Streptococcus uberis, an environmental organism also associated with dairy animals, is a common and persistent cause of bovine mastitis. New approaches to control these infections need to be identified. One such strategy may be the application of bacteriocins; proteinaceous antimicrobials elaborated by bacteria that typically inhibit the growth of strains closely related to the producer organism. The well-characterized lactococcal bacteriocin nisin is the active ingredient in two commercial products currently in use for the prevention of mastitis. However, reports of resistance development have prompted the investigation of alternative bacteriocins to be used in conjunction with nisin in 'bacteriocin cocktails' designed to have more comprehensive inhibitory activity against mastitis pathogens. The bacteriocins of gram-positive bacteria have been divided into four distinct classes: (I) lantibiotics, (II) non-lantibiotic peptides, (III) large proteins, and (IV) circular peptides. Although it has been known for more than twenty years that S. uberis commonly produce bacteriocin-like inhibitory substances (BLIS), none had been characterised prior to the present study. The first step in the current investigation was a survey of the BLIS activities of a set of fifteen S. uberis and S. bovis strains against a set of standard indicators as well as common gram-positive mastitis pathogens. Additional tests using a deferred antagonism agar plate-based assay showed that some of the BLIS activities were heat-sensitive and their production was influenced by the presence of either blood or a fermentable carbohydrate source in the test medium. On the basis of the results obtained from these tests it became apparent that S. uberis and S. bovis may commonly produce more than a single inhibitory agent. S. uberis 42 became the focus of this study because (a) it had broad inhibitory activity against mastitis-associated bacteria, (b) it did not display cross-resistance to nisin, and (c) from the preliminary screening results it appeared to produce both heat-stable and heat-labile inhibitory agents. Acid extracts of S. uberis 42 cells yielded inhibitory activity that, when fractionated by reversed-phase HPLC, yielded a peptide of 3029 Da. Although this peptide was blocked to Edman degradation at position 2, following propanethiol-modification a 20-amino acid sequence was obtained. Degenerate primers to lantibiotic biosynthesis gene homologs were used to initiate inverse PCR and primer walking, ultimately yielding a 15-kb contiguous sequence encompassing 11 genes typical of those involved in lantibiotic synthesis, regulation and immunity. Due to the close similarities to nisin of the S. uberis 42 lantibiotic precursor (78%), and the organisation and composition of the locus, this inhibitor was named nisin U. Nucleotide sequences homologous to insertion sequences were detected in the vicinity of the nisin U locus, and indicate a possible mechanism of acquisition of this locus by S. uberis. The locus was detected in ten other S. uberis, and also in two S. agalactiae and two S. thoraltensis strains, and in one S. porcinus and one S. pluranimalium strain. The amino acid sequences of some of these differed in one or two amino acids, and these variants were named nisin U2 and nisin U3 accordingly. Nisin U, the two nisin U variants, and nisin A exhibited cross-immunity (i.e. all of the producer strains were insensitive to each form of nisin) and cross-inducibility (i.e. all of the producer strains displayed enhanced production when exposed to each form of nisin). Nisin U did not contribute to the entire spectrum of inhibitory activity of S. uberis 42. Freeze thaw extracts of S. uberis 42 agar cultures yielded heat-labile inhibitory activity that was inhibitory to L. lactis A5, a producer of nisin Z. Subsequent purification by cation-exchange chromatography, gel filtration, and reversed-phase HPLC yielded a peptide of mass 7048 Da, which was resistant to Edman degradation. Digestion with chymotrypsin released an 819 Da peptide fragment of sequence NH₂-KAQAVIW-COOH. Tn916 mutagenesis of S. uberis 42 enabled the identification of the genetic locus of the inhibitor, comprising six genes potentially involved in its biosynthesis and immunity. The detection of a pair of flanking 159-bp direct repeats indicates possible acquisition of the locus by 'long target duplication'. The inhibitor was inferred to be a circular peptide, on the basis of its behaviour to Edman degradation, and by comparison of its locus with that of other circular bacteriocins. On the basis that the purified peptide appears to induce lysis in sensitive bacteria, although by an as-yet unidentified mechanism, the inhibitor was named uberolysin. The uberolysin structural gene was detected in eight other strains of S. uberis, however not all of these appeared to be producing active inhibitor. No bacteriocins closely resembling the two reported in this thesis have been demonstrated previously to be produced by members of the genus Streptococcus. The remarkable diversity in the structures, activity spectra and basic modes of action of these two bacteriocins produced by a single strain of S. uberis, combined with the observation of apparent greater heterogeneity in properties of a preliminary sampling of BLIS-producing strains, indicates that these bacteria may be an important source of novel antimicrobials of potential value for the treatment of mixed bacterial infections and for minimising potential resistance development.

Bovine mastitis is one of the most costly dairy-based diseases worldwide. Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. New strategies need to be developed to control this pathogen. However, a fundamental understanding of the complex relationships that exist between the cow, the pathogen and the environment are required in order to advance the development of prevention strategies. Microarray technology was used to evaluate the complex transcriptional changes which occur in the bovine mammary gland following the onset of clinical S. uberis mastitis. A 22,000 bovine cDNA microarray indicated that S. uberis mastitis led to the up-regulation of 1,283 genes and the down-regulation of 1,237 genes by greater than 1.5 fold. Gene ontology analysis demonstrated that S. uberis mastitis was typically associated with the up-regulation of genes that are involved in the immune response and homeostasis and a down-regulation of genes involved in lipid metabolism. Quantitative real-time analyses for a selection of genes associated with the immune response validated the microarray data. Mammary epithelial cell cultures did not show an increase in the expression of any of these immune factors in response to the same S. uberis strain used to induce clinical mastitis. This indicates that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not S. uberis. The application of bacteriocins, proteinaceous antimicrobials produced by bacteria which typically inhibit the same or closely-related species to that of the producer organism, has been suggested as one possible approach in the control of mastitis. S. uberis have been previously found to commonly produce bacteriocin-like inhibitory substances (BLIS). The BLIS activities of a set of fifteen S. uberis and S. bovis strains were assessed. The results confirmed the prolific and varied nature of BLIS production by S. uberis and S. bovis and also indicated that these strains may commonly produce more than one inhibitory agent. This survey of BLIS production led to the detection and characterisation of a novel circular bacteriocin, uberolysin, produced by S. uberis strains 233 and 42. The structural gene of uberolysin was subsequently identified in nine (64%) of the fifteen test strains. Multiplex PCR analysis showed that 93% of 158 New Zealand S. uberis isolates contained the structural genes of at least one of the four known S. uberis bacteriocins (uberolysin, nisin U, ubericin A and ubericin 63). However, no apparent direct association was identified between any one of these bacteriocin-related loci and apparent ability to cause mastitis on New Zealand dairy farms. The uberolysin structural gene was detected in 91% of the isolates and this widespread distribution prompted the advancement and evaluation of a potential role for uberolysin in immunomodulation within the bovine mammary gland. Two different preparations of uberolysin were found to have different stimulatory effects on monocytes, neutrophils and epithelial cells. The less highly purified preparation appeared to diminish the production of TNF-α by monocytes in the presence of a bacterial stimulus and to decrease neutrophil phagocytosis. By contrast, the relatively more highly purified preparation of uberolysin itself induced a significant immune response by monocytes. Consistent with this, the purer preparation of uberolysin induced an increase in C3, IL-1β, IL-6, IL-8, the β-defensin LAP, the acute-phase protein MSAA, the calcium-binding protein S100A12 and TLR2 by quantitative real-time analysis. Although currently only two S. uberis bacteriocins (uberolysin and nisin U) have been fully characterised, the present study has shown that this species may be an important source of novel antimicrobials. Furthermore, bacteriocin production by S. uberis may have an immunomodulation role within the mammary gland. A better understanding of the complex immune response initiated at the onset of clinical S. uberis mastitis and of the role that bacteriocins have in S. uberis pathogenesis may lead to development of improved strategies to combat this disease.

Graduation date: 1993

Thesis (Ph.D.)--University of Waikato, 2008. / Title from PDF cover (viewed September 18, 2008) Includes bibliographical references (p. 324-347)

An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.

No description available.

Lactic acid bacteria produce pediocin-like bacteriocins designated as Class Ha. These antimicrobial peptides are antagonistic against Listeria monocytogenes and other closely related Gram-positive bacteria Self-protection of the producer organism is attributed to the immunity proteins, encoded by genes that are eo-transcribed with the structural gene that encode the bacteriocin. The lactic acid bacterium, Leuconostoc gelidum UAL 187-22 is immune to its own bacteriocin, leucocin A. This is accredited to its immunity protein and the possible absence of a receptor on its cytoplasmic membrane. Leucocin A was purified from the supernatant of 1. gelidum to 90% purity by ion-exhange chromatography and C18 reverse phase High Pressure Liquid Chromatography (RP-HPLC) eluted with an acetonitrile, 0.1% Triflouroacetic acid (TFA) gradient. The immunity gene was isolated from the same producer using the polymerase chain reaction from the recombinant plasmid pJF 5.5 using primers EAL-2 and EAL-3. The amplicon was truncated into versions A and B by removing the C- and N-terminals, with HaeIII and ClaI restriction enzymes, respectively. The amplicon and the truncated fragments A and B were cloned into pMALc2 to construct recombinant plasmids pKPl, pKPIA and pKPIB, correspondingly, which were transformed into Escherichia coli (E. coli) strain JMI03. Clones were confirmed by colony PCR and Southern blot hybridization. The recombinant clones were subsequently expressed as MBP-IP, MBP-IPA and MBP-IPB fusion proteins that were verified by Western blot using the anti-MBP antibody. Factor Xa protease was used to cleave MBP from the proteins of interest. The resulting pure immunity protein versions had an approximate molecular weight of slightly more that 10 kDa. The binding interactions of the purified immunity protein constructs and leucocin A were compared on the Biacore 2000 instrument with surface plasmon resonance. None of the immunity constructs interacted with leucocin A, however, the N-terminal region of the immunity protein interacted with the cytoplasmic extract. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-07-29T13:36:35Z No. of bitstreams: 1 2011 - Bruno Rocha Pribul.pdf: 1463809 bytes, checksum: ba7b3b0f00c0b4469b45fe02ec9f8a7f (MD5) / Made available in DSpace on 2016-07-29T13:36:35Z (GMT). No. of bitstreams: 1 2011 - Bruno Rocha Pribul.pdf: 1463809 bytes, checksum: ba7b3b0f00c0b4469b45fe02ec9f8a7f (MD5) Previous issue date: 2011-02-24 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / PRIBUL, Bruno Rocha. Evaluation of Vancomycin Resistance and Susceptibility Profile Bacteriocins of staphylococci isolated from bovine mastitis in Southern State of Rio de Janeiro. 41p Thesis (Master of Veterinary Science). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Bacteria of the genus Staphylococcus are among the most implicated in the etiology of mastitis. An additional difficulty in controlling bacterial infections by this genus is represented by its frequent resistance to antibiotics. The glycopeptides (eg vancomycin) appear as an alternative therapy because of the increasing strains of staphylococci resistant to beta-lactams, which are the drugs of choice in these infections. Thus, glycopeptide resistance represents a threat to the future of antimicrobial therapy in humans and animals. This study aimed to evaluate the profile of vancomycin resistance pheno-genotypically in Staphylococcus spp. milk samples from cows with mastitis. All 150 isolates (50 isolates of Staphylococcus aureus, 50 isolates of Staphylococcus intermedius and 50 isolates of Coagulase-negative Staphylococcus spp. (CNS) were evaluated using the tests of disk diffusion, agar microdilution and detection of vanA e vanB genes. The antimicrobial susceptibility test showed high resistance to beta-lactam antibiotics for all groups. In the disk diffusion test, 24% of coagulase-positive isolates showed resistance to teicoplanin (24/100) and 23% to vancomycin (23/100). In the agar microdilution test for detection of CIM, 16% of the isolates were resistant to vancomycin (16/100). Among the resistant isolates in the agar microdilution testing, 56.25% of the isolates (9 / 16) had MIC values ranging from 4-8?g/ml. Staphylococcus coagulase-negative showed no resistance to the evaluated glycopeptides. Genes for resistance to vancomycin were detected in 37.5% of vancomycin-resistant isolates (6 / 16). To seek a correlation between the presence of glycopeptide resistance and the ability of producing biofilm, tests were performed to characterize growth in Congo red agar and also detection of the biofilm adherence test in microplates. It were detected 66.66% and 90, 00%, respectively, of isolates producing biofilm. The production of "slime" presented no statistical correlation with resistance to glycopeptides. The sensitivity profile of Staphylococcus spp. was assessed against the bacteriocins produced by Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus fermentum. Lactobacillus paracasei bacteriocins presented the highest inhibition capacity. / PRIBUL, Bruno Rocha. Avalia??o da Resist?ncia a Vancomicina e do Perfil de Suscetibilidade a Bacteriocinas de Staphylococcus spp Isolados de Mastite Bovina da Regi?o Sul do Estado do Rio de Janeiro. 41p Disserta??o (Mestrado em Ci?ncias Veterin?rias). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. As bact?rias do g?nero Staphylococcus est?o entre as mais implicadas na etiologia das mastites. Uma dificuldade adicional no controle das infec??es bacterianas por este g?nero ? representada por sua freq?ente resist?ncia aos antimicrobianos. Os glicopept?deos, como a vancomicina, aparecem como uma alternativa terap?utica devido ao crescente aumento de cepas estafiloc?cicas resistentes aos betalact?micos, que s?o os f?rmacos de elei??o nestas infec??es. Desse modo, a resist?ncia aos glicopept?deos representa uma amea?a ao futuro da terapia antimicrobiana em humanos e animais. O presente trabalho buscou avaliar o perfil de resist?ncia ? vancomicina em isolados de Staphylococcus spp. oriundos de amostras de leite de vacas com mastite. Todos os 150 isolados estudados (50 Staphylococcus aureus, 50 Staphylococcus intermedius e 50 Staphylococcus spp. coagulase negativos), foram avaliados atrav?s dos testes de difus?o em disco simples, microdilui??o em ?gar e detec??o dos genes vanA e B. O teste de suscetibilidade antimicrobiana revelou elevada resist?ncia aos beta-lact?micos para todos os grupos avaliados. No ensaio de difus?o em disco, os isolados coagulasepositivos apresentaram um perfil de resist?ncia de 24% a teicoplanina (24/100) e 23% a vancomicina (23/100). No teste de microdilui??o em ?gar para detec??o da CIM, 16% dos isolados apresentaram resist?ncia ? vancomicina (16/100). Entre os isolados resistentes no teste de microdilui??o em ?gar, 56,25% dos isolados (9/16) apresentaram valores de CIM que variaram entre 4-8?g/ml. Os Staphylococcus coagulase-negativos n?o apresentaram resist?ncia aos glicopeptideos. Os genes de resist?ncia ? vancomicina foram detectados em 37,5% dos isolados vancomicina-resistentes (6/16). Para se buscar uma correla??o entre a presen?a de resist?ncia aos glicopeptideos e a capacidade de forma??o de biofilme foram realizados os testes de caracteriza??o de crescimento em ?gar vermelho congo e detec??o de biofilme pelo teste de ader?ncia em microplacas, onde foram detectados 66,66% e 90,00% respectivamente, de isolados produtores de biofilme. A produ??o de ?slime? n?o apresentou correla??o estat?stica com a resist?ncia aos glicopept?deos. O perfil de sensibilidade dos isolados de Staphylococcus spp. foi avaliado frente as bacteriocinas produzidas pelos Lactobacillus paracasei, Lactobacillus acidophillus e Lactobacillus fermentum. As bacteriocinas que apresentaram maior capacidade de inibi??o frente aos Saphylococcus spp. testados foram as produzidas pelos Lactobacillus paracasei

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.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Infection is one of the major causes of increased morbidity and the escalating costs associated with orthopedic surgery. The areas that are infected are often difficult to reach and thus difficult to treat. In some surgeries antibiotic-loaded bone cements are used to control infection. Polymethylmethacrylate (PMMA) and calcium phosphate-based bone cements (CPC) are usually used as bone fillers. CPC are bioresorbable and biocompatible (unlike PMMA cements), but can only be used in non- or low-load bearing areas and are thus more applicable in cranio-and maxilla-facial surgeries. Several in vitro and in vivo trials have been conducted on the incorporation of antibiotics and other therapeutic agents into CPC and the release of these agents. As with any solid matrix, release is defined by specific parameters, i.e. matrix porosity, solubility of the drug and interaction of the drug with the cement. The increase in antibiotic-resistant pathogens, mainly as a result of overuse of antibiotics, has a major impact on the choice of antibiotics that are used in the treatment of bacterial infections. The search for alternative antimicrobial compounds that are active against resistant pathogens, is thus of utmost importance. Antimicrobial peptides (bacteriocins) produced by lactic acid bacteria may pose a possible alternative to antibiotics. Some of these peptides are active against antibiotic-resistant pathogens. Bacteriocins are small cationic, hydrophobic, or amphiphilic peptides active against a narrow range of target organisms. Most of these peptides are active in the nanomolar range. It may then be advantageous to incorporate bacteriocins into CPC to evaluate if they may be used as an alternative to antibiotics. The aim of the project was to evaluate if bacteriocins could be successfully incorporated into self seting brushite bone cement and remain effective in vivo without altering basic cement characteristics. Incorporation of bacteriocins into CPC is a novel concept. The low setting temperature and pH of CPC renders it the ideal matrix for incorporation of antimicrobial peptides. In this study, peptide ST4SA, a class IIa broad-spectrum bacteriocin, has been incorporated into brushite bone cement and characterized in vitro. Incorporation of the peptide did not have a significant effect on the crystal entanglement or setting reaction of the cement. Peptide ST4SA was rapidly released and inhibited the growth of the target strain effectively. In another experiment, peptide ST4SA was suspended in poly (lactide-co-glycolide) and electrosprayed to form micro particles that were entrapped in brushite cement. Association of the peptide with microparticles resulted in a delayed release from the cement, followed by a constant release. Nisin F, a class Ia bacteriocin was also incorporated into brushite cement and its activity studied in vitro and in vivo. Similar results were observed in vitro as recorded with peptide ST4SA incorporated into brushite cement. Small cylinders of brushite cement loaded with nisin F were implanted into subcutaneous pockets in mice and each pocket infected with a bioluminescent strain of Staphylococcus aureus (Xen 36). Nisin F in the bone cement prevented the growth of S. aureus in the wound and controlled infection. With this study we have shown that antimicrobial peptides that differ in structure (classes I and II) could be incorporated into bone cement and control the growth of S. aureus in vivo and in vitro. The mode of action of these peptides differs from antibiotics in that they form a permanent pore in the cell membrane of the target organism. This minimizes the chance of a strain becoming resistant to the peptide. Incorporation of antimicrobial peptides into bone cement may be a possible alternative to antibiotics in the control of bacterial infections associated with implants. / AFRIKAANSE OPSOMMING: Infeksie is een van die grootste bydraende faktore tot sterftes en verhoogde kostes in ortopediese chirurgie. Geinfekteerde areas is dikwels moeilik bereikbaar en dus ook moeilik om te behandel. In sommige operasies word antibiotika-gelaaide beensement gebruik om infeksie te beheer. Polymetielmetakrilaat (PMMS) en kalsium fosfaat gebaseerde beensement (KFS) word gebruik as been vullers. KFS is bioverenigbaar en bio-absorberend (in teenstelling met PMMS), maar kan slegs in geen- of liggewig-draende areas gebruik word en is dus van groter toepassing in skedel-, kaak- gesig- en mondchirurgie. Verskeie in vitro en in vivo toetse is al gedoen op die inkorporering van antibiotika en ander terapeutiese middels in KFS en die vrystelling daarvan uit die matriks. Soos met enige soliede matriks is vrylating van die geinkorporeerde bestanddeel afhanklik van sekere parameters, onder andere porositeit, oplosbaarheid van die middel, en die interaksie van die middel met beensement. Die toename in antibiotika-weerstandbiedende patogene plaas geweldige druk op die keuse van antibiotika wat gebruik word in die beheer van bakteriese infeksie. Die soeke na alternatiewe antimikrobiese middels aktief teen bestande patogene is dus van kardinale belang. Antimikrobiese peptiede (bakteriosiene) gepproduseer deur melksuur bakteriee mag dalk . alternatief tot antibiotika wees. Sommige van hierdie peptiede is aktief teen verskeie weerstandbiedende patogene. Bakteriosiene is kationiese, hidrofobiese of amfifiliese peptiede wat naverwante bakteriee inhibeer of doodmaak. Die meeste van hierdie peptiede is aktief op nanoskaal vlak. Dit mag dalk dus voordelig wees om bakteriosiene in been sement te evalueer as moontlike alternatiewe tot antibiotika. Die doel van die proejek was om te evaleer of bakteriosiene suksesfol in "brushite" sement geïnkorporeer kan word en steeds effektief in vivo bly sonder om die basiese eienskappe van die sement te verander. Inkorporasie van bakteriosiene in KFS is 'n nuwe konsep. Die lae stollingstemperatuur en pH van KFS maak dit moontlik om bakteriosiene daarin te inkorporeer. In hierdie studie is peptied ST4SA, . klas IIa wye-spektrum bakteriosien, in "brushite" sement geïnkorporeer en in vitro bestudeer. Die toevoeging van die peptied het nie 'n beduidende effek op die stolreaksie of kristal verstrikking van die sement gehad nie. Peptied ST4SA is effektief vrygelaat en het die groei van die teikenorganisme suksesvol onderdruk. In 'n ander eksperiment is peptied ST4SA in poli (D,L-laktied-ko-glikolied) gesuspendeer en met behulp van elektrosproeiing tot mikropartikels omvorm en is in "brushite" sement geïnkorporeer. Assosiasie van die peptied met mikropartikels het die inisiële vrylating van die peptied vertraag, gevolg deur 'n konstante vrylating. Nisien F, . klas Ia lantibiotikum, is ook in "brushite" sement geïnkorporeer en die aktiwiteit daarvan in vitro en in vivo bestudeer. Die in vitro eienskappe is soortgelyk aan die eienskappe wat vir peptied ST4SA-gelaaide sement waargeneem is. Klein stafies "brushite" sement, waarin nisien F geïnkoproreer is, is in onderhuidse sakkies in muise geplaas en die area met 'n bio-liggewende bakterie (S. aureus Xen 36) geïnfekteer. Nisien F in die beensement het die groei van S. aureus in die wond onderdruk en infeksie beheer. Met hierdie studie het ons bewys dat bakteriosiene wat struktureel van mekaar verskil (klasse I en II) in beensement geïnkorporeer kan word en die groei van S. aureus in vitro en in vivo kon beheer. Die wyse waarop hierdie peptiede die groei van sensitiewe organismes inhibeer verskil van die van antibiotika deurdat dit porieë in die selmembraan vorm. Die moontlikheid dat organismes weerstandbiedend raak tot die peptied is dus heelwat skraler. Die insluit van antimikrobiese peptiede in beensement mag dalk 'n alternatief tot antibiotika wees in die voorkoming van bakteriële infeksie geassosieer met ortopediese chirurgie.