Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalis
FAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivo
and in vitro study. During initial tests peptide AS-48 showed no significant activity
towards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity was
obtained only after purification with Triton X-114 phase partitioning, followed by cation
exchange chromatography. Titers for the purified peptide varied between 3200 and 12800
AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae and
Streptococcus dysgalactiae, but not against Escherichia coli.
The size of peptide AS-48 was determined at 7150 Da, based on electronspray mass
spectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained by
adding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the lag
growth phase. When the same concentration of peptide AS-48 was added to a culture of
S. aureus in mid-exponential growth, a slight decrease in viable cell numbers was
recorded, which lasted for only 30 min. Cell growth commenced thereafter.
In situ experiments in cows were done with purified peptide AS-48, encapsulated in
liposomes. These in vivo studies were conducted by administering peptide AS-48 (6400
AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreated
with 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 the
peptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatment
trial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viable
cell numbers of S. aureus was recorded. In the same quarters, a reduction in SCC
amounted to almost 80%.
A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptide
AS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which produces
enterocins 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 transconjugant
FA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greater
than that recorded for strains FA2/Ent and FAIRE 92, respectively and included E.
faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus
curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri,
Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus
carnosus 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 in
fermentation and gene expression will be needed before the transconjugant E. faecalis
FA2/Ent/AS-48 may be used in the treatment of mastitis. / AFRIKAANSE OPSOMMING: Die effek van die bakteriosien-agtige, peptied AS-48, geproduseer deur Enterococcus
faecalis FAIRE 92, is gedurende ‘n in vivo en in vitro studie teen ‘n mastitiese
Staphylococcus aureus-isolaat getoets. Aanvanklike toetse met peptied AS-48, selfs
tienvoudig gekonsentreerde selvrye supernatant, het geen beduidende aktiwiteit teen S.
aureus getoon nie. Aktiwiteit is eers verkry na suiwering met Triton X-114 fase-skeiding
gevolg deur katioon uitruilingschromatografie. Titers vir die gesuiwerde peptied het
tussen 3200 en 12800 AE/ml gewissel. Die gesuiwerde peptied het ook aktiwiteit teen
Streptococcus agalactiae en Streptococcus dysgalctiae getoon, maar nie teen Escherichia
coli nie.
Peptied AS-48 het ‘n molekulêre massa van 7150 Da, soos bepaal met elektronsproeimassa
spektrometrie en SDS-PAGE. Totale inhibisie van selgroei is verkry deur 1 ml
gesuiwerde peptied AS-48 (3200 AE/ml) by ‘n 100 ml kultuur van S. aureus in die
sloerfase te voeg. Dieselfe konsentrasie peptied AS-48, toegevoeg tydens die mideksponensiële
groeifase, het egter slegs ‘n klein vermindering in die aantal lewende selle
teweeg gebring en het ook vir slegs ‘n 30 min geduur. Selgroei het hierna weer normaal
voort gegaan.
In situ eksperimente op koeie is uitgevoer met gesuiwerde peptied AS-48, geenkapsuleerd
in liposome. Hierdie In vivo studies is onderneem deur peptied AS-48
(6400 AE/ml) in verskillende kwarte van die uier, kunsmatig of reeds geïnfekteerd met S.
aureus, toe te dien. In ‘n voorkomings-eksperiment waar kwarte vooraf met peptied AS-
48 behandel is, is ‘n verlaging van byna 90% in die lewende seltelling van S. aureus
relatief tot die kontrole kwarte, sonder behandeling met peptied AS-48, verkry. ‘n 50%
verlaging in die somatiese seltelling (SST) is verkry. In die behandelings-eksperiment,
waar geïnfekteerde kwarte met peptied AS-48 behandel is, is ‘n verlaging van byna 90%
in lewende S. aureus selle gevind. In dieselfde kwarte is ‘n verlaging van byna 80% in
die SST genoteer.‘n Rekombinante ras is gekonstrueer deur plasmied 92 (p92), wat kodeer vir peptied AS-
48, vanaf Enterococcus faecalis FAIRE 92 na E. faecalis FA2/Ent, wat enterosien 1071A
en 1071B produseer, te konjugeer. Southern-klad hibridisasie het die teenwoordigheid
van plasmied p92 in die ontvanger ras, sonder die verlies van plasmied pEF1071 wat
enterosien 1071A en 1071B kodeer, getoon. Al drie antimikrobiese peptiede, nl.
enterosien 1071A, enterosien 1071B en peptied AS-48, is deur die transkonjugant
FA2/Ent/AS-48 geproduseer. Die spektum van antimikrobiese aktiwiteit van die
transkonjugant vand die transkonjugant is breër as dié van rasse FA2/Ent en FAIRE 92,
onderskeidelik en het ook E. faecalis, Bacillus cereus, Lactobacillus acidophilus,
Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus
plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris,
Leuconostoc pentosaceus, Staphylococcus carnosus en S. aureus ingesluit. Hierdie
organismes word nie deur ras FA2/Ent geïnhibeer nie. Lae vlakke van peptied AS-48 is
egter deur ras FA2/Ent/AS-48 geproduseer. Verdere navorsing in fermentasie en geenuitdrukking
is nodig voordat E. faecalis FA2/Ent/AS-48 in die behandeling van mastitis
gebruik kan word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16296 |
| Date | 04 1900 |
| Creators | Davidse, Elton (Elton Kurt) |
| Contributors | Dicks, L.M.T., University of Stellenbosch. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | ix, 122 leaves |
| Rights | University of Stellenbosch |
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