Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Antimicrobial peptides continue to be one of the most important classes of food additives. The food industry is especially interested in the application of naturally occuring and biologically derived preservatives. Among the metabolites of industrial importance produced by propionibacteria are peptides called bacteriocins. Bacteriocins are ribosomally synthesized peptides with antagonistic activity against closely related microorganisms. Many microorganisms associated with food produce bacteriocins, which have stimulated interest in the use of these peptides as natural food preservatives. Numerous bacteriocins are produced by lactic acid bacteria, but only a few have been reported for propionibacteria. Since propionic acid bacteria have GRAS (generally regarded as safe) status, their metabolic compounds should be safe for human consumption. Propionibacterium thoenii 447, isolated from Emmentaler cheese, produces a bacteriocin-like peptide, named thoeniicin 447, with a narrow spectrum of activity. The peptide displays a bactericidal mode of action against Lactobacillus delbrueckii subsp. bulgaricus and a bacteriostatic action against Propionibacterium acnes. Optimal bacteriocin production was detected during the early stationary growth phase. The peptide is resistant to heat treatments of 60°C and 80°C for 15 and 30 min and to 100°C for 15 min, but loses 80% of its activity after autoclaving (10 min at 121°C). Thoeniicin 447 remains active after incubation in buffers with pH values ranging from 1-10. The peptide is inactivated by pepsin, pronase, a-chymotrypsin, trypsin and Proteinase K. Thoeniicin 447 was partially purified by ammonium sulfate precipitation, followed by SP-Sepharose cation exchange chromatography. The estimated size of thoeniicin 447, according to tricine-SDSPAGE, is approximately 6 kDa. Based on DNA sequencing, the mature peptide is 7130 Da in size and homologous to propionicin Tl produced by P. thoenii strain 419. Thoeniicin 447 is a relatively small, cationic and heat-stable peptide and can therefor be classified as a member of class II bacteriocins. These features are very similar to those of bacteriocins produced by lactic acid bacteria. However, no unique classification system has been proposed for bacteriocins of propionibacteria. As a member of the genus Propionibacterium, P. thoenii 447 is generally regarded as safe. This, together with the narrow spectrum of activity, particularly the action against P. acnes, heat tolerance of thoeniicin 447 and its activity over a wide pH range renders the peptide suitable for possible pharmaceutical applications. / AFRIKAANSE OPSOMMING: Antimikrobiese middels sal deurgaans beskou word as een van die belangrikste klasse van voedsel bymiddels. Die voedselindustrie is veral geïnteresseerd in die toepassing van preserveermiddels van 'n meer natuurlike en biologiese oorsprong. Onder die metaboliese produkte van industriële belang wat deur propionibakterieë geproduseer word is antimikrobiese peptiede (bakteriosiene). Bakteriosiene is ribosomaal-gesintetiseerde peptiede met 'n antagonistiese aktiwiteit teenoor naverwante bakterieë. Verskeie bakteriosiene word deur melksuurbakterieë geproduseer, terwyl slegs enkele vir propionibakterieë beskryf is. Baie van hierdie propionibakterieë word in die algemeen as veilig beskou en het GRAS status. Die metaboliete wat hulle produseer behoort dus veilig vir menslike gebruik te wees. Propionibacterium thoenii 447 is uit Emmentaler kaas geisoleer en produseer 'n bakteriosien-agtige peptied, naamlik thoeniicin 447 met 'n beperkte spektrum van aktiwiteit. Die peptied het 'n bakteriosidiese werking teenoor Lactobacillus delbrueckii subsp. bulgaricus en 'n bakteriostatiese werking teenoor Propionibacterium acnes. Optimum bakteriosien produksie is verkry tydens die vroeë stationêre groeifase. Die peptied is bestand teen hittebehandelings van 60°C en 80°C vir 15 en 30 min, asook 100°C vir 15 min, maar verloor 80% van sy aktiwiteit na outoklavering (lOmin by 121°C). Die peptied blyaktief na inkubasie in buffers van pH 1-10. Die peptied word deur pepsien, pronase, uchymotripsien, tripsien en Proteinase K geïnaktiveer. Thoeniicin 447 is met behulp van ammoniumsulfaat-presipitasie, gevolg deur SPSepharose katioon-uitruilchromatografie gedeeltelik gesuiwer. Skeiding op "n trisien-SDS poliakrielarnied-jel het 'n aktiewe band van ongeveer 6 kDa getoon. Volgens die DNA volgorde bepaling is thoeniicin 447, 7130 Da in grootte en homoloog aan Propionicin Tl, geisoleer vanaf P. thoenii stam 419. Thoeniicin 447 is 'n relatiewe klein, kationiese en hitte-bestande peptied en kan op grond hiervan as 'n lid van die klas II bakteriosiene geklassifiseer word. Hierdie eienskappe is soortgelyk aan die eienskappe van bakteriosiene geproduseer deur melksuurbakterieë. Tot op hede is geen klassifikasiesisteem vir die bakteriosiene van propionibakterieë voorgestel nie. As 'n lid van die genus Propionibacterium, word P. thoenii 447 in die algemeen as veilig beskou. Dit, tesame met die nou spektrum van aktiwiteit, veral teenoor P. acnes, die hittetoleransie van thoeniicin 447, asook die aktiwiteit oor 'n wye pH-grens, maak die peptied geskik vir moontlike farmaseutiese toepassings.

Graduation date: 1994

No / Propionibacterium acnes is the target of antimicrobial treatments for acne vulgaris. Acquired resistance to erythromycin, clindamycin and tetracyclines has been reported in strains from diverse geographical loci, but the molecular basis of resistance, via mutations in genes encoding 23S and 16S rRNA, respectively, has so far only been elucidated for isolates from the U.K. The study set out to determine whether similar or different resistance mechanisms occur in resistant P. acnes isolates from outside the U.K. The phenotypes and genotypes of 73 antibiotic-resistant strains of P. acnes obtained from the skin of acne patients in the U.K., U.S.A., France, Germany, Australia and Japan were compared. Antibiotic susceptibilities were determined by minimum inhibitory concentration (MIC) measurements, and polymerase chain reaction and DNA sequencing were used to identify mutations in genes encoding rRNA. Most erythromycin-resistant isolates (MIC90¿ 512 ¿g mL¿1) were cross-resistant to clindamycin but at a much lower level (MIC90¿ 64 ¿g mL¿1). As in the U.K., resistance to erythromycin was associated with point mutations in 23S rRNA in 49 of 58 strains. An A¿G transition at Escherichia coli equivalent base 2058 was present in 24 strains. This gave a unique cross-resistance phenotype against a panel of macrolide, lincosamide and type B streptogramin antibiotics. Two further point mutations (at E. coli equivalent bases 2057 and 2059) were identified (in three and 22 isolates, respectively) and these were also associated with specific cross-resistance patterns originally identified in isolates from the U.K. However, nine of 10 erythromycin resistant-strains from Germany did not exhibit any of the three base mutations identified and, in six cases, cross-resistance patterns were atypical. Consistent with previous U.K. data, 34 of 38 tetracycline-resistant strains carried a base mutation at E. coli 16S rRNA equivalent base 1058. Tetracycline-resistant isolates displayed varying degrees of cross-resistance to doxycycline and minocycline, but isolates from the U.S.A. had higher MICs for minocycline (4¿16 ¿g mL¿1) than isolates from other countries and, in particular, Australia. All the P. acnes isolates resistant to one or more of the commonly used antiacne antibiotics were sensitive to penicillin, fusidic acid, chloramphenicol and the fluoroquinolone, nadifloxacin. All but one isolate (from the U.K.) were sensitive to trimethoprim. This study shows that 23S and 16S mutations identified in the U.K. conferring antibiotic resistance in P. acnes are distributed widely. However, resistant strains were isolated in which mutations could not be identified, suggesting that as yet uncharacterized resistance mechanisms have evolved. This is the first report of high-level resistance to minocycline and is of concern as these strains are predicted to be clinically resistant and are unlikely to remain confined to the U.S.A. Epidemiological studies are urgently required to monitor how resistant strains are selected, how they spread and to ascertain whether the prevalence of resistance correlates with antibiotic usage patterns in the different countries.