Protective effects of metabolites from lactic acid bacteria against infections of mastitis pathogen in bovine cells

Abstract

Bovine mastitis poses significant economic challenges for dairy farms globally. Metabolites from lactic acid bacteria (LAB) offer promising alternative substances for preventing bovine mastitis. This study demonstrated the inhibitory activity of metabolite production from seven LAB isolates in the supernatant medium. Four isolates, namely Lactiplantibacillus plantarum TISTR 2070, Lacticaseibacillus casei TISTR 1340, Enterococcus faecalis TCAN02, and Lactiplantibacillus plantarum AD73, exhibited antibacterial activity against bacterial pathogens causing mastitis, including Staphylococcus haemolyticus, Staphylococcus capitis subsp. ureolyticus, Streptococcus agalactiae O4, Streptococcus dysgalactiae, and Streptococcus uberis. The minimum inhibitory concentration (MIC) values ranged from 6.25 to 25 mg/mL, with minimum bactericidal concentration (MBC) values of 12.5 to 25 mg/mL. Furthermore, analysis of the physical and chemical properties revealed active metabolites in LAB supernatants, particularly lactic acid that was detected in all samples. Additionally, this study presents novel findings on infection of CPAE bovine endothelial cells in vitro by mastitis bacterial pathogens and demonstrating cytopathic effects such as vacuolation, magalocytosis, and cytotoxicity after bacterial infection on the bovine endothelial cells. Moreover, metabolites from LAB supernatant exhibited a protective effect against bacterial colonization and infection of bacterial pathogens causing mastitis on CPAE bovine endothelial cells and leading to increase cell viability. The results of this study suggest that metabolites from LAB samples exert potential candidates as alternative therapeutic agents for bovine mastitis.

Keywords: Antibacterial activity; Bovine mastitis; Colonization; Infection; Lactic acid bacteria.

Fig. 1

The growth curves of the seven LAB strains; E. faecalis TCAN02, E. faecalis TCAN03, E. faecalis TCAN04, E. faecalis TCAN13, L. lactis TCAN01, L. lactis TCAN16 and L. plantarum AD73.

Fig. 2

HPLC chromatogram of lactic acid (A) detected in the supernatants of L. plantarum TISTR 2070 (B), L. casei TISTR 1340 (C), E. faecalis TCAN02 (D), L. plantarum AD73 (E).

Fig. 3

Giemsa staining of mastitis bacterial pathogens on CPAE bovine endothelial cells after being treated with the LAB supernatants of L. plantarum TISTR 2070, L. casei TISTR 1340, E. faecalis TCAN02 and L. plantarum AD73, and comparing with the untreated control. Adhesion of bacteria on CPAE cells observed under oil immersion microscope (100X) after staining with Giemsa stain.

Fig. 4

The morphological changes of CPAE bovine endothelial cells after infection with mastitis bacterial pathogens at a MOI of 50 and 100 bacteria per cells for 4 h compared to uninfected control cells.

Fig. 5

Efficacy of LAB supernatants for inhibition of infection with mastitis bacterial pathogens at a MOI of 100 into CPAE bovine endothelial cells for 4 h when compared to infected endothelial cell control.

Fig. 6

Effect of LAB supernatants on the viability of CPAE bovine endothelial cells after infection with mastitis bacterial pathogens at a MOI of 100 into CPAE bovine endothelial cells for 4 h and compared to infected endothelial cells control. Percentage viability of cells is expressed as a mean ± standard deviation. Significance is determined at ∗p < 0.05 when compared to bacteria-infected cells.