Control of bacterial pathogens associated with mastitis in dairy cows with natural antimicrobial peptides produced by lactic acid bacteria

[摘要] Mastitis is considered to be the most costly disease affecting the dairy industry.Management strategies involve the extensive use of antibiotics to treat and prevent thisdisease. Prophylactic dosages of antibiotics used in mastitis control programmes could selectfor strains with resistance to antibiotics. In addition, a strong drive towards reducingantibiotic residues in animal food products has lead to research in finding alternativeantimicrobial agents.Streptococcus macedonicus ST91KM, isolated from bulgarian goat yoghurt, producesthe bacteriocin macedocin ST91KM with a narrow spectrum of activity against Grampositivebacteria. These include mastitis pathogens Streptococcus agalactiae, Streptococcusdysgalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidisas well as Lactobacillus sakei and Micrococcus varians. Macedocin ST91KM is, accordingto tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. The activity of macedocin ST91KMremained unchanged after 2 h of incubation at pH 2.0 to 10.0 and 100 min at 100 °C. Thepeptide was inactivated after 20 min at 121 °C and when treated with pronase, pepsin andtrypsin. Treatment with α-amylase had no effect on activity, suggesting that the mode ofaction does not depend on glycosylation. Precipitation with 60 % saturated ammoniumsulphate, followed by Sep-Pak C18 separation recovered 43 % of macedocin ST91KM.Amplification of the genome of strain ST91KM with primers designed from the sequence ofthe macedocin prescursor gene (mcdA) produced two fragments (approximately 375 and 220bp) instead of one fragment of 150 bp recorded for macedocin produced by S. macedonicusACA-DC 198. Strain ACA-DC 198 was not available. However, the DNA fragmentamplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACADC198, revealed 99 % homology to the mcdA of S. macedonicus ACA-DC 198 (accession number DQ835394). Macedocin ST91KM may thus be a related bacteriocin described for S.macedonicus.The peptide adsorbed equally well (66 %) to L. sakei LMG13558 and insensitivecells, e.g. Enterococcus faecalis BFE 1071 and FAIR E92, and Streptococcus caprinusATCC 700066. Optimal adsorption of macedocin ST91KM was recorded at 37 °C and 45 °Cand at pH of 8 - 10. Addition of solvents decreased adsorption by 50%, suggesting that thereceptors to which the bacteriocin binds have lipid moieties. The addition of MgCl2, KI andNa2CO3 completely prevented adsorption of macedocin ST91KM to the target cells, possiblydue to competitive ion adsorption on the bacterial cell surface. The peptide has abacteriocidal mode of action, resulting in lysis and the release of DNA and β-galactosidase.Atomic force microscopy of sensitive cells treated with macedocin ST91KM have showndeformation of the cell structure and developing of irregular surface areas.Antimicrobial susceptibility patterns were evaluated against eighteen mastitispathogens. All isolates tested were resistant to methicillin and oxacillin, but had minimuminhibitory concentrations (MICs) falling in the intermediate and susceptible range againsterythromycin. S. agalactiae and S. epidermidis had the highest sensitivity to macedocinST91KM. A teat seal preparation containing macedocin ST91KM effectively releasedbacteriocin inhibiting the growth of the bacterial pathogen. Macedocin ST91KM could formthe basis for an alternative dry cow therapy to prevent mastitis infections in dairy cows, as itis effective against pathogens that display resistance to conventional antibiotic therapy.

[发布机构] Stellenbosch University