Stability and lytic activity assessment in milk of bacteriophages targeting Escherichia coli 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 characterizing isolated phages in milk is a crucial initial step. This study aimed to compare the stability and lytic activity of 10 phages targeting E. coli in milk and assessing their stability at different temperatures and pH.
Ten bacteriophages isolated from wastewater were spotted on 53 E. coli strains isolated from bovine mastitis to evaluate their host range. The stability of the phages was evaluated across different pH (2-12) and T°C (25-60°C). Stability in milk was assessed after 6h of incubation at 37°C. The optimal multiplicity of infection (MOI) was determined for each phages and used in subsequent lytic activity assays. These assays involved inoculating milk with E. coli and phages at the determined MOI and tracking bacterial titers at different timepoints in raw, heat-treated and UHT milk.
A narrow host spectrum was observed for the phages and stability was maintained at pH ranging from 4 to 10 and temperatures ranging from 25 to 45°C. At 60°C, only 5/10 phages persisted but with a significant degradation. Stability analysis in milk showed that all phages remained stable in raw and heat-treated milk. Lytic activity assays demonstrated a bacterial decrease with all phages, but for 5/10 phages, bacterial regrowth occurred after 5h of incubation.
In conclusion, milk components are not an obstacle for phage therapy to control bovine mastitis. However, bacterial regrowth suggests the presence of resistances that could be bypassed with the use of phage cocktails. DNA sequencing of the phages will be performed to ensure their safety and sequencing of the bacterial collection will allow us to determine their serotypes, which are linked to the phages host range.
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), the Care-FEPEX (ULiège, Liège, Belgium) and the “Association intercommunale pour le démergement et l’épuration des communes de la province de Liège” (AIDE, Liège, Belgium).