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

Bovine mastitis is a major production disease in dairy cattle and complementary treatments to antimicrobials are urgently needed. Intramammary phage therapy is a promising approach but milk components 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 in milk was assessed after 6h incubation at 37°C. The lytic activity was established by inoculating milk with S. aureus and phages at a MOI of 1000 and titrating the phages at different timepoints to highlight their replication. The optimal MOI was assessed for each phages and used for further lytic tests where bacteria were titrated. A broad host spectrum was observed for 24 out of 28 phages. Stability analysis showed that all phages presented a decrease in titer which was significantly higher in raw milk. However, phages were still active in raw and pasteurized milk with stability rates of respectively 7% and 10%. Regarding the lytic activity, half of the phages were able to replicate in milk with a lower rate in raw milk. When using optimal MOI, bacterial reduction was observed. In conclusion, heat-sensitive components in milk alter the phages properties. Further tests in milk components should be performed to assess their effect on phages. The resistance of bacteria against phages should be investigated and the use of phage cocktails should be evaluated to circumvent the phage resistant bacteria emergence.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)