Isolation of bacteriophages specific to bovine mastitis-causing bacteria and characterization of their lytic activity in pasteurized milk.

Abstract

Bovine mastitis is one of the most serious issues in dairy production. It is caused by contagious and coliform pathogens such as Staphylococcus spp., Escherichia coli, and Klebsiella pneumoniae. In addition, the emergence of drug-resistant bacteria raises urgent concerns in the field of drug treatment, thus requiring the exploration of alternative treatments. Bacteriophage therapy has been shown to be a promising alternative approach for the control of antibiotic-resistant pathogens. In this study, we aimed to isolate phages specific to contagious mastitis and coliform mastitis, characterize the isolated phages, and examine their ability to lyse bacteria in pasteurized milk samples. The Staphylococcus phage vB_Sau-RP15 isolated from raw milk in our previous study was used in this study. Other three phages, vB_Eco-RN12i1, vB_Kpn-RN14i1, and vB_Ssc-RN20i3, were isolated from wastewater using E. coli 5823, K. pneumoniae 194, and Staphylococcus sciuri MM01 as hosts, respectively. The host range and efficiency of plating (EOP) were determined following phage isolation. Moreover, the lysis activities of these phages against their hosts were investigated in pasteurized milk using a multiplicity of infections (MOIs) of 10 and 100 at 37°C. Phages were applied using individual and combination phages. According to the EOP results, all phages showed high specificity to their respective hosts. They are tailed phages with distinct morphologies. Individual phage treatments in spiked pasteurized milk with their respective bacterial hosts significantly reduced the bacterial counts in both MOI conditions during the first 2 h of the treatment (approximately 1-8 log reduction compared to the control). Although these phages specifically infected only their hosts, the phage cocktail resulted in a better result compared to the use of individual phage. However, bacterial regrowth was observed in all experiments, which may be related to the development of phage-insensitive mutants. Our findings suggest that the application of phages could be used to treat bovine mastitis. Phage cocktail is suitable to promote the efficacy of phage treatment in pasteurized milk. However, when considering the use of phages in dairy cows, certain phage properties in raw milk and in vivo and ex vivo should be highlighted to ensure their effectiveness as biocontrol agents for bovine mastitis treatment.