Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / 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 this
disease. Prophylactic dosages of antibiotics used in mastitis control programmes could select
for strains with resistance to antibiotics. In addition, a strong drive towards reducing
antibiotic residues in animal food products has lead to research in finding alternative
antimicrobial agents.
Streptococcus macedonicus ST91KM, isolated from bulgarian goat yoghurt, produces
the bacteriocin macedocin ST91KM with a narrow spectrum of activity against Grampositive
bacteria. These include mastitis pathogens Streptococcus agalactiae, Streptococcus
dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis
as well as Lactobacillus sakei and Micrococcus varians. Macedocin ST91KM is, according
to tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. The activity of macedocin ST91KM
remained unchanged after 2 h of incubation at pH 2.0 to 10.0 and 100 min at 100 °C. The
peptide was inactivated after 20 min at 121 °C and when treated with pronase, pepsin and
trypsin. Treatment with α-amylase had no effect on activity, suggesting that the mode of
action does not depend on glycosylation. Precipitation with 60 % saturated ammonium
sulphate, followed by Sep-Pak C18 separation recovered 43 % of macedocin ST91KM.
Amplification of the genome of strain ST91KM with primers designed from the sequence of
the macedocin prescursor gene (mcdA) produced two fragments (approximately 375 and 220
bp) instead of one fragment of 150 bp recorded for macedocin produced by S. macedonicus
ACA-DC 198. Strain ACA-DC 198 was not available. However, the DNA fragment
amplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACADC
198, 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 insensitive
cells, e.g. Enterococcus faecalis BFE 1071 and FAIR E92, and Streptococcus caprinus
ATCC 700066. Optimal adsorption of macedocin ST91KM was recorded at 37 °C and 45 °C
and at pH of 8 - 10. Addition of solvents decreased adsorption by 50%, suggesting that the
receptors to which the bacteriocin binds have lipid moieties. The addition of MgCl2, KI and
Na2CO3 completely prevented adsorption of macedocin ST91KM to the target cells, possibly
due to competitive ion adsorption on the bacterial cell surface. The peptide has a
bacteriocidal 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 shown
deformation of the cell structure and developing of irregular surface areas.
Antimicrobial susceptibility patterns were evaluated against eighteen mastitis
pathogens. All isolates tested were resistant to methicillin and oxacillin, but had minimum
inhibitory concentrations (MICs) falling in the intermediate and susceptible range against
erythromycin. S. agalactiae and S. epidermidis had the highest sensitivity to macedocin
ST91KM. A teat seal preparation containing macedocin ST91KM effectively released
bacteriocin inhibiting the growth of the bacterial pathogen. Macedocin ST91KM could form
the basis for an alternative dry cow therapy to prevent mastitis infections in dairy cows, as it
is effective against pathogens that display resistance to conventional antibiotic therapy.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2323 |
| Date | 03 1900 |
| Creators | Pieterse, Renee |
| Contributors | Dicks, L. M. T., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | Stellenbosch University |
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