Stability and lytic activity assessment of bacteriophages targeting Staphylococcus aureus causing bovine mastitis in milk

Bovine mastitis is a major cause of culling in dairy cattle and the antimicrobial treatment of the infection contributes to the emergence and the spread of antimicrobial resistances. Phage therapy could be a promising approach but the biological and physicochemical properties of milk can affect the phages properties. The objective of this study was to compare the stability and lytic activity of phages targeting Staphylococcus aureus in raw and pasteurized milk.A total of 28 bacteriophages previously isolated against S. aureus were spotted on 44 S. aureus strains isolated from bovine mastitis to evaluate the phage host range. The phage stability was assessed by titrating them in milk prior and after 6h incubation at 37°C. The lytic activity was assessed by inoculating milk with S. aureus and phages at a MOI of 1000. Bacterial and phage titration at different timepoints allowed to compare the lytic activity of the phages and their replication.A broad host spectrum was observed for 24/28 phages. Stability analysis showed that all phages were still active after 6h incubation in both raw and pasteurized milk with an average stability rate of 8%. Regarding the lytic activity test, 16/21 phages were able to replicate in milk but no decrease in bacterial count was measured, which could be linked to resistant bacteria. Heat-treated milk allowed significatively better stability and replication of the phages.These results show that heat-sensitive components in milk alter the phages properties. Further tests in milk fractions should be performed to assess their effect on phages attributes. The resistance of bacteria against phages should be investigated and the use of phage cocktails could be evaluated in the lytic activity assay. Acknowledgements: This program benefited from a financial support of Wallonia in the frame of a BioWin’s Health Cluster and Wagralim’s Agri-food Innovation Cluster program (Vetphage). The authors acknowledge the “Agence Régionale de Santé et d’Identification Animale” (ARSIA, Ciney, Belgium) and the Care-FEPEX (ULiège, Liège, Belgium).