products are considered to be major vehicles of Salmonella pathogenesis to humans. The use of broad-host range bacteriophages as biocontrol agents has recently emerged as a novel approach as it offers numerous advantages compared to traditional methods. Here, we isolated and assessed the host range of 33 Salmonella phages against 22 Salmonella Enterica serovars using a high throughput turbidimetric assay. Two O-antigen dependent phages, one BtuB dependent phage, one rough-specific phage, and one OmpC dependent phage were selected for cocktail composition based on their host range, bacterial receptor recognition and ease of propagation. Application of the phage cocktail in liquid culture in vitro at MOI of 103 completely inhibited the growth of the top ten Salmonella serovars circulating in Canada, while delaying the growth of another six serovars for more than five hours. Furthermore, Salmonella Enteritidis was used to study the emergence of bacteriophage insensitive mutants (BIMs) and the growth inhibition at 25°C and 15°C for 48 and 96 hours, respectively. The phage cocktail exhibited a low BIM frequency of 6.16x10-4±1.9x10-4. BIMs isolated after cocktail treatment only developed resistance to one phage, while remaining susceptible to the rest of the phages suggesting no cross-resistance to all phages in S. Enteritidis. Bacteria challenge experiment using difference cocktail concentrations showed complete growth inhibition at 25°C and 15°C for 48 and 96 hours, respectively. These results suggest that using phage candidates that bind to different receptors to formulate the phage cocktail is a promising strategy to ensure mitigating Salmonella contamination risk during poultry processing.

Publication date

2021-09-13

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