Prevention and treatment of mastitis in dairy cows with bacteriocins produced by Enterococcus faecalis

[摘要] ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalisFAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivoand in vitro study. During initial tests peptide AS-48 showed no significant activitytowards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity wasobtained only after purification with Triton X-114 phase partitioning, followed by cationexchange chromatography. Titers for the purified peptide varied between 3200 and 12800AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae andStreptococcus dysgalactiae, but not against Escherichia coli.The size of peptide AS-48 was determined at 7150 Da, based on electronspray massspectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained byadding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the laggrowth phase. When the same concentration of peptide AS-48 was added to a culture ofS. aureus in mid-exponential growth, a slight decrease in viable cell numbers wasrecorded, which lasted for only 30 min. Cell growth commenced thereafter.In situ experiments in cows were done with purified peptide AS-48, encapsulated inliposomes. These in vivo studies were conducted by administering peptide AS-48 (6400AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreatedwith peptide AS-48, a reduction close to 90% in the viable cell numbers of S.aureus was recorded relative to the control quarters, which were not treated with thepeptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatmenttrial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viablecell numbers of S. aureus was recorded. In the same quarters, a reduction in SCCamounted to almost 80%.A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptideAS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which producesenterocins 1071A and 1071B. Southern blot hybridization experiments revealed thepresence of plasmid p92 in the recipient strain without the loss of plasmid pEF1071,which encodes enterocins 1071A and 1071B. All three antimicrobial peptides, i.e.enterocin 1071A, enterocin 1071B and peptide AS-48, were produced in transconjugantFA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greaterthan that recorded for strains FA2/Ent and FAIRE 92, respectively and included E.faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacilluscurvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri,Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcuscarnosus and S. aureus. These organisms are not inhibited by strain FA2/Ent. However,low levels of peptide AS-48 was produced by strain FA2/Ent/AS-48. Further research infermentation and gene expression will be needed before the transconjugant E. faecalisFA2/Ent/AS-48 may be used in the treatment of mastitis.

[发布机构] Stellenbosch University