Dissertations / Theses on the topic 'Bacteriocins'

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2006.
Kefir is a traditional fermented milk that is carbonated, has a sharp acidic taste, yeasty flavour and contains a low percentage alcohol (less than 2% (v/v)). The beverage is manufactured by fermenting milk with Kefir grains, comprised of microorganisms, polysaccharides and milk proteins. The microbial population of Kefir grains primarily include lactic acid bacteria (LAB), namely lactococci and lactobacilli, yeasts, Acetobacter and filamentous fungi. Kefir exhibits antimicrobial activity in vitro against some fungi, and Grampositive and Gram-negative bacteria. Although the exact cause of this inhibition in Kefir is not known, the ability of LAB to inhibit the growth of closely related bacteria is well known. This inhibition of pathogenic and spoilage microbes may be due to the production of organic acids, hydrogen peroxide, acetaldehyde, diacetyl, carbon dioxide or bacteriocins. Acid is not the only contributor to the antimicrobial activity of Kefir and Kefir grains, and bacteriocins may play a role in the inhibitory activity. The bacteriocin producer Lactobacillus plantarum ST8KF, isolated from Kefir and Kefir grains, produces a bacteriocin 3.5 kDa in size. The mode of activity of bacteriocin ST8KF (bacST8KF) is thought to be bacteriostatic in exponential cultures of Enterococcus faecalis E88, Lactobacillus casei LHS, Lactobacillus curvatus DF38, Lactobacillus sakei DSM 20017, Lactobacillus salivarius 241 and Listeria innocua F and LMG 13568. The peptide is sensitive to proteolytic enzymes and does not adsorb to the surface of the producer cell. The bacteriocin is stable between pH 2.0 and 10.0, and for 20 min at 121°C. Maximum bacteriocin activity was observed in modified MRS medium supplemented with glucose or saccharose, meat extract, KH2PO4, glycerol, thiamine or cyanocobalamin, or in modified MRS medium without tri-ammonium citrate. Maximum levels of adsorption of bacST8KF (80%) to Lb. casei LHS and Lb. sakei DSM 20017 were recorded. Adsorption (80%) of the bacteriocin to Lactobacillus paraplantarum ATCC 700211T and Streptococcus caprinus ATCC 700066, which are not sensitive to the bacteriocin was also recorded. Optimal adsorption to E. faecalis E88 was recorded at 25°C at pH 2.0, and to L. innocua LMG 13568 at 4°C, 10°C and 25°C at pH 6.0. Potassium ions, MgCl2, Tris, NH4- citrate, Na-acetate, Na2CO3, EDTA and SDS led to decreased adsorption to both sensitive strains, while NaCl and mercaptoethanol resulted decreased adsorption to E. faecalis E88, but not to L. innocua LMG 13568. Methanol resulted in lower levels of adsorption to L. innocua LMG 13568 but not to E. faecalis E88. Triton X-100 and Triton X-114 increased the adsorption of bacST8KF by 40%, and ethanol and chloroform had no effect on bacteriocin adsorption. The growth of Lb. plantarum ST8KF and L. innocua LMG 13568 in a mixed culture resulted in an increase of bacST8KF production. Cells treated with bacST8KF secreted DNA and galactosidase. As bacST8KF remains stable under a variety of conditions, the bacteriocin may have application, if awarded GRAS (generally regarded as safe) status, in various food products as a natural additive or preservative. The genes encoding bacteriocin production are located on a 3.9 kilo base (kb) plasmid. Curing of the plasmid resulted in a mutant strain of Lb. plantarum ST8KF, and the Lb. plantarum strains ST8KF(+) and ST8KF(-) differed with regards to antibiotic resistance and carbohydrate fermentation reactions. The wild type and the cured strain were incorporated into Kefir grains during mass cultivation. The survival of the bacST8KF sensitive Enterococcus mundtii ST4SA added to the milk during Kefir production using the enriched mass cultured grains was monitored using fluorescent in situ hybridization. Enterococcus mundtii ST4SA was present in higher numbers in the ST8KF(-) Kefir system when compared to the ST8KF(+) system. It can, therefore, be concluded that Lb. plantarum ST8KF(+) contributes to the antimicrobial activity of Kefir through the production of bacteriocin ST8KF.

Orientador: Ana Lúcia Barretto Penna
Coorientador: Sabrina Neves Casarotti
Banca: Neuza Jorge
Banca: Aline Teodoro de Paula
Resumo: As bactérias acidoláticas (BAL) são bastante utilizadas em processos fermentativos na indústria de laticínios, porém algumas delas agem não somente como fermentadoras, com a produção de ácidos orgânicos a partir dos carboidratos presentes, mas também podem produzir substâncias que colaboram para a segurança microbiológica do produto fermentado ou compostos benéficos à saude. Em estudos in vitro anteriores, foi constatado que Leuconostoc mesenteroides subsp. mesenteroides SJRP55 apresenta potencial probiótico e ação bacteriostática sobre bactérias patogênicas, como Listeria monocytogenes e Escherichia coli. Neste trabalho foi avaliado o efeito de Leuconostoc mesenteroides subsp. mesenteroides SJRP55 em creme fermentado, em co-cultura com outras BAL, e estudar as características físico-químicas e microbiológicas do creme, além de avaliar a capacidade de bioconservação pela produção de bacteriocinas, ácidos orgânicos e propriedade funcional pela produção de ácido linoleico conjugado (CLA) e pela atividade antioxidante por inibição de radicais livres. Foi utilizado creme de leite UHT homogeneizado padronizado em 20% de gordura e fermentado conforme quatro tratamentos: T1 - cultura mista de Lactococcus lactis subsp. lactis e Lc. lactis subsp. cremoris; T2 - cultura mista de Lc. lactis subsp. lactis e Lc. lactis subsp. cremoris + Listeria monocytogenes ATCC 15313; T3 - Cultura mista de Lc. lactis subsp. lactis e Lc. lactis subsp. cremoris + Ln. mesenteroides subsp. mesenteroides...
Abstract: Lactic acid bacteria (LAB) are widely used in fermentation processes in the dairy industry, however some of them act not only as starters, with the production of organic acids from the carbohydrates, but they can also produce substances that contribute to the microbiological safety of the fermented product or produce health benefic compounds. In previous in vitro studies, it was found that Leuconostoc mesenteroides subsp. mesenteroides SJRP55 presents probiotic potential and bacteriostatic action on pathogenic bacteria, such as Listeria monocytogenes and Escherichia coli. In this study it was evaluated the effect of Leuconostoc mesenteroides subsp. mesenteroides SJRP55 in fermented cream, in co-cultivation with other BAL, and to study the physicochemical and microbiological characteristics of the cream, besides evaluating the capacity of bioconservation by the production of bacteriocins, organic acids and functional property by the production of conjugated linoleic acid (CLA) and antioxidant activity through the inhibition of free radicals. UHT milk cream standardized at 20% fat was fermented according to four treatments: T1 - Mixed culture of Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris, T2 - Mixed culture of Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris + Listeria monocytogenes ATCC 15313, T3 - Mixed culture of Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris + Ln. mesenteroides subsp. mesenteroides SJRP55, and T4 - Mixed ...
Mestre

An aim of this work was to compare interactions between bacteria, and to correlate them with increased or decreased biofilm formation. A better understanding of the interactions occurring within biofilms may lead to more effective control strategies. As the strains used in this study were closely related Enteric species, considerable bacteriocin activity occurred. Bacteriocin-producing strains were found to have a competitive advantage over bacteriocin-sensitive strains, both in gaining a foothold into a new community, and discouraging the attachment of potential competitors. Bacteriocins and bacteriocin-producing strains may be used as a novel strategy to control biofilm growth, and discourage the attachment of pathogenic strains. In general, a decrease in biofilm size and stability, and an increase in sensitivity to disinfectants was exhibited by bacteriocin-producing mixed species biofilms. There were, however, exceptions: certain biofilms of Enterobacter agglomerans/Ent when antagonised with a second, competitive strain produced a signal to repress bacteriocin synthesis in the competing strain, leading to a co-operative state. These biofilms were thicker, more stable and demonstrated an increased resistance to disinfectants. There is also the possibility that bacteriophage can be used to control biofilm formation. Studies indicated that small titres of phage were more successful in the removal of Enterobacter cloace/5920 biofilms. However, infection of three phages, φ1.15, Winchburgh and Blackburn phage, was required to completely eradicate the biofilms. The triple-combination of phage was also found to selectively remove a single bacterial species form a mixed species biofilm. The role of EPS in biofilm resistance and the adaptation of biofilms to increasing concentrations of disinfectant were also investigated. While the involvement of EPS was found to be transient, it was thought that repeated exposure to an antimicrobial agent may select for a more resistant phenotype, leading to biofilm resistance. For example, biofilms responded to increasing concentrations of triclosan by producing a triclosan mutant, and it was thought that increasing concentrations of benzalkonium chloride selected for strains utilising increased expression of multi-drug efflux pumps.

Thesis (PhD)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Worldwide, bacteriocins, particularly those produced by food-related lactic acid bacteria, are receiving attention due to the possible use of these peptides as natural preservatives in food, replacing potentially harmful chemical preservatives. Bacteriocins are ribosomally synthesized proteins or peptides that inhibit closely related microorganisms. Most bacteriocins produced by lactic acid bacteria are small, heat resistant peptides that inhibit other Gram-positive bacteria, including food-borne pathogens such as Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus, but do not inhibit Gram-negative bacteria, molds or fungi. Bacteriocins are produced as inactive prepeptides that become active after the N-terminal leader peptide is cleaved off. Small heat resistant bacteriocins are either lantibiotics (Class I), containing unusual posttranslationally modified amino acids, or peptides that are non-Ianthionines (Class II). The Class II bacteriocins are further divided into four different groups: Class lIa, the anti-listerial bacteriocins containing the YGNGV consensus sequence in the N-terminal of the protein, Class lib, bacteriocins consisting of two peptides, Class IIc, bacteriocins that are secreted via the sec pathway, and Class lid, bacteriocins that do not belong in the previous three subgroups. A bacteriocin producing lactic acid bacterium was isolated in our laboratory from traditionally home fermented South African sorghum beer. The producing bacterium was found to be a facultative heterofermentative Lactobacillus sp. and was identified as Lactobacillus plantarum or Lactobacillus pentosus by using the API 50 CHL carbohydrate fermentation system and numerical analysis of total soluble cell protein patterns. RAPD-PCR analysis identified the strain as L. plantarum, but 16S rRNA sequencing confirmed its identification as L. pentosus. The bacteriocin, first designated plantaricin 423 and later bacteriocin 423, was identified as a Class lIa small heat resistant anti-listerial bacteriocin containing the YGNGV consensus motif. Bacteriocin 423 inhibited a variety of Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp. and Listeria spp. The bacteriocin was inactivated by proteolytic enzymes and active over a wide pH range (pH 1-10). Bacteriocin 423 lost 50 % of its activity after autoclaving for 15 min at 121°C, but was not affected by lesser heat treatments. Bacteriocin production was increased by optimizing the growth medium, which consisted of glucose, tryptone, yeast extract, potassium phosphate, sodium acetate, ammonium citrate, manganese sulphate, Tween 80 and casamino acids. The bacteriocin was found to be plasmid-encoded. Genetic analysis of the bacteriocin operon indicated a high percentage of homology to the operon of another Class lIa bacteriocin, pediocin PA-1, although the structural genes of the two bacteriocins were markedly different. The structural gene of bacteriocin 423 was amplified by PCR and cloned into a yeastJE. coli vector between the ADH1 promoter and terminator sequences and fused in-frame to the MFa1 secretion signal sequence. Saccharomyces cerevisiae transformed with this plasmid expressed the bacteriocin. The sequence of prebacteriocin 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVN WGOAFSCSVSHLANFGHGKC) is similar, but not identical to any other reported Class lIa anti-listeria I peptide.
AFRIKAANSE OPSOMMING: Bakteriosiene, veral dié wat deur melksuurbakterieë geproduseer word, wek belangstelling as gevolg van die moontlike gebruik van hierdie natuurlike antimikrobiese proteiëne as preserveermiddels in voedselprodukte, in plaas van potensieël gevaarlike chemiese preserveermiddels. Bakteriosiene is ribosomaal-vervaardigde proteiëne wat naverwante bakterieë inhibeer. Die meeste bakteriosiene wat deur melksuurbakterieë geproduseer word, is klein en hittebestand. Hierdie bakteriosiene inhibeer ander Gram-positiewe bakterieë, insluitend patogene soos Listeria monocytogenes, Bacillus cereus, Clostridium perfringens en Staphylococcus aureus, maar inhibeer nie Gram-negatiewe bakterieë, giste of swamme nie. Bakteriosiene word as onaktiewe prepeptiede geproduseer, wat ge-aktiveer word wanneer die N-terminale leierpeptied afgesplits word. Klein hittebestande bakteriosiene is óf lantibiotika (Klas I), met ongewone aminosure, óf normale peptiede (Klas II). Laasgenoemde klas kan verder in vier groepe verdeel word. Klas lIa is anti-listeriese bakteriosiene met fn YGNGVaminosuurvolgorde in die N-terminale kant van die peptied. Klas lib sluit in bakteriosiene wat uit twee peptiede bestaan. Klas lie is sec-afhanklike bakteriosiene, en Klas lid sluit in al die bakteriosiene wat nie in die eerste drie groepe geklassifiseer kan word nie. 'n Bakteriosien-produserende melksuurbakterie is uit tradisionele tuisgefermenteerde Suid- Afrikaanse sorghumbier geïsoleer. Die bakterie is as 'n fakultatief heterofermentatiewe Lactobacillus sp. geïdentifiseer. Die bakterie is verder as 'n Lactobacillus plantarum of Lactobacillus pentosus geïdentifiseer deur middel van die API 50 CHL-koolhidraat fermentasiesisteem en numeriese analiese van totale oplosbare selproteiënprofiele. Met RAPD-PCR analiese is die organisme as L. plantarum geïdentifiseer, maar 168 rRNA nukleotiedopeenvolging het die identiteit van die organisme as L. pentosus bevestig. Bakteriosien 423, aanvanklik geklassifiseer as plantaricin 423, is fn klein Klas lIa, hittebestande en anti-listeriese bakteriosien met die YGNGV motief, wat verskeie Grampositiewe bakterieë inhibeer. Bakteriosien 423 het verskeie Gram-positiewe organismes geïnhibeer, onder andere Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp., en Listeria spp. Proteolitiese ensieme inaktiveer die bakteriosien. Die peptied was oor 'n pH reeks van 1-10 aktief. Outoklavering vir 15 min by 121°C het die aktiwiteit van die peptied halveer, maar die bakteriosien is nie geïnaktiveer met ander hittebehandelings nie. Produksie van die bakteriosien is verhoog deur die groeimedium te optimiseer. Die groeimedium het bestaan uit glukose, triptoon, gisekstrak, kaliumfosfaat, natriumasetaat, ammoniumsitraat, mangaansulfaat, Tween 80 en casaminosure. Die bakteriosien se genetiese determinante is op In plasmied gesetel. Genetiese analiese van die bakteriosien operon het 'n hoë homologie met In ander Klas lIa bakteriosien, pediocin PA-1, getoon, maar die strukturele gene van die twee bakteriosiene verskil merkbaar. Die strukturele geen van bakteriosien 423 is met PKR ge-amplifiseer en in 'n gistE. coli-vektor tussen die ADH1 promotor- en termineerderopeenvolgings, in leesraam met die MFa1 sekresiesein, gekloneer. Saccharomyces cerevisiae wat met hierdie plasmied getransformeer is, het bakteriosien 423 uitgedruk. Die aminosuurvolgorde van prebakteriosien 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVNWGOAFSCSVSHLANFGHGKC) is verwant aan, maar nie identies aan, ander Klas lIa anti-listeriese peptiede.

Orientador: Ary Fernandes Júnior
Banca: Maria de Lourdes Ribeiro da Cunha
Banca: Rosemeire Cristina Lianhri Rodrigues Pietro
Resumo: A pesquisa por novas drogas antimicrobianas tem aumentado, seja na indústria farmacêutica e também na indústria de alimentos. Isso acontece devido ao aumento no número de bactérias resistentes aos antimicrobianos, e a busca por conservantes alimentares que possibilitem o aumento na vida de prateleira dos alimentos. O interesse por alimentos mais saudáveis, especialmente aqueles sem adição ou com quantidades reduzidas de aditivos químicos, vem aumentando constantemente. Os produtos naturais, especialmente os de origem microbiana e de espécies vegetais são considerados fontes importantes para o desenvolvimento de novos antimicrobianos. O trabalho teve como objetivo avaliar a ação antibacteriana de óleos essenciais de plantas, seus compostos majoritários, a ação antibacteriana da nisina (bacteriocina produzida por Lactococcus lactis) e a ação antibacteriana da combinação desses compostos majoritários com a nisina em meio de cultura e no leite. Inicialmente foi avaliado o potencial antibacteriano com a determinação da concentração inibitória mínima (CIM) dos óleos essenciais de orégano (Origanum vulgare), tomilho (Tymus vulgaris), cravo da índia (Syzygium aromaticum) e canela (Cinnamomun zeylanicum) e respectivos compostos majoritários carvacrol, timol, eugenol e cinamaldeído, e da nisina sobre cepas padrões ATCC de bactérias de importância na área de alimentos: Staphylococcus aureus ATCC 25923, Escherichia coli O157 ATCC 43895, Salmonella Enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 10100, Listeria monocytogenes ATCC 15313, Aeromonas hydrophila ATCC 7966 e Lactobacillus rhamnosus ATCC 9595 utilizando a metodologia da microdiluição em meio de Mueller Hinton Caldo (MHC). Em alguns casos a atividade inibidora dos óleos essenciais foi maior que a atividade do seu respectivo composto isolado, e em outros a atividade inibidora do composto isolado foi maior que ...
Abstract: The research for new antimicrobial drugs has increased in a pharmaceutical industry as well as in the food industry. This happens due to the increase in the number of bacteria resistant to antimicrobial agents, and the search for food preservatives which make possible the increase in the shelf life of foods. The interest in healthier foods, especially those without addition or with reduced amounts of chemical additives is increasing constantly. The natural products, especially of microbial origin and plant species are considered important sources for the development of new antimicrobial. This study purpose to evaluate the antibacterial activity of essential oils from plants, their major compounds, the antibacterial action of nisin (bacteriocin produced by Lactococcus lactis) and the antibacterial activity of the combination of these major compounds with nisin in culture medium and in milk. First was evaluated antimicrobial activity with determination of minimum inhibitory concentration (MIC) of the essential oils of oregano (Origanum vulgare), thyme (Tymus vulgaris), clove (Syzygium aromaticum) and cinnamon (Cinnamomun zeylanicum) and their major compounds carvacrol , thymol, eugenol and cinnamaldehyde, and nisin on ATCC strains of bacteria of importance in the food industry: Staphylococcus aureus ATCC 25923, Escherichia coli O157 ATCC 43895, Salmonella Enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 10100, Listeria monocytogenes ATCC 15313, Aeromonas hydrophila ATCC 7966 and Lactobacillus rhamnosus ATCC 9595 using the microdilution method in Mueller Hinton Broth medium (MHC). In some cases the inhibitory activity of essential oils has been higher than the activity of the respective isolated compound, and in others the inhibitory activity of the compound isolate was greater than that of the respective essential oils, whereas nisin was active against Gram-positive bacteria and a lower inhibitory ...
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Bacteriocins (BCNs) are ribosomally synthesized polypeptides or proteins with antimicrobial activity, produced by different groups of bacteria. Many lactic acid bacteria (LAB) produce BCNs with broad spectra of inhibition. The antimicrobial activity of BCNs against spoilage organisms (SPOs) has raised considerable interest in their application in juice preservation. The objectives of the study were to: (i) isolate, identify and screen BCN producing bacteria for antimicrobial activity against spoilage bacteria and fungi, (ii) optimize production of BCN from selected producers and (iii) investigate the industrial application of the BCN as a preservative in fruit juice. Eleven LAB strains of BCN producers were screened for antimicrobial activity. BCNs from Lactobacillus plantarum and Pediococcus pentosaceus 34 were the most effective against juice spoilage bacteria and fungi. The effect of medium components on bacteriocin production in L. plantarum and P. pentosaceus 34 was also determined. Clementine:Valencia (1:1) juice was used for the first time as the growth medium for L. plantarum and P. pentosaceus 34. The BCN from L. plantarum showed the highest activity and was, therefore, chosen for juice fermentation studies. The identification of L. plantarum was confirmed by biochemical tests, polymerase chain reaction (PCR) and sequencing of the recA gene. The highest BCN activity was observed for L. plantarum grown in De Man-Rogosa-Sharpe (MRS) and a combination of all supplements (i.e. peptone, MnSO4.H2O, Tween 80, glucose and whey), followed by MRS and Tween 80, peptone, MnSO4.H2O and MRS alone. MRS was a better medium for BCN production than juice [Clementine:Valencia (1:1)]. Size exclusion chromatography (SEC) was used to isolate the active L. plantarum BCN fraction which corresponded to an approximate molecular weight of 3.2 kDa and was proteinaceous in nature. Plantaricin structural genes (plnEF, plnJ, plnK, plnN) were detected in the L. plantarum strain by PCR and sequenced, and were chromosomally encoded as no plasmids could be detected. This implies that the BCN from L. plantarum is most likely a type of class IIa plantaricin which is responsible for the broad inhibitory activity observed. For the industrial application studies, L. plantarum BCN-containing cell free supernatant (BCNsup) added to “Ready to Drink” (RTD) Clementine:Valencia (1:1) juice at concentrations of 3 600 - 500 000 ppm decreased growth of SPOs, Lactobacillus acidophilus and Streptococcus thermophilus. At 250 000 ppm, the L. plantarum BCNsup achieved 5.3 and 6.8 log reductions of the L. acidophilus, after 24 and 48 h, respectively, which is larger than the USFDA (2001) requirement of a 5 log reduction in SPO activity, for preservation of fruit juices. However, there was a decrease in the activity when the BCNsup was applied to industrial (Valor) RTD juice (mango-orange) at decreasing concentrations of 100 000, 50 000 and 25 000 ppm. Organoleptic tests showed that the BCN did not alter flavor or taste of the juice and did not cause toxicity or allergic reactions. A food safety risk assessment was conducted in order to determine the Critical Control Point(s) [CCP(s)] at which the BCN could be applied to control identified microbiological hazards, and a Hazard Analysis and Critical Control Point (HACCP) plan was developed. This is the first report on the optimisation of L. plantarum BCN production in juice [Clementine:Valencia (1:1)], followed by inoculation into RTD juice (mango-orange), including a HACCP plan for the application of the BCN as a preservative in juice.

Dental caries is the most common bacterial disease of humans and occurs when oral bacteria produce acids, following their fermentation of dietary carbohydrates. This acid can then cause a localised demineralisation of the tooth surface. A group of seven species of bacteria, collectively known as the mutans streptococci, have been predominantly implicated in the onset of dental caries. In particular, Streptococcus mutans and Streptococcus sobrinus have been shown to be the main aetiological agents of this disease in humans. Most attempts to control the microbial component of caries target these bacteria. The past 50 years has provided considerable information about the pathogenesis of dental caries, the likely route and time of transmission of cariogenic bacteria to susceptible hosts and possible ways of either treating or controlling the onset of this disease. In regards to the latter, many techniques (such as the use of tooth brushes, mouth washes, dental floss and tooth paste) for the control of plaque build-up exist and the examples listed are generally part of a daily routine. However, these techniques need to be applied regularly, and as such only highly-motivated individuals generally experience improved oral health. Therefore, the search for more effective less labour-intensive approaches continues. One area of research is into the potential application of small ribosomally-synthesised antimicrobial peptides, known as bacteriocins. Bacteriocins generally inhibit closely-related species that occupy the same ecological niche. Their relatively-specific targeting, plus the fact that many are remarkably heat and chemically-stable molecules, makes them excellent candidates for possible anti-caries applications. Numerous bacteriocins produced by the lactic acid bacteria have now been identified. Most can be broadly categorised into one of four main classes, of which Class I, the lantibiotics and Class II, the small (<10 kDa) non-modified peptides, contain the most examples. Many screens for anti-mutans streptococcal (MS) bacteriocins have been carried out and it appears that the best source of anti-MS bacteriocins are the mutans streptococci themselves. Research in this laboratory has identified examples of anti-mutans streptococcal bacteriocins produced by both mutans streptococci and non-mutans streptococci. The present study investigated the anti-MS inhibitors produced by two streptococcal strains, S. mutans N and Streptococcus sanguis K11. During the course of this study a third strain, S. mutans UA159, was also studied for its bacteriocinogenic properties. Although S. sanguis K11 produces anti-mutans streptococcal inhibitory activity, this appears only effective against Streptococcus rattus. In addition however, the inhibitory activity of this strain is also directed against all tested strains of Streptococcus agalactiae and ca. 50% of Streptococcus pyogenes. In the present study a 5069 Da novel inhibitory agent (sanguicin K11) was characterised and shown responsible for this unusual inhibitory spectrum. Through reverse genetics the sanK11 locus was identified and shown to encode a Class II type bacteriocin, the first shown to be produced by S. sanguis. Following screens of additional S. sanguis, sanK11 was shown to be present only in strains producing the same type of inhibitory pattern (P-type) as strain K11. The cysteine residues at positions 7 and 38 of the sanguicin K11 propeptide were shown to form a disulphide bridge essential for sanguicin K11 inhibitory activity. S. mutans N and eight other S. mutans strains have been found to have what appears to be the same inhibitory spectrum, which includes members of the mutans streptococci and several other oral streptococcal species. One strain (UA140) of the eight has previously been shown to produce the lantibiotic mutacin I and the non-lantibiotic mutacin IV. S. mutans N was known to produce the non-lantibiotic mutacin N. The current study set out to investigate how two strains, apparently producing completely different bacteriocins could have the same inhibitory spectrum. Reverse genetics identified the mutacin N structural gene (mutN) and mutagenesis studies showed that this bacteriocin was responsible only for the inhibitory activity against mutans streptococci. Further sequencing around the mutN locus identified a second bacteriocin-like locus (mutO) adjacent to mutN. mutO was also identified to have anti-mutans streptococcal inhibitory activity and because of the close proximity of mutO and mutN and given the homology they share with other known two-peptide bacteriocins it seemed probable that mutacins O and N are components of a new member of this special class of bacteriocins (Class IIb, the two peptide bacteriocins) in which the optimal inhibitory activity is dependent on the co-operative activity of the two peptides. Further investigations of strain N examined the expression of mutacins O and N. During a search for a suitable heterologous non-mutacinogenic S. mutans strain to act as an expression host, the genome reference strain, S. mutans UA159 was given consideration. However, contrary to previous reports, this strain was found to exhibit bacteriocin-like inhibitory activity. During a follow-up investigation, strain UA159 was found to inhibit 84 strains representing 11 different species of bacteria, but no inhibition of mutans streptococci was detected. The locus (nlmAB) encoding the two-peptide bacteriocin mutacin IV was identified within the UA159 genome. Using genetic dissection of nlmA and nlmB, the contribution of each peptide was examined and it was found that only the NlmA* propeptide appears to be active, raising doubts as to whether mutacin IV is a bona fide two-peptide bacteriocin. Deletion of the entire nlmAB locus created a mutant strain that exhibited a loss of inhibitory activity against the same 64 strains as was found for the nlmA mutant. A BLASTP search for the consensus leader sequence that precedes the propeptide of Class II bacteriocins, identified ORFs encoding 9 more putative bacteriocin-like peptides. Further genetic dissection identified the SMU.1914c locus as being responsible for the inhibitory activity against a further 15 strains not already sensitive to mutacin IV. SMU.1914c was renamed mutacin V. However, it appears that another as yet unidentified mutacin(s) is also produced by strain UA159 given that three indicator strains still remained sensitive to a double mutant [UA[Delta](1914/NlmAB)] in which both the mutacin IV and putative mutacin V loci were inactivated. Export of Class II bacteriocins has been found to occur by either a SEC-dependent system or via a dedicated peptide ATP Binding Cassette (ABC) transporter. Three potential ABC transporter ORFs were identified in S. mutans UA159. Two (comA and cslA) had the characteristic accessory factor ORF (comB and cslB respectively) located adjacent to the main ABC transporter ORF, while the third ORF763 appeared to lack this. Mutagenesis of each of these five ORFS was carried out and confirmed cslAB to be the ABC transporter involved in the export of the competence stimulating factor, while the function of ORF763 could not be established in this study. Mutagenesis of either comA or comB resulted in a complete cessation of bacteriocin production by the respective mutant strains. Historically, comA and comB is the nomenclature used for loci encoding the exporter of the competence inducing factors in streptococci. In light of this new information, comA and comB were renamed nlmT and nlmE respectively, to account for the newly defined role of this ABC transporter. The present study investigated four bacteriocins two of which (sanguicin K11 and mutacin ON) appear to have some potential for application to anti-caries control, and the others (mutacins IV and V) being shown to be produced by the genome reference strain (UA159). All three mutacins were shown to be exported from their respective producer cells by the NlmTE ABC transporter, while sanguicin K11 is predicted to be exported by a peptide ABC transporter located adjacent to sanK11. Bacteriocins may yet provide a novel alternative for the treatment and control of dental caries. In their favour is that fact that they have relatively narrow defined inhibitory spectra and thus are unlikely to produce widespread changes to plaque ecosystems. Potential uses include as topical agents where bacteriocin preparations could be incorporated into dentrifices such as toothpastes or mouthwashes. Alternatively, streptococci producing anti-mutans streptococcal bacteriocins could be implanted into the oral cavity in strain replacement therapy strategies. There are pros and cons to each technique and the most effective anti-caries control appears more likely to result from "cocktail therapy" where bacteriocins are combined with a number of other anti-mutans streptococcal agents to achieve long-lasting protection against mutans streptococcus proliferation.

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A pesquisa por novas drogas antimicrobianas tem aumentado, seja na indústria farmacêutica e também na indústria de alimentos. Isso acontece devido ao aumento no número de bactérias resistentes aos antimicrobianos, e a busca por conservantes alimentares que possibilitem o aumento na vida de prateleira dos alimentos. O interesse por alimentos mais saudáveis, especialmente aqueles sem adição ou com quantidades reduzidas de aditivos químicos, vem aumentando constantemente. Os produtos naturais, especialmente os de origem microbiana e de espécies vegetais são considerados fontes importantes para o desenvolvimento de novos antimicrobianos. O trabalho teve como objetivo avaliar a ação antibacteriana de óleos essenciais de plantas, seus compostos majoritários, a ação antibacteriana da nisina (bacteriocina produzida por Lactococcus lactis) e a ação antibacteriana da combinação desses compostos majoritários com a nisina em meio de cultura e no leite. Inicialmente foi avaliado o potencial antibacteriano com a determinação da concentração inibitória mínima (CIM) dos óleos essenciais de orégano (Origanum vulgare), tomilho (Tymus vulgaris), cravo da índia (Syzygium aromaticum) e canela (Cinnamomun zeylanicum) e respectivos compostos majoritários carvacrol, timol, eugenol e cinamaldeído, e da nisina sobre cepas padrões ATCC de bactérias de importância na área de alimentos: Staphylococcus aureus ATCC 25923, Escherichia coli O157 ATCC 43895, Salmonella Enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 10100, Listeria monocytogenes ATCC 15313, Aeromonas hydrophila ATCC 7966 e Lactobacillus rhamnosus ATCC 9595 utilizando a metodologia da microdiluição em meio de Mueller Hinton Caldo (MHC). Em alguns casos a atividade inibidora dos óleos essenciais foi maior que a atividade do seu respectivo composto isolado, e em outros a atividade inibidora do composto isolado foi maior que ...
The research for new antimicrobial drugs has increased in a pharmaceutical industry as well as in the food industry. This happens due to the increase in the number of bacteria resistant to antimicrobial agents, and the search for food preservatives which make possible the increase in the shelf life of foods. The interest in healthier foods, especially those without addition or with reduced amounts of chemical additives is increasing constantly. The natural products, especially of microbial origin and plant species are considered important sources for the development of new antimicrobial. This study purpose to evaluate the antibacterial activity of essential oils from plants, their major compounds, the antibacterial action of nisin (bacteriocin produced by Lactococcus lactis) and the antibacterial activity of the combination of these major compounds with nisin in culture medium and in milk. First was evaluated antimicrobial activity with determination of minimum inhibitory concentration (MIC) of the essential oils of oregano (Origanum vulgare), thyme (Tymus vulgaris), clove (Syzygium aromaticum) and cinnamon (Cinnamomun zeylanicum) and their major compounds carvacrol , thymol, eugenol and cinnamaldehyde, and nisin on ATCC strains of bacteria of importance in the food industry: Staphylococcus aureus ATCC 25923, Escherichia coli O157 ATCC 43895, Salmonella Enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 10100, Listeria monocytogenes ATCC 15313, Aeromonas hydrophila ATCC 7966 and Lactobacillus rhamnosus ATCC 9595 using the microdilution method in Mueller Hinton Broth medium (MHC). In some cases the inhibitory activity of essential oils has been higher than the activity of the respective isolated compound, and in others the inhibitory activity of the compound isolate was greater than that of the respective essential oils, whereas nisin was active against Gram-positive bacteria and a lower inhibitory ...

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE
Staphylococcus aureus has been considered a major public health problem worldwide due mainly to the ability to develop resistance to antibiotics. The prevalence of methicillin resistant strains of S. aureus (MRSA) in both hospital and community settings has been increasing, with cases of infections and deaths reported in healthy children and adults. Bacteriocins have been identified as promising alternatives for the control of this pathogen. However, besides the therapeutic use of these peptides still not approved, some studies indicate the possibility of selection of resistant strains. Therefore, prior to the therapeutic use of bacteriocins, studies must be carried out to understand the effect that bacteriocin may have on the selection of resistant strains to avoid the current problem with lineages resistant to traditional antibiotics. In view of the above, the objective of this study was to verify the antimicrobial activity of nisin against strains of MRSA and MSSA isolated from the oropharynx of health professionals and verify the selection of strains resistant to bacteriocin. For the genotypic characterization of the MRSA and MSSA strains, the nucA, LuckPV, mecA and mecC genes were tested. The MSSA lines were positive for amplification of the nucA gene only, confirming the identification of genus and species of these strains. For MRSA strains, the only gene that was not detected was mecC, confirming the methicillin resistance phenotype and that these strains are of human origin and of community environments. The results obtained by the agar diffusion test demonstrated that 80% of the MSSA strains (n = 30) were inhibited by nisin and only one MRSA line (n = 6) showed no sensitivity to bacteriocin. The MIC of MSSA strains ranged from 97.7 to 1250 IU / mL and from MRSA strains of 937.50 to 5000 IU / mL. The DBM for MSSA strains varied from 97.7 IU / mL to values greater than 50,000 IU / mL, and for MRSA, this variation was between 5000 IU / mL at values greater than 10,000 IU / mL depending on the lineage. For most lineages DBM was higher than MIC, showing that the effect of bacteriocin depends on bacteriocin concentration: low concentrations exert bacteriostatic effect and high concentrations bactericidal effect. The addition of increasing concentrations of the bacteriocin to the BHI medium generally resulted in the increase in the lag phase and decrease in the specific growth rate and maximal DO reached by the MSSA and MRSA cultures. MSSA and MRSA strains were transferred for approximately 30 generations in the presence of bacteriocin and a decrease in sensitivity was observed with a consequent increase in nisin MIC for all strains tested. The bacteriocin resistance phenotype has been shown to be a stable trait for these strains and may be associated with a genetic factor. It has also been observed that the use of nisin may trigger cross-resistance to some antibiotics. The results obtained demonstrated that nisin is efficient in controlling the growth of MSSA and MRSA. However, the fact that these lines demonstrate resistance to bacteriocin after transfer in the presence of the same indicates the need to develop strategies to avoid in the future the current problem of resistance to antibiotics. The best way to use bacteriocins therapeutically is to suggest that it is in combination with traditional antibiotics.
Staphylococcus aureus têm sido considerado um dos maiores problemas de saúde pública a nível mundial devido, principalmente, a habilidade de desenvolver resistência a antibióticos. A prevalência de linhagens de S. aureus resistentes a meticilina (MRSA) tanto em ambiente hospitalar quanto em ambientes comunitários têm aumentado, sendo descritos casos de infecções e relatos de mortes em crianças e adultos saudáveis. Bacteriocinas tem sido apontadas como alternativas promissoras para o controle deste patógeno. Entretanto, além do uso terapêutico destes peptídeos ainda não ser aprovado, alguns estudos indicam a possibilidade de seleção de linhagens resistentes. Portanto, antes do uso terapêutico de bacteriocinas, estudos precisam ser realizados para o entendimento do efeito que a bacteriocina possa ter na seleção de linhagens resistentes para evitar o problema da atualidade com linhagens resistentes aos antibióticos tradicionais. Diante do exposto, o objetivo deste trabalho foi verificar a atividade antimicrobiana da nisina contra cepas de MRSA e MSSA isoladas da orofaringe de profissionais da área de saúde e verificar a seleção de linhagens resistentes à bacteriocina. Para a caracterização genotípica das linhagens MRSA e MSSA, foram testados os genes nucA, LuckPV, mecA e mecC. As linhagens MSSA foram positivas para amplificação apenas do gene nucA, confirmando a identificação de gênero e espécie destas linhagens. Para as linhagens MRSA, o único gene que não foi detectado foi o meC, confirmando o fenótipo de resistência a meticilina e que estas linhagens são de origem humana e de ambientes comunitários. Os resultados obtidos pelo teste de difusão em ágar demonstraram que 80% das linhagens MSSA (n=30) foram inibidas pela nisina e apenas uma linhagem MRSA (n=6) não apresentou sensibilidade à bacteriocina. A CIM das linhagens de MSSA variou de 97,7 a 1250UI/mL e das linhagens MRSA de 937,50 a 5000 UI/mL. A DBM para as linhagens de MSSA variou de 97,7 UI/mL a valores superiores a 50000 UI/mL, e para MRSA esta variação ficou entre 5000 UI/mL a valores maiores que 10000 UI/mL dependendo da linhagem. Para a maioria das linhagens a DBM foi maior que a CIM, mostrando que o efeito da bacteriocina depende da concentração da mesma: baixas concentrações exercem efeito bacteriostático e altas concentrações efeito bactericida. A adição de concentrações crescentes da bacteriocina ao meio BHI resultaram, de maneira geral, no aumento da fase lag e diminuição da velocidade específica de crescimento e DO máxima atingida pelas culturas MSSA e MRSA. Linhagens de MSSA e MRSA foram transferidas por aproximadamente 30 gerações na presença da bacteriocina e foi observada diminuição na sensibilidade com consequente aumento da CIM da nisina para todas as linhagens testadas. O fenótipo de resistência à bacteriocina demonstrou ser uma característica estável para estas linhagens, podendo estar associado a um fator genético. Foi observado também que a utilização da nisina pode desencadear resistência cruzada a alguns antibióticos. Os resultados obtidos demonstraram que a nisina é eficiente em controlar o crescimento de MSSA e MRSA. Entretanto, o fato destas linhagens demonstrarem resistência a bacteriocina após transferência na presença da mesma indica a necessidade de desenvolver estratégias para evitar no futuro o problema atual de resistência a antibióticos. A melhor maneira de usar bacteriocinas terapeuticamente sugere-se que seja em combinação com antibióticos tradicionais.
São Cristóvão, SE

Thesis (MScAgric)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: The winemaking process involves a number of microorganisms, each with its own role. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are Gram-positive bacteria associated with must and wine and perform the malolactic fermentation (MLF), while the acetic acid bacteria (AAB) are Gram-negative bacteria converting ethanol to acetic acid. These microorganisms are present in the cellar and fermentation tanks and can be seen either as beneficial or as wine spoilage microorganisms because, under certain circumstances, they affect the wine quality if they should grow in the wine or must. Strict measures need to be implemented in the cellar during the winemaking process to ensure microbiological stability. This can be achieved through good microbiological practices and, additionally, chemical preservatives. Sulphur dioxide (S02) is widely used as the primary preservative in winemaking. However, consumer resistance has been building up against the use of chemical preservatives, due to the possible health risks and a decrease in nutritional value and sensorial quality of certain foods and beverages. Biopreservation as an alternative to the traditionally-used chemical preservation is a new approach and has been attracting much attention. This implies the use of the natural microflora and/or their antibacterial products, such as bacteriocins and bacteriolytic enzymes (e.g. lysozyme). Bacteriocins from LAB are proteins or protein complexes, produced by Gram-positive bacteria, with antibacterial activity against closely-related Gram-positive species. Lysozyme occurs in substances such as hen egg white and has lytic activity against Gram-positive bacteria. ' The bacteriocins nisin, of the class I lantibiotics, and pediocin PA-1 and leucocin BTA 11a, of the class lIa Listeria-active bacteriocins, have been investigated for the biopreservation of wine. Nisin, however, is the only bacteriocin that has been approved for use as a preservative, while pediocin is likely to follow in approval. Lysozyme has been approved for use in winemaking by the Office International de la Vigne et du Vin (OIV). The main objectives of this study were to determine whether these substances showed any antimicrobial action against wine-associated microorganisms, namely LAB, AAB and yeasts. The stability and suitability of the bacteriocins and lysozyme as antimicrobials in wine was researched, especially when used in combination. Possible synergistic or antagonistic interactions between the bacteriocins were also investigated by means of the microtitre broth dilution method and scanning electron microscopy, as well as at what concentration and combinations the bacteriocins were most effective against increasing LAB concentrations. It was found that nisin, pediocin and leucocin were effective to varying degrees against a test panel of LAB type and reference strains, as well as wine isolates. Nisin repeatedly had the highest level of inhibition against all the LAB tested, followed by pediocin and leucocin. There was no inhibition of the wine-associated AAB and yeasts tested. Pediocin stability was evaluated in simulated wine must and proved to be stable for at least 20 days, without being affected by the sulphur or alcohol content. A low pH, however, led to a more rapid decrease in activity. The same was found for nisin and leucocin in other studies. Combinations of bacteriocins at increasing concentrations were evaluated against increasing concentrations of a LAB wine isolate. When used in pairs (namely, nisinleucocin, nisin-pediocin and pediocin-Ieucocin), the combinations were most effective against lower concentrations of bacteria, namely 102 and 104 cfu/ml. At lower concentrations, the pairs of bacteriocins were much less effective against the higher bacterial concentrations of 106 and 108 cfu/ml. Leucocin-pediocin combinations were the least effective, while nisin-Ieucocin combinations were marginally more effective than the nisin-pediocin combinations. The most pronounced effect was observed when all three the bacteriocins were used together. Combinations of bacteriocins had no inhibitory effect against AAB. Pediocin and lysozyme was used in combination against the same wine isolate, but no conclusive conclusions could be drawn in this experiment. __ Scanning electron microscopy was used to investigate any disturbances in cell morphology when bacteriocins were added to LAB. The above-mentioned LAB was subjected to bacteriocins used singularly and also in combinations of equal amounts of bacteriocins. The action of the bacteriocins led to major disturbances in cell morphology. Once again, the combination of leucocin-pediocin was the least effective, even less so than when the single bacteriocins were used. The nisin-pediocin and nisin-Ieucocin combinations seemed to be more effective in causing cell disturbances and perturbations. The microtitre broth dilution methodwas used to further characterise the nature of the interaction of the pairs of bacteriocins. This test showed clearly that the bacteriocins had definite interactions. By adding one bacteriocin to varying concentrations of another bacteriocin, the inhibitory action of the second bacteriocin was affected, either increasing or decreasing its effectiveness. The most important factor to consider seems to be the ratio at which the bacteriocins should be used together, leading either to synergism or antagonism, and this also implies a very complex interaction. This project indicated that it is indeed possible to use both bacteriocins and lysozyme in "Vine preservation, both being stable in wine environments and effective against LAB without affecting the yeast fermentation. Bacteriocins could also be used in combination, to broaden the inhibition spectrum, as well as possibly increasing the inhibitory potential of the individual antimicrobials. The underlying interactions in such combinations should be carefully researched, however, when considering using combinations of antimicrobials in food and beverage products. Further attention can also be given to finding biopreservatives against the Gram-negative AAB, as well as to research the interaction of the pairs of bacteriocins over time. Another point to consider would be the engineering of yeasts or bacteria to produce these antibacterial substances in situ as part of their metabolism.
AFRIKAANSE OPSOMMING: Daar is 'n verskeidenheid mikroorganismes in die wynrnaakproses betrokke, elkeen met sy eie rol. Giste is vir die alkoholiese fermentasie verantwoordelik, die Gram-positiewe melksuurbakterieë (MSB) wat in mos en wyn voorkom, is vir die appelmelksuurgisting (AMG) verantwoordelik, terwyl die Gram-negatiewe asynsuurbakterieë (ASB) etanol in asynsuur omskakel. Hierdie mikroorganismes is in die wynkelder en fermentasietenke teenwoordig en kan as óf gunstig óf ongunstig beskou word, afhangende van die toestande waaronder hulle groei en hoe die wyn daardeur beïnvloed word. Om mikrobiologiese stabiliteit in wyn te verseker, moet daar streng higiëniese maatreëls in die kelder toegepas word en word daar ook van addisionele chemiese preserveermiddels gebruik gemaak. Swaweidioksied (S02) word tans algemeen as pnmere preserveermiddel in die wynbedryf gebruik. Weens die moontlike gesondheidsrisiko's wat S02 mag inhou en die moontlike verlaging van die voedingswaarde en sensoriese gehalte waarmee dit in sommige voedsel- en drankprodukte geassosieer word, bou daar tans verbruikersweerstand teen die gebruik daarvan as chemiese preserveermiddelop. Biopreservering is 'n alternatief tot hedendaagse chemiese preservering en het reeds baie belangstelling ontlok. Hierdie metode impliseer die gebruik van die natuurlike mikroflora en/of die antimikrobiese produkte van hierdie rnikroërqanisrnes, soos bakteriosiene en bakteriolitiese ensieme (bv. lisosiem). Bakteriosiene van MSB is proteïene of proteïenkomplekse met antimikrobiese aktiwiteit teen naby-verwante Grampositiewe spesies. Lisosiem kom in produkte soos hoendereierwit voor en het litiese aktiwiteit teen Gram-positiewe bakterieë. Die bakteriosiene nisien, wat tot die klas I lantibiotiese bakteriosiene behoort, en pediosien PA-1 en leukosien B-TA11a, wat tot die klas lIa Listeria-aktiewe bakteriosiene behoort, is as moontlike biopreserveringsagense in wyn ondersoek. Nisien is egter tot op hede die enigste bakteriosien wat amptelik vir gebruik as 'n preserveermiddel in voedsel goedgekeur is, terwyl pediosien moontlik sal volg. Lisosiem is vir gebruik in wynmaak deur die Office International de la Vigne et du Vin (OIV) goedgekeur. Die hoofdoelwitte van hierdie studie was om te bepaal of die bogenoemde stowwe antimikrobiese werking teen wyngeassosieerde mikroorganismes het, soos die ongewenste MSB, ASB en giste. Die stabiliteit en geskiktheid van dié bakteriosiene en lisosiem as antimikrobiese middels in wyn is ook ondersoek, veral wanneer hulle in kombinasie vir preservering gebruik is. 'n Mikrotiterverdunningsboeljon-metode en skanderingselektronmikroskopie is gebruik om moontlike sinergistiese en antagonistiese interaksies tussen bogenoemde bakteriosienpare te ondersoek. Terselfdertyd is die effektiefste konsentrasies en kombinasies van bakteriosiene teen stygende MSB-getalle bepaal. Daar is bevind dat nisien, pediosien en leukosien in verskillende mates teen 'n toetspaneel van MSB tipe- en verwysingsrasse, asook MSB-wynisolate, effektief is. Nisien was herhaaldelik die effektiefste teen dié MSB, gevolg deur pediosien en dan leukosien. Die bakteriosiene was nie teen die wyngeassosieerde ASB of giste wat getoets is, effektief nie. Daar is ook bewys dat pediosien vir tot 20 dae stabiel in 'n gesimuleerde wynomgewing was, sonder dat die alkohol- of die swaweldioksiedkonsentrasie 'n invloed op die aktiwiteit gehad het nie. 'n Lae pH het geblyk om die grootste invloed op die afname in aktiwiteit te hê. Hierdie bevindinge ten opsigte van pediosien het die resultate van nisien en leukosien in ander, soortgelyke ondersoeke, bevestig. Die werking van toenemende konsentrasies van bakteriosienkombinasies (as pare van nisien-Ieukosien, nisien-pediosien, leukosien-pediosien, en al drie saam as nisienpediosien- Ieukosien) teen toenemende getalle van In wyngeïsoleerde MSB is geëvalueer. Wanneer die bakteriosiene in pare gebruik is, was die kombinasies die effektiefste teen laer MSB selgetalle (102 en 104 kfe/ml), terwyl dit baie minder effektief teen hoër selgetalle (106 en 108 kfe/ml) was, veral wanneer lae bakteriosienkonsentrasies gebruik is. Die nisien-Ieukosien kombinasiewas tot 'n geringe mate meer effektief as die nisien-pediosien kombinasie. Die leukosien-pediosien kombinasie het die laagste effektiwiteit van.al die pare bakteriosiene wat gebruik is, getoon. Die sterkste werking is waargeneem toe al drie die bakteriosiene saam teen bogenoemde MSB gebruik is. Die bakteriosien kombinasies het geen effek teen ASB gehad nie. Pediosien en lisosiem is ook in kombinasie teen dieselfde wynisolaat gebruik, maar geen oortuigende afleidings kon van hierdie eksperiment gemaak word nie. Skanderingselektronmikroskopie is gebruik om enige morfologiese verandering in die MSB-wynisolaat waar te neem wanneer bakteriosiene daarby gevoeg is. Dieselfde wynisolaat is weer gebruik en bakteriosiene is by die bakterieë gevoeg, enkelvoudig asook in kombinasies (soos voorheen gebruik) teen gelyke hoeveelhede. Die werking van die bakteriosien het gelei na merkbare veranderinge in selmorfologie, en die kombinasie van pediosien-Ieukosien was weereens die minste effektief. Die mikrotiterverdunningsboeljon-metode is gebruik om die aard van die bakteriosieninteraksies verder te karakteriseer. Die toetse het duidelik aangedui dat die bakteriosiene op mekaar reageer. Deur een bakteriosien tot variërende konsentrasies van 'n ander bakteriosien te voeg, is die inhibitoriese werking van die tweede bakteriosien geaffekteer deurdat die effektiwiteit daarvan toegeneem of afgeneem het. Dit het ook geblyk dat die belangrikste faktor wat hier in ag geneem moet word die verhouding is waarteen die bakteriosiene met mekaar gebruik word, aangesien dit tot óf sinergisme óf antagonisme kan lei. Dft dui op 'n baie komplekse interaksie. Die resultate van hierdie projek het dus daarop gedui dat dit inderdaad moontlik is om beide bakteriosien en lisosiem in wynpreservering te gebruik, aangesien beide nie net stabiel in 'n wynomgewing is nie, maar ook effektief is teen MSB sonder dat die gisfermentasies geaffekteer word. Bakteriosiene kan ook in kombinasie gebruik word om die inhibisie spektrum te verbreed, en om ook moontlik die inhibisiepotensiaal van die individuele peptiede te verhoog. Onderliggende interaksies by sulke kombinasies moet egter sorgvuldig ondersoek word wanneer daar oorweeg word om kombinasies van hierdie antimikrobiese middels in voedsel- en drankprodukte te gebruik. Verder moet daar ook aandag geskenk word om biopreserveermiddels te vind wat ook teen die Gram-negatiewe ASB effektief is, asook aan die aard van die verloop van interaksies van pare van bakteriosiene oor tyd. Nog 'n punt om te oorweeg is die manipulasie van giste of bakterieë omdie antimikrobiese peptiede in situ, as deel van hulle metabolisme, te produseer.

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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

A aplicação das bacteriocinas de bactérias láticas como agentes de bioconservação de alimentos é dependente de vários fatores, com destaque para composição química do alimento que pode interferir na eficácia da atividade antimicrobiana. Esse estudo objetivou avaliar a influência de ingredientes da matriz alimentar na multiplicação e produção das bacteriocinas por Lactobacillus sakei subsp. sakei 2a e na atividade das bacteriocinas produzidas, trabalhando-se com um modelo composto de extrato de carne, extrato de levedura, proteose peptona e amido, adicionado de 4% de NaCl e/ou 0,5% de glicose e mantido em refrigeração (4° C) por até 10 dias. O estudo foi realizado com os seguintes materiais contendo as bacteriocinas: 1. sobrenadante concentrado livre de células obtido a partir da cultura de L. sakei 2a em caldo MRS; 2. extrato ácido neutralizado obtido do sedimento da cultura em caldo MRS e 3. extrato ácido neutralizado e semi-purificado por cromatografia líquida. Listeria monocytogenes Scott A foi empregada como microrganismo indicador da atividade antimicrobiana e Lactobacillus sakei ATCC 15521 como controle não produtor de bacteriocinas. Os resultados indicaram que nenhum dos ingredientes do modelo avaliado teve papel importante na multiplicação de L. sakei 2a e na bacteriocinogênese. No entanto, 4% de NaCl interferiu negativamente na produção das bacteriocinas, enquanto 0,5% de glicose auxiliou a produção, mesmo na presença de 4% de NaCl. Em relação à atividade das bacteriocinas, verificou-se que nem os ingredientes do modelo, nem 4% de NaCl e nem 0,5% de glicose apresentaram interferência marcante ao longo dos dez dias estudados, independentemente do material contendo as bacteriocinas testado.
The application of bacteriocins produced by lactic acid bacteria as biopreservatives in foods depends of several factors, such as the chemical composition of the food, which can interfere with the antimicrobial activity. This study aimed to evaluate the influence of food ingredients on the growth and production of bacteriocins by Lactobacillus sakei subsp. sakei 2a, and on the activity of the bacteriocins produced, by means of a food model prepared with meat extract, yeast extract, proteose-peptone and starch, added of 4% 4% de NaCl and/or glucose, maintained under refrigeration (4° C) for up to 10 days. The study was carried out with the following bacteriocins-containing materials: 1. the concentrated cell-free supernatant from an overnight culture of L.sakei 2a in MRS broth; 2. acid extract obtained from the sediment of the overnight culture of L. sakei 2a in MRS broth and 3. acid extract submitted to a semi-purification by HPLC. Listeria monocytogenes Scott A was used as the antimicrobial activity indicator microorganism and Lactobacillus sakei ATCC15521 as the non-bacteriocinogenic negative control. Results indicated that none of the ingredients presented a significant role in the growth of L.sakei 2a, and in the production and activity of the bacteriocins. However, 4% of NaCl caused a reduction on the production of bacteriocins, while 0,5% of glucose stimulated their production, even in the presence of 4% of NaCl. The interference of the ingredients of the model, 4% of NaCl and/or 0,5% of glucose on the activity of the bacteriocins was not marked, regardless the materials used in the tests.

Orientador: Saulo Santesso Garrido
Banca: Saulo Santesso Garrido
Banca: Daniela Cardoso Umbelino Cavallini
Banca: Wilton Rogério Lustri
Resumo: Devido ao crescente aumento de doenças transmitidas por alimentos, a segurança microbiológica se torna uma questão de saúde pública pelas suas características de endemicidade, alta morbidade e pela dificuldade da adoção de medidas de controle desses microrganismos. Diante deste fato, o objetivo deste trabalho foi sintetizar e caracterizar os análogos peptídicos LeuB e LeuC-1 derivados de bacteriocinas naturais denominadas Leucocinas. Os peptídeos foram sintetizados manualmente pelo método de síntese em fase sólida, submetidos à desproteção total e clivagem, com liberação dos peptídeos brutos. Foram realizadas as análises comparativas usando HPLC e ESI-MS, e com os respectivos peptídeos puros foram feitos os ensaios antimicrobiano, enzimático, permeabilização, antioxidante, hemolítico e de espectroscopia de dicroísmo circular. Com isso, observou-se que o método de síntese dos análogos foi adequado e o processo de purificação possibilitou a obtenção dos peptídeos com alto grau de pureza. O peso molecular teórico dos peptídeos foi confirmado por espectrometria de massas. O LeuB apresentou uma maior capacidade em inibir o crescimento de Escherichia coli O157:H7 e em Salmonella sorovar Typhimurium, enquanto que LeuC-1 apresentou efeito de inibição de crescimento de Listeria monocytogenes e também de S. sorovar Typhimurium. É importante destacar que todas essas bactérias são de interesse na área de alimentos, já que são as causadoras da maioria dos casos de infecção alimentar. O en... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Due to the increase of foodborne diseases, microbiological safety becomes a public health issue due to its characteristics of endemicity, high morbidity and the difficulty of adopting control measures of these microorganisms. In view of this fact, the objective of this work was to synthesize and characterize LeuB and LeuC-1 peptidics analogues derived from natural bacteriocins called Leucocins. The peptides were synthesized manually by the solid phase synthesis method, subjected to total deprotection and cleavage, with release of the crude peptides. Comparative analyzes were performed using HPLC and ESI-MS, and with the respective pure peptides the antimicrobial, enzymatic, permeabilization, antioxidant, hemolytic and circular dichroism spectroscopy. With this, it was observed that the method of synthesis of the analogs was adequate and the purification process allowed to obtain the peptides with high purity. The theoretical molecular weight of the peptides was confirmed by mass spectrometry. LeuB showed a greater capacity to inhibit the growth of Escherichia coli O157: H7 and Salmonella serovar Typhimurium, whereas LeuC-1 presented inhibition effect of growth of Listeria monocytogenes and also of S. serovar Typhimurium. It is important to highlight that all these bacteria are interesting in the area of food, since they are the cause of most cases of food infection. The enzyme inhibition assay with DNA gyrase and Topoisomerase IV showed that only the LeuB peptide has the ability to inhibit these bacterial enzymes, suggesting a possible mechanism of action of this Leucocin derivative. This peptide also showed the ability to permeabilize bacterial membrane mimetics composed of POPC/POPG (75/25). On the other hand, the LeuC- 1 peptide did not present significant capacity to inhibit the activity of the enzymes DNA gyrase and Topoisomerase IV and also did not present... (Full abstract, click on electronic access below)
Mestre

Dysgalacticin is a large, 21.5 kDa bacteriocin that belongs to subgroup B of the class III bacteriocins. It is ribosomally produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 and exerts inhibitory activity mainly against the medically important pathogen Streptococcus pyogenes by a "non-lytic" mechanism. Despite numerous studies of the mechanisms of action of a wide variety of bacteriocins and of the basis of their producer strain self-immunity, relatively little is known about the "non-lytic" class of bacteriocins. The structural gene encoding for dysgalacticin (dysA) was known to be carried on a small, rolling circle plasmid pW2580 (3.04 kb) (Heng et al., 2006). However, the dysgalacticin immunity gene (dysI) had not been identified prior to the present study. The aims of this research were to elucidate the mechanism of action of dysgalacticin against S. pyogenes and to identify the genetic basis and the mechanism of producer strain self-immunity. Recombinantly-produced dysgalacticin was used to determine the mode of action against S. pyogenes. Dysgalacticin was bactericidal for S. pyogenes, increasing the permeability of the cytoplasmic membrane and ultimately leading to leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [�⁴C]serine uptake, a membrane potential-dependent process in S. pyogenes. Interestingly, dysgalacticin inhibited glucose fermentation by non-growing cell suspensions and blocked transport of both glucose and the nonmetabolisable analogue 2-deoxyglucose. This finding indicates that dysgalacticin may target the phosphophenolpyruvate (PEP)-dependent glucose and mannose phosphotransferase system (PTS) of S. pyogenes. Taken together, these data suggest that dysgalacticin targets the glucose-PTS and/or mannose-PTS as a receptor, leading to inhibition of sugar uptake, and a subsequent dissipation of the membrane potential leading to cell death. Complementation studies demonstrated that dysI is located on pW2580. RNA analysis showed that dysI is co-transcribed with genes encoding for the plasmid copy control protein, copG and replication initiation protein, repB. S. pyogenes transformed with a plasmid containing dysI displayed a markedly higher dysgalacticin MIC (1024 nM) than the corresponding dysgalacticin-sensitive, plasmid-negative strain (8 nM). Further studies of this DysI-expressing S. pyogenes showed that membrane integrity, glucose fermentation and [�H]2DG uptake were not affected by dysgalacticin treatment. These findings are consistant with a mechanism whereby the immunity peptide binds to the target-binding site of dysgalacticin, effectively blocking access by the bacteriocin. H₆DysI was found to localise to the cytoplasmic membrane, further indicating that DysI may bind to the proposed target of dysgalacticin, i.e., the membrane-bound glucose-PTS and mannose-PTS. Thus both the mode of action and the producer strain self-immunity of dysgalacticin are likely to be cytoplasmic-membrane based. Homology searching revealed that the bacteriocin SA-M7 produced by M-type 57 S. pyogenes has structural similarities to dysgalacticin, as do two hypothetical proteins, EF1097 and YpkK, of Enterococcus faecalis and Corynebacterium jeikeium, respectively (Heng et al., 2004, 2006). These proteins were all predicted to contain relatively unstructured N-termini and helix-loop-helix structured C-termini. In each case the C-termini contain two conserved cysteine residues that are predicted to form a disulphide bridge. Heterologous expression of SA-M57, EF1097 and YpkK in Escherchia coli demonstrated that all three proteins have antimicrobial activity, but of differeing activity spectra. Reductive-alkylation of SA-M57, EF1097 and YpkK confirmed that their predicted disulphide bonds were essential for biological activity. These proteins were later renamed streptococcin A-M57, enterococcin V583 and corynicin JK respectively. The outcome of preliminary domain-swapping experiments supported the existence of functional domain-type segments in streptococcin A-M57, enterococcin V583, corynicin JK and dysgalacticin. The N-terminal domain of each of these proteins and also the C-terminal domain of corynicin JK were successfully expressed in E. coli. The failure to express the C-termini of the remaining proteins was thought possibly due to toxicity of thses pepetides for the E. coli host. Nevertheless, the C-terminus of corynicin JK displayed an inhibitory spectrum apparently identical to that of the full-length corynicin, indicating that the N-terminus may not always be required for target binding of this class of antimicrobials. Preliminary mode of action studies revealed that streptococcin A-M57, enterococcin V583 and corynicin JK all resemble dysgalacticin in that they exert inhibitory activity by non-lytic means. These results, in combination with the protein structural predictions indicate that dysgalacticin, streptococcin A-M57, enterococcin V583 and corynicin JK are all members of the same basic class of "non-lytic" bactericoicns.

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.

O aumento na demanda por alimentos saudáveis e minimamente processados impulsiona a busca por novos agentes antimicrobianos. As bacteriocinas são peptídeos produzidos via ribossomo por algumas espécies de bactérias, podendo ser usadas na conservação e garantia da inocuidade de alimentos, não apresentando as possíveis ações tóxicas de conservadores clássicos amplamente utilizados na indústria alimentícia. Carnobacterium maltaromaticum C2 foi isolado de peixe defumado brasileiro (Surubim, Pseudoplatystoma sp.), e apresenta grande capacidade de inibir a multiplicação de Listeria monocytogenes, demonstrando seu potencial para aplicação na bioconservação de alimentos. Em estudos anteriores, foi demonstrado que essa linhagem bacteriana produz compostos antimicrobianos de origem proteica. Neste trabalho, foram avaliados aspectos gerais da produção de bacteriocinas por C. maltaromaticum C2, assim como sua purificação e caracterização. C. maltaromaticum C2 produz bacteriocinas entre 5 e 25ºC, com ótimo entre 20 e 25ºC. Do mesmo modo, a produção desses compostos foi maior em caldo APT (All purpose Tween), entretanto para as etapas de purificação foram utilizados o caldo BHI (Brain heart infusion) e CAA (Casamino acids), por causarem menos interferência no processo. Lactobacillus sakei e L. monocytogenes foram inibidos pelas bacteriocinas parcialmente purificadas produzidas por C. maltaromaticum C2, e seus peptídeos antimicrobianos apresentaram moderada estabilidade térmica quando expostos a 100ºC por 30 minutos. Foram utilizadas duas técnicas para extração e purificação das bacteriocinas, a técnica de adsorção-dessorção às células produtoras, e a purificação com a resina XAD-16, baseada em interações hidrofílicas e hidrofóbicas com os peptídeos, seguida de extração em fase sólida, sendo que este último processo de purificação resultou em um extrato com alto teor de pureza, como observado durante as análises por cromatografia líquida de alta eficiência em coluna de fase reversa. Com o auxílio de técnicas de espectrometria de massas, foi detectado nos extratos obtidos a presença das carnobacteriocinas BM1 e B1, assim como o peptídeo antimicrobiano CbnX. Este trabalho é pioneiro na purificação de CbnX, pois anteriormente havia somente a descrição de seu gene, mas não havia sido descrita a purificação do peptídeo. Neste sentido, a linhagem estudada é única até o momento e poderá favorecer estudos de expressão gênica de bacteriocinas, bem como a otimização de processos de bioconservação.
The high demand for healthy and minimally processed foods has increased the search for new antimicrobial agents. Bacteriocins are ribosomally synthesized peptides produced by some bacteria, and are useful for biopreservation and food safety, without the possible toxic effects of classical preservatives widely used in food industry. Carnobacterium maltaromaticum C2 was isolated from Brazilian smoked fish (Surubim, Pseudoplatystoma sp.), and it inhibits Listeria monocytogenes, an important foodborne pathogen. In previous studies, it was demonstrated that this bacterial strain produces bacteriocins. In this study, general aspects of the production of bacteriocins by C. maltaromaticum C2 were evaluated, as well as their purification and characterization. C. maltaromaticum C2 produces bacteriocins between 5 and 25ºC, with the optimum incubation temperature between 20 and 25ºC. Similarly, the production of these compounds was higher in APT (All-purpose Tween) broth. However, for the purification steps, BHI (Brain heart infusion) broth and CAA (Casamino acids) broth were used due to their low interference with the processes. Lactobacillus sakei and L. monocytogenes were inhibited by the partially purified bacteriocins produced C. maltaromaticum C2, and their antimicrobial peptides showed moderate thermal stability when tested at 100ºC by 30 minutes. Two techniques for extraction and purification of the antimicrobial peptides were used, the adsorption-desorption of bacteriocins to the producer cells, and the purification with XAD-16 resin, based on hydrophilic and hydrophobic interactions with the peptides, followed by a step of solid phase extraction. The latter resulted in an extract with high purity, as observed by the analysis with reverse-phase high performance liquid chromatography. With mass spectrometry techniques, carnobacteriocins BM1 and B1 were detected, as well as the antimicrobial peptide CbnX. This is an innovative work because the purification of CbnX had never been reported, except its gene. In this respect, this C. maltaromaticum strain is unique until this moment, and may promote researches on gene expression, as well the optimization of biopreservation processes.

Além do aspecto nutricional de suma importância, é notória a contribuição do leite humano para o processo de desenvolvimento da microbiota intestinal do recémnascido, um importante mecanismo de defesa do organismo contra doenças infecciosas. O papel do leite humano como fonte de bactérias probióticas, principais constituintes da microbiota intestinal, tem sido tópico de pesquisas recentes. Este trabalho foi desenvolvido com o objetivo de determinar e comparar a composição da microbiota de oito amostras de leite humano e verificar o potencial de utilização desse produto como fonte de bactérias probióticas. Para tanto, utilizaram-se cinco meios de cultivos seletivos para contagem presuntiva de gêneros normalmente encontrados em leite humano: lactococos, enterococos, bifidobactérias e propionibactérias. A análise quantitativa da microbiota demonstrou tendência de diminuição da contagem em função do aumento do tempo de lactação. A análise qualitativa confirmou a presença de distintos gêneros de bactérias lácticas potencialmente probióticas com algumas variações entre as amostras de leite humano. Na segunda etapa 800 colônias isoladas a partir dos cinco meios de cultivos e caracterizadas como bactérias lácticas foram selecionadas quanto às suas propriedades probióticas (produção de bacteriocina, tolerância à acidez e a sais biliares, resistência à antibióticos, capacidade de adesão a chapas de aço inoxidável) e tecnológicas (capacidade de crescimento e sobrevivência em leite). Verificou-se que apenas 15 (1,9%) linhagens produziram bacteriocinas com atividade contra Listeria innocua L11 e Micrococcus luteus ATCC®4698, linhagens utilizadas como indicadoras, por meio do método de antagonismo simultâneo em poços, usando ágar MRS. Treze dessas linhagens também apresentaram atividade contra Bacillus cereus CTC 011, Listeria monocytogenes ATCC®7644, Lactococcus lactis subsp. lactis CTC 204 e Lactobacillus helveticus ATCC®15009. As duas linhagens remanescentes demonstraram atividade principalmente contra Listeria monocytogenes ATCC®7644. Nenhuma das quinze culturas produtoras de bacteriocinas apresentou atividade contra as bactérias Gram-negativas Escherichia coli ATCC® 2074 e Salmonella thyphimuirim ATCC® 2364. Por outro lado, Staphylococcus aureus ATCC® 1602 foi resistente as quinze bacteriocinas selecionadas neste trabalho. Seis linhagens de bactérias lácticas (BALs) foram selecionadas para avaliação das demais propriedades probióticas. Observou-se que uma dessas linhagens diferenciou-se por apresentar sobrevivência a pH 2,0 e a pH 3,0, enquanto as demais mostraram tolerância apenas a pH 3,0. Todas as linhagens selecionadas apresentaram a capacidade de tolerância a 0,3% de sais biliares, de se aderir à superfície de aço inoxidável e de resistência à clindamicina, eritromicina e gentamicina. Quanto às propriedades tecnológicas, todas as seis linhagens apresentaram capacidade de crescimento em leite e não produziram odor desagradável ou pós-acidificação do leite fermentado durante a estocagem a 4ºC por 28 dias. Notou-se, entretanto, diminuição, de, aproximadamente, 2,0 Log UFC.mL-1, na contagem de células viáveis ao final do período de estocagem. Finalmente, por meio da avaliação dos perfis de fermentação de carboidratos e de outras reações bioquímicas, duas das linhagens isoladas de leite humano foram identificadas como Enterococcus durans e quatro como Enterococcus avium. Os 12 resultados permitem concluir que o leite humano é fonte potencial de bactérias com potencial probiótico para aplicação industrial.
In addition to the nutritional aspect of paramount importance, it is clear the contribution of human milk for the development process of the intestinal tract of the newborn, an important mechanism of defense against infectious diseases. The role of human milk as a source of probiotic bacteria, major constituents of the intestinal microbiota, has been the topic of recent research. This work was carried out to determine and compare the composition of the microbiota of eight human milk samples and verify the potential use of this product as a source of probiotic bacteria. For this purpose, we used five selective culture media for counts of presumptive genera commonly found in human milk: lactococcal, enterococci, bifidobacteria and propionibactérias. The quantitative analysis of microbes showed a trend of decreasing counts as a function of time increased lactation. The qualitative analysis confirmed the presence of different kinds of potentially probiotic lactic acid bacteria with some variations between samples of human milk. In the second stage 800 colonies isolated from the five culture media and characterized as lactic acid bacteria were selected for their probiotic properties (bacteriocin production, tolerance to acid and bile salts, antibiotic resistance, adhesion to stainless steel plates) and technology (ability to grow and survive in milk). It was found that only 15 (1.9%) produced bacteriocin strains with activity against Listeria innocua L11 and Micrococcus luteus ATCC 4698®, strains used as an indicator, by the method of antagonism wells simultaneously, using MRS agar. Thirteen of these strains also showed activity against Bacillus cereus CTC 011, Listeria monocytogenes ATCC® 7644, Lactococcus lactis subsp. CTC 204 lactis and Lactobacillus helveticus ATCC 15009®. The two remaining strains showed activity mainly against Listeria monocytogenes ATCC 7644®. None of the fifteen cultures producing bacteriocins active against Gram-negative Escherichia coli ATCC 2074® and Salmonella thyphimuirim ATCC 2364®. Moreover, Staphylococcus aureus ATCC 1602® was resistant the fifteen bacteriocins selected in this work. Six strains of lactic acid bacteria (BALs) were selected for analysis of other probiotic properties. It was observed that one of these strains distinguished by presenting survival at pH 2.0 and pH 3.0 while the other showed only tolerance to pH 3.0. All strains were selected for the ability tolerance of 0.3% bile salts, of adhering to stainless steel surface and resistance to clindamycin, erythromycin and gentamycin. The technological properties, all six strains were capable of growth in milk and produced no unpleasant odor or post-acidification of the fermented milk during storage at 4°C for 28 days. It was noted, however, decreased from approximately 2,0 Log UFC.mL-1 in the viable cell count at the end of storage period. Finally, by evaluating the profiles of fermentation of carbohydrates and other biochemical reactions, two of the strains isolated from human milk have been identified as Enterococcus durans and Enterococcus avium as four. The results indicate that human milk is a potential source of probiotic bacteria with potential for industrial application.

ABSTRACT CHARACTERIZATION AND IDENTIFICATION OF BACTERIOCINS FROM TWO LACTOCOCCUS LACTIS SUBSP. LACTIS STRAINS Akç
elik, Oya M.S., Department of Biotechnology In this study, bacteriocins from two L. lactis subsp. lactis isolates of Turkey origin designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after &
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-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of partially purified bacteriocins were determined by SDS-polyacrylamide gel electrophoresis. PCR amplification was carried out with different primers for the detection of structural genes of lactococcal bacteriocins. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Association of the bacteriocin production with plasmid DNA was examined by using acriflavine as a plasmid curing agent. Plasmid profiles of the wild type and its non-bacteriocin producing mutants were determined by using the alkali lysis method followed by agarose gel electrophoresis. The genetic nature of industrially important characteristics of Lactococcus lactis strains were investigated through gene transfer studies via conjugation. According to the results of plasmid curing and conjugal transfer trials, it was concluded that in Lactococcus lactis subsp. lactis OC1 strain a 39,7 kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity. In Lactococcus lactis subsp. lactis OC2 strain, on the other hand, a 16 kb plasmid appeared to be responsible for lacticin 481 production and lactose fermentation.

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.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to its aroma and flavour. However, they can also be responsible for many wine spoilage problems that compromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics to the sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect the wholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethyl carbamate. Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilage micro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains the benefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand to reduce SO2 levels due to the increasing health related risks and other factors. All these considerations have increased the interest in research to look for new preservation strategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in the last decade. Various types of bacteriocins have been identified and characterized. However, there are few reports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finished wine. The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocin production. The isolates originated from South African red wines undergoing spontenous malolactic fermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibition zones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum of antimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important in winemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocins they produce could possibly find application in the food fermentation industry. The physiological results, biochemical tests and sugar fermentation profiles all gave the same results for the seven isolates, which were indicative of enterococci. The identification through 16S rRNA gene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCR fingerprinting gave the same profile for the seven strains confirming that they were all identical on genetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below 4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search results confirmed that all eight strains contained the enterocin P gene from Ent. faecium. The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at 121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enable enterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococci producing bacteriocins have potential applications in various foods and fermented products. The pH tests showed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH range make them potentially more suitable as natural preservatives of foods and fermented products where products are acidified or pH decreases due to natural LAB present. They also have potential applications in oenological process where pH levels are as low as 3 and 4. Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibit enterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydrate moieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicated that the compound was proteinaceous in nature. Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with the exponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication of primary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours and was maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell free supernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314 was clearly demonstrated by complete inhibition of growth for most of the experimental period, while the control increased exponentially throughout the experiment. In conclusion, this study has confirmed the isolation and identification of Ent. faecium strains from wine, a genus that is rarely found in the wine environment. Although one can speculate on the origin of this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolates produced enterocin P which have antimicrobial action against wine-related LAB genera and therefore have a potential role in wine spoilage control.
AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) speel ‘n belangrike rol in die redusering van die suurgehalte van wyn en dra ook by tot die aroma en smaak daarvan. Hulle kan egter ook verantwoordelik wees vir vele wynbederfprobleme wat die gehalte en waarde van wyn negatief beïnvloed. Hoewel Oenococcus oeni positiewe karaktertrekke aan die sensoriese eienskappe van wyn verleen, kan sekere spesies van die genus, Lactobacillus en Pediococcus, die heilsaamheid van wyn beïnvloed deur ongewenste verbindings, soos biogeniese amienes en etielkarbamaat, te produseer. Chemiese preserveermiddels, soos swaweldioksied (SO₂), word gebruik om die groei van bederfmikro-organismes tydens die wynbereidingsproses te voorkom. SO₂ fungeer ook as ‘n reduseermiddel en onderhou die voordele van die antioksidant eienskappe van die poli-fenole van wyn. Daar is egter ‘n wêreldwye vraag na die redusering van SO₂-vlakke as gevolg van die toename in gesondheidsverwante risiko’s en ander faktore. Al hierdie oorwegings het belangstelling in die navorsing van nuwe preserveringstrategieë laat toeneem en MSB-geproduseerde bakteriosiene lyk na ‘n potensiële alternatief wat in die laaste dekade ondersoek word. Verskeie tipes bakteriosiene is geïdentifiseer en getipeer. Daar is egter nog weinig gerapporteer oor bakteriosiene wat deur MSB van wynkundige oorsprong geproduseer is of oor bakteriosiene wat in afgeronde wyn teenwoordig is. Die huidige studie het 155 MSB isolate van die Instituut vir Wynbiotegnologie se kultuurversameling vir bakteriosien-produsering gegradeer. Agt stamme (5%) is as produseerders geïdentifiseer, soos gestaaf is deur sterk inhibisiesones wat teen sensitiewe organismes op agarplate gevorm het. Die produseerders het ‘n breë spektrum van antimikrobiese aktiwiteit by inhiberende Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes en Pediococcus pentosaceus stamme gedemonstreer. Sommige van hierdie bakteriese genera is belangrik in wynbereiding, omdat dit potensiële wynbederfbakterieë is. Hierdie isolate en/of die bakteriosiene wat dit produseer, kan dus moontlik toepassing in die voedselfermentasiebedryf vind. Die fisiologiese resultate, biochemiese toetse en suikerfermentasieprofiele het almal dieselfde resultate vir die sewe isolate, wat indikatief van enterococci was, gelewer. Die identifisering deur 16S rRNA-basispaaropeenvolging het onthul dat die sewe getoetste isolate almal Enterococccus faecium was. RAPD-PKR-vingerafdrukke het dieselfde profiel vir die sewe rasse gelewer, wat bevestig dat die rasse almal identies op genetiese vlak was. Deur die molekulêre gewig vas te stel deur middel van SDSPAGE, het dit getoon dat die peptiede kleiner as 4.6 kDa in grootte is. PKR-amplifikasie van die enterosien-P geen, die bepaling van basispaaropeenvolging en BLAST-soekresultate het bevestig dat al agt rasse die enterosien-Pgeen van Ent. faecium bevat. Die enterosien wat in hierdie studie getoets is, was hitte-stabiel teen 100°C (30 min), maar het 50% van sy aktiwiteit teen 121°C (15 min) verloor. Faktore soos bakteriosienproduksie en hittebestandheid, is van die vele faktore wat enterococci in staat stel om dominant in gefermenteerde produkte, soos suiwelprodukte of vleis te wees. Enterococci wat bakteriosiene produseer het dus potensiële toepassings in verskeie kossoorte en gefermenteerde produkte. Die pH-toetse het getoon dat enterosien-P oor ‘n breë pH spektrum (2-10) aktief was. Enterosienaktiwiteit oor ‘n wye pH spektrum maak dit potensieel meer geskik as natuurlike preserveermiddels vir kossoorte en gefermenteerde produkte waar produkte versuur word of die pH afneem as gevolg van natuurlike MSB wat teenwoordig is. Dit het ook potensiële toepassings in enologiese prosessering waar pH-vlakke so laag as 3 en 4 is. Proteolitiese ensiembehandelings met lisosiem, lipase, litikase en katalase kon nie enterosienaktiwiteit inhibeer nie. Daar is getoon dat hul antimikrobiese aktiwiteit onafhanklik was van lipiede, koolhidraatdele óf waterstofperoksied. α-Chymotripsien en proteïenase-K het enterosien onaktief gemaak, wat getoon het dat die samestelling proteïenagtig van nature is. Bakteriosienproduksie wat in twee van die stamme #16.3 en 128.1 getoets is, het ooreengestem met die eksponensiële groeifase wat na 6 ure van inkubasie teen 30°C plaasgevind het, en wat ‘n aanduiding is van primêre metabolitiese kinetika. Die hoogste produksie van 400 AU/ml is na agt ure waargeneem en is vir etlike ure (46 uur) in die stasionêre fase gehandhaaf. Die bakterie-dodende effek van die selvrye supernatant van #16.3 en 128.1 teenoor die sensitiewe kultuur van Lactobacillus pentosus DSM 20314 is duidelik gedemonstreer deur totale inhibisie van groei vir die grootste deel van die eksperimentele periode, terwyl die kontrole eksponensieel deur die hele eksperiment toegeneem het. Hierdie studie het dus die isolering en identifisering van Ent. faecium-stamme, ‘n genus wat baie selde gevind word in ‘n wynomgewing, vanuit wyn bevestig. Alhoewel daar gespekuleer kan word oor die oorsprong van hierdie bakterie in wyn bv. menslike hantering en besmette water, het hierdie rasse wel enterosien geproduseer en daarom die potensiaal om ‘n rol te speel in beheer teen verskeie bederf-MSB-genera.
TIA, NRF and THRIP

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007.
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine. The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation. The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni. The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected. The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours. The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed. This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.

Enterococcus spp. pertencem ao grupo das bactérias láticas e estão presentes em solos, águas, plantas, microbiota autóctone de vários alimentos e como membros da microbiota intestinal de humanos e animais. Esses microrganismos foram considerados por muito tempo como comensais, mas o aumento da severidade das infecções nosocomiais causadas por enterococos mutirresistentes a antimicrobianos e, a falta de conhecimento sobre seus fatores de virulência geram insegurança na utilização de cepas deste gênero na produção de alimentos como culturas fermentadoras e/ou probióticas. A diferença entre uma cepa de enterococos com potencial patogênico e outra aparentemente segura para uso em processamento de alimentos não é clara, e a probabilidade de que esta última adquira fatores de virulência merece investigação. O objetivo do presente projeto foi determinar características fenotípicas e genotípicas de Enterococcus spp. isolados de amostras de alimentos e águas correlacionando sua presença com indicadores clássicos de higiene e contaminação fecal. De 812 colônias indicativas do gênero enterococos obtidas a partir de 120 amostras de alimentos, 299 isolados (37%) foram presuntivamente caracterizados como Enterococcus spp. Após identificação por PCR, 139 (46,5%) E. faecium, 80 (26,8%) E. faecalis, 36 (12%) E. casseliflavus e 8 (2,7%) E. gallinarum. Produção de gelatinase foi detectada apenas em isolados de E. faecalis (60%). Um isolado de E. faecium (0,7%) e 31 isolados de E. faecalis (38,7%) apresentaram perfil β-hemolítico. Produção de bacteriocina contra Lactobacillus sakei e/ou Listeria monocytogenes foi observada para 10% dos isolados de E. faecalis e 23% dos isolados de E. faecium. Hidrólise de sais biliares foi observada para 100% dos isolados de E. gallinarum, 86% E. casseliflavus, 65% E. faecalis e 62,6% de E. faecium. Alguns isolados de E. faecium apresentaram resistência à vancomicina, eritromicina e tetraciclina. Entre os isolados de E. faecalis não houve resistência à vancomicina, mas foi observada resistência à tetraciclina, eritromicina e alta concentração de gentamicina. Houve uma maior prevalência dos genes de virulência (esp, gel, ace, as, efaA e cylA) entre os isolados de E. faecalis quando comparado a E. faecium. Além disso, os isolados de E. faecalis, resistentes a antibióticos, mostraram forte adesão a células Caco-2 e capacidade de formação de biofilme em superfície abiótica. RAPD-PCR individualizou 14 cepas de E. faecium e 17 cepas de E. faecalis dentre os 52 isolados Enterococcus spp. resistentes a antibióticos. A variabilidade dos resultados impediu o estabelecimento de uma correlação entre a presença ou contagem de coliformes, E. coli e enterococos nas amostras analisadas. Os dados deste trabalho sobre marcadores fenotípicos e genotípicos de virulência, e a presença de cepas resistentes a antibióticos evidenciam a necessidade da avaliação cuidadosa de linhagens de enterococos para aplicações em alimentos.
Enterococcus spp. belong to the group of lactic acid bacteria widely distributed in soil, plants, foods, animals and humans. In the past, these microorganisms were considered commensals but the increase of antibiotic-resistant enterococci and the lack of knowledge about their virulence markers, had raised concerns regarding the safety of using strains of this genus in the food production as fermentative or probiotic cultures. Besides this, literature data suggests the use of enterococci as sanitary indicator for foods. Differences between enterococci strains with pathogenic potential and an apparently safe ones is unclear and there is a concern about virulence markers transfer. The aim of this work was to determine phenotypic and genotypic characteristics of Enterococcus spp. isolated from foods and water and also to correlate their presence with classical indicators of sanitary quality. Out of 812 presumptive enterococci colonies obtained from 120 food samples, 299 isolates (37%) were presuntively characterized as Enterococcus spp. Isolates were identified by PCR: 139 (46.5%) E. faecium, 80 (26.8%) E. faecalis, 36 (12.0%) E. casseliflavus and 8 (2.7%) E. gallinarum. Only E. faecalis isolates (60%) produced gelatinase. One E. faecium (0.7%) and 31 E. faecalis (38.7%) were β-haemolytic. Bacteriocin activity against Lactobacillus sakei and/or Listeria monocytogenes was observed for 10% of the E. faecalis and for 23% of the E. faecium isolates. All E. gallinarum isolates, 86% of the E. casseliflavus, 65% of the E. faecalis and 62.6% of the E. faecium isolates showed bile salt hydrolysis activity. Some E. faecium isolates were resistant to vancomycin, erythromycin and tetracycline. Vancomycin resistance was absent among the E. faecalis but, resistance to tetracycline, erythromycin and highlevel gentamicin was observed. There was a higher prevalence of virulence genes (esp, gel, ace, as, efaA e cylA) among the E. faecalis isolates when compared to the E. faecium. Antibiotic resistant E. faecalis isolates strongly adhered to Caco-2 cells and formed biofilm on abiotic surface. Using RAPDPCR 14 E. faecium and 17 E. faecalis strains could be individualized from the 52 antibiotic resistant enterococci. It was not possible to correlate the presence of total coliforms, E. coli and Enterococcus spp. in the samples of food and water analysed due to results variability. Data obtained regarding phenotypic and genotypic virulence markers and the presence of antibiotic resistant enterococci raise the needs of a carefully evaluation of the Enterococcus spp. strains before future applications in foods.

O charque é um produto cárneo tipicamente brasileiro, salgado e seco ao sol, ainda produzido de maneira artesanal. Durante sua produção há uma etapa de fermentação, realizada pela microbiota naturalmente presente na matéria-prima, o que dificulta a padronização do produto, e pode influenciar negativamente em suas características sensoriais e qualidade microbiológica. O controle da etapa de fermentação do charque seria uma alternativa para minimizar este problema e, neste contexto, as bactérias láticas produtoras de bacteriocinas se enquadram de forma interessante. A microbiota autóctone de charque inclui principalmente bactérias láticas e micro-organismos halofílicos e halotolerantes, sendo assim, este produto apresenta potencial como fonte para o isolamento de novas bactérias láticas produtoras de bacteriocinas. Assim, este trabalho teve por objetivo isolar e identificar culturas de bactérias láticas produtoras de bacteriocinas naturalmente presentes no charque, caracterizar parcialmente as bacteriocinas produzidas por essas culturas, avaliar seu potencial de aplicação neste produto para a melhoria de sua qualidade microbiológica e avaliar seu efeito na ecologia microbiana do charque, nas diferentes etapas de sua fabricação. Através da técnica de tripla camada em ágar foi isolada uma cepa de Lactococcus lactis subsp. lactis apresentando o gene codificador para nisina Z e com capacidade de inibir, in vitro, micro-organismos medianamente e altamente halotolerantes isolados de charque, além de outros micro-organismos deteriorantes e patogênicos importantes em alimentos, como Lactobacillus spp., Listeria monocytogenes e Staphylococcus aureus. A bacteriocina produzida pela cepa isolada neste estudo também possui características interessantes para sua aplicação na bioconservação de alimentos, como resistencia ao calor, presença de agente químicos e altos teores de NaCl, além de não ser afetada pelo pH. A aplicação dessa cepa em charque modelo resultou na redução de até 2 ciclos log na população de micro-organismos halotolerantes, indicando apresentar um potencial de aplicação como agente de bioconservação do produto. Os ensaios de avaliação da ecologia microbiana, empregando DGGE, indicaram que a fermentação natural do charque ocorreu com a participação de bactérias láticas dos gêneros Lactobacillus, Streptococcus, Lactococcus e de micro-organismos halotolerantes do gênero Staphylococcus. Além disso, os estudos referentes à dinâmica populacional demonstraram que a adição da cepa bacteriocinogênica ao charque não influenciou, de forma qualitativa, as populações presentes no produto.
Charqui is a Brazilian traditional meat product, salted and sun-dried, still manufactured without control of the fermentation step, which is performed by the indigenous microbiota. This fact interferes on the standardization of the product and can negatively affect the sensorial properties and microbiological quality. The application of a known microbiota would be an alternative to minimize this problem and the bacteriocin-producing lactic acid bacteria can can fit in this purpose. The charqui indigenous microbiota mainly includes lactic acid bactéria and halophilic and halotolerant microorganisms, therefore, this product presents a potencial as a source for the isolation of new bacteriocin-producing lactic acid bacteria. The aim of the present work was to isolate and identify bacteriocin-producing lactic acid bacteria from charqui, characterize the bacteriocins produced by the isolated culture, evaluate its potential as biopreservative in charqui and its influence on the microbial populations during the manufacture of the product. A bacteriocinogenic Lactococcus lactis subsp. lactis strain was isolated from charqui through the triple-layer agar technique. This strain produces a nisin-like bacteriocin capable to inhibit in vitro medium and highly halotolerant bacteria isolated from charqui and other food-borne pathogenic and spoilage microorganisms. The application of this strain for charqui manufacturing caused a reduction of up to 2 log in the halotolerant bacteria population, evidencing its potential application for charqui biopreservation. Studies in the populational dynamics using DGGE indicated that the presence of the bacteriocinogenic strain did not affect the microbial populations in the product.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Lactic acid bacteria can produce bacteriocins and have been explored for applications as natural preservatives in foods. Bacteriocins are peptides and proteins with antimicrobial activity and can inhibit the growth of pathogenic bacteria such as Listeria monocytogenes and other spoilage microorganisms. L. monocytogenes can be present in many types of food due to its ability to survive in a wide temperature range, and causes listeriosis by ingestion of contaminated food. This study aimed at the isolation and identification of lactic acid bacteria (LAB) with bacteriocinogenic potential, the characterization of the cell-free supernatants (CFS) regarding: the antimicrobial activity; the spectrum of action; thermal stability and at different pHs; resistance to chemicals and NaCl concentrations; the mode of action of the bacteriocins produced; the adsorption capacity to the target cells in different temperatures, pH and concentrations of chemicals and NaCl; perform the molecular identification of the LAB. Three strains (LS1, LS2 and LS3) bacteriocinogenic of raw goat's milk with anti-listeria activity were isolated. In particular Lactococcus lactis (LS2), which produced bacteriostatic, active bacteriocin at low pH and 4 ° C to 80 ° C, which can be an alternative for the control of Listeria in fermented dairy products.
Bactérias láticas podem produzir bacteriocinas e têm sido exploradas em aplicações como conservadores naturais nos alimentos. Bacteriocinas são peptídeos e proteínas com atividade antimicrobiana e podem inibir a multiplicação de bactérias patogênicas como Listeria monocytogenes e micro-organismos deterioradores. L. monocytogenes pode estar presente em diversos tipos de alimentos, devido à sua capacidade de sobreviver em ampla faixa de temperatura e causar a listeriose através da ingestão de alimentos contaminados. Este estudo objetivou o isolamento e identificação de bactérias láticas (BAL) com potencial anti-Listeria de leite de cabra cru, a caracterização dos sobrenadantes livres de células (SLC) quanto: à atividade antimicrobiana; ao espectro de ação; à estabilidade térmica e em diferentes valores de pH; à resistência a agentes químicos e a diferentes concentrações de NaCl; ao modo de ação; à capacidade de adsorção à listeria em diferentes condições de temperatura, pH e na presença de agentes químicos. Também visou realizar a identificação molecular dos isolados a fim de aplicar as BAL ou as bacteriocinas por elas produzidas como estratégia de bioconservação de alimentos. Foram isoladas três culturas (LS1, LS2 e LS3) bacteriocinogênicas de leite de cabra cru com atividade anti-listeria. Em particular Lactococcus lactis (LS2), que produziu bacteriocina bacteriostática, ativa em pH baixo e em temperatura de 4 °C a 80 °C, que pode ser uma alternativa para o controle de listeria em produtos lácteos fermentados.

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.

Batches of fromage blanc, a soft white cheese were prepared from whole pasteurized cowâ s milk. Processing and formulation methods were used in cheese making to reduce Listeria monocytogenes in artificially contaminated cheese. Treatments implemented included use of additional starter culture in formulation (25% more starter culture than original formulation), use of a higher temperature draining process (at 45oC instead of 22oC), addition of the anti-listerial bacteriocin nisin (Danisco Nisaplin) in formulation at different levels (125 ppm, 250 ppm, 400 ppm), and combinations of these treatments. Characteristics including pH, fat content, protein content, and color were evaluated for each treatment cheese. Statistically significant differences (p<0.0001) were found between the population (log CFU/g) values of L. monocytogenes in the different treatment cheeses and control cheese. Treatments using additional starter culture or higher temperature draining alone were not successful in significantly reducing numbers of L. monocytogenes, but when combined, a 1 log reduction resulted. Of the different concentrations of nisin used in cheese formulation, the level of 250 ppm nisin was used in combination treatments. The treatments using 250 ppm nisin were able to reduce numbers of L. monocytogenes by 2 log 24h after addition. Combination treatments with 250 ppm nisin and additional starter culture in formulation reduced the level of L. monocytogenes by only 1 log, while combination treatments coupling 250 ppm nisin with a higher temperature draining and treatments with 250 ppm nisin, additional starter culture, and a higher temperature draining were able to reduce the pathogen by 2 log. There were statistically significant (p<0.0001) differences found between cheese treatments for values of pH, fat content, and protein content. This soft cheese could be standardized for each of these parameters by the processor before packaging and sale of cheese. There were no statistically significant (p>0.05) differences found between colorimetric values for different cheese treatments.
Master of Science

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.

The colicin and lectin-like bacteriocins are a broad class of antimicrobial proteins produced by Gram-negative bacteria. They are generally narrow spectrum, killing or inhibiting the growth of closely related bacteria. Numerous Gram-negative bacteria that are important pathogens of both animals and plants produce and are susceptible to these bacteriocins. As such, these proteins represent an attractive alternative to traditional small molecule antibiotics for controlling bacterial infection. Very little is known about bacteriocins produced by Gram-negative plant pathogens and so the aim of this work was to discover novel bacteriocins active against globally important plant pathogens from the genera Pectobacterium and Pseudomonas. The bacteriocins discovered in this study were then structurally and functionally characterised and assessed for their ability to impart disease resistance when expressed in a model transgenic system. This study presents the discovery and characterisation of the bacteriocins syringacin M, syringacin L1 and pyocin L1 from the genus Pseudomonas, As well as the discovery and characterisation of the unusual ferredoxin containing pectocins from the genus Pectobacterium. Also presented is the discovery of a novel virulence related ferredoxin/iron-uptake system in Pectobacterium, which is parasitised by the pectocins for cell entry. Additionally, the transgenic expression of the bacteriocin putidacin L1 in both Arabidopsis thaliana and Nicotiana benthamiana was shown to provide these plants with resistance to infection by strains of the plant pathogen P. syringae.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Os produtos lácteos possuem uma microbiota autóctone bastante diversificada, na qual o grupo das Bactérias Ácido Lácticas (BAL) é de notável relevância devido às suas características benéficas, tecnológicas e bioconservantes, atraindo o interesse para sua utilização em diversos segmentos biotecnológicos, em especial na indústria de alimentos. O objetivo deste trabalho foi isolar e identificar BAL bacteriocinogênicas de queijos artesanais, caracterizando aspectos ligados à produção e purificação das bacteriocinas, inocuidade, potencial benéfico dos isolados e propriedades inibitórias contra Listeria spp. As cepas bacteriocinogênicas Enterococcus hirae ST57ACC e Pediococcus pentosaceus ST65ACC foram isoladas a partir da técnica de tripla camada e identificadas por metodologias fenotípicas e moleculares. As bacteriocinas produzidas por E. hirae ST57ACC e P. pentosaceus ST65ACC demostraram estabilidade em ampla faixa de pH e temperatura, e foram inativadas após tratamento com enzimas proteolíticas, comprovando sua natureza proteica. Tratamentos com EDTA, SDS, NaCl e Tween 80 não afetaram a atividade das bacteriocinas. Os sobrenadantes de ambos os isolados foram capazes de inibir Listeria innocua e diversas cepas de L. monocytogenes pertencentes à diferentes sorogrupos e obtidas de fontes distintas, inibindo completamente o desenvolvimento de L. monocytogenes após 12 h. Em co-culturas das cepas bacteriocinogênicas com a cepa indicadora L. monocytogenes 422 em leite desnatado, observou-se que E. hirae ST57ACC foi capaz de controlar a multiplicação do patógeno após 48 h. E. hirae ST57ACC e P. pentosaceus não apresentaram resultados positivos para 25 genes relacionados a bacteriocinas conhecidas, indicando que podem produzir novas bacteriocinas. As cepas de E. hirae ST57ACC e P. pentosaceus ST65ACC foram também avaliadas quanto ao seu potencial benéfico e segurança: ambos os isolados permaneceram viáveis após tratamento em condições gastrointestinais simuladas, exibindo altos níveis de auto e co-agregação com L. monocytogenes e níveis variados de hidrofobicidade, demonstrando que E. hirae ST57ACC e P. pentosaceus ST65ACC podem prevenir potencialmente o estabelecimento de infecções pelo patógeno. Por meio da metodologia de agar-spot, avaliou-se a possibilidade de interferência de 33 medicamentos comerciais, de diferentes grupos sobre a multiplicação de E. hirae ST57ACC e P. pentosaceus ST65ACC, revelando que apenas antiinflamatórios e medicamentos contendo loratadina e cloridrato de propranolol apresentaram atividade inibitória sobre as cepas. Testes fenotípicos para determinação da susceptibilidade antimicrobiana demonstraram que E. hirae ST57ACC e P. pentosaceus ST65ACC foram resistentes à vancomicina, oxacilina e sulfa/trimetoprim dentre os 11 antibióticos testados pelo método de disco difusão. Com relação à PCR, poucos genes relacionados à resistência a antibióticos foi foram identificados. Nenhum dos isolados amplificou genes de produção de aminas biogênicas e nem apresentou produção das mesmas. A expressão de diferentes elementos do sistema de transporte ABC e metabolismo de açúcares foi identificada para ambos os isolados. Variações na proporção de inóculo não influenciaram a taxa de multiplicação de E. hirae ST57ACC nem de P. pentosaceus ST65ACC, no entanto, a produção de bacteriocinas foi detectada apenas 9 horas após a inoculação das cepas, quando inoculadas nas proporções de 5% e 10%. Adicionalmente, verificou-se que a densidade celular das cepas bacteriocinogênicas esteve correlacionada à produção de bacteriocinas em sistemas de fermentação tradicional e fermentação com controle de pH a 5,5 e agitação. E. hirae ST57ACC e P. pentosaceus ST65ACC foram capazes de se multiplicar e produzir bacteriocinas na presença de xilo-oligossacarídeos após 6 horas de incubação, porém em níveis reduzidos quando comparados ao cultivo em meio MRS. Por fim, as bacteriocinas produzidas por E. hirae ST57ACC e P. pentosaceus ST65ACC foram purificadas a partir de diferentes metodologias. A bacteriocina produzida por P. pentosaceus ST65ACC foi purificada em duas etapas, com rendimento final de 101,33 revelando- se um peptídeo com massa molecular de 3,5 a 8,5 kDa, determinado por SDS-PAGE. Em contrapartida, um protocolo de três etapas foi empregado na purificação da bacteriocina produzida por E. hirae ST57ACC, com rendimento final de 3,05. Adicionalmente, uma fração semi-purificada foi testada com a linhagem celular HT- 29, demonstrando que a bacteriocina não apresenta efeitos citotóxicos contra células humanas, sendo considerada segura neste aspecto. Os dados obtidos neste trabalho indicam que os isolados E. hirae ST57ACC e P. pentosaceus ST57ACC podem ser considerados importantes ferramentas biotecnológicas na produção de bacteriocinas de interesse ao controle de L. monocytogenes e na biopreservação de alimentos.
Dairy products present a rich and diverse autochthonous microbiota, in which Lactic Acid Bacteria (LAB) are relevant, due to their beneficial, technological and biopreservative features, attracting the interest for their biotechnological application, in food industry, pharmaceutic area and human and veterinary medicine fields. The aim of this study was to isolate and to identify bacteriocinogenic LAB from artisanal cheeses, characterizing some aspects linked to bacteriocin production and purification, safety and beneficial potential of the isolates, as well as their inhibitory properties against Listeria spp. Bacteriocinogenic strains Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC were isolated by using the triple- layer technique and identified by phenotypical and molecular methods. Bacteriocins produced by E. hirae ST57ACC and P. pentosaceus ST65ACC were stable in a wide range of pH and temperature, losing their activity after treatment with proteolytic enzymes, confirming their proteinaceous nature. Treatments with EDTA, SDS, NaCl and Tween 80 did not affect bacteriocin activity. Cell-free supernatants from both isolates were able to inhibit Listeria innocua and several L. monocytogenes strains, from different serogroups obtained from diverse sources, eliminating L. monocytogenes after 12 h. In co-culture experiments conducted in skimmed milk with the bacteriocinogenic isolates and the target strain L. monocytogenes 422, E. hirae ST57ACC controlled the target strain growth after 48 h. E. hirae ST57ACC and P. pentosaceus ST65ACC did not present positive results for 25 known bacteriocin related genes, indicating that they might express new bacteriocins. E. hirae ST57ACC e P. pentosaceus ST65ACC were also evaluated for their beneficial and safety features: both isolates remained viable after treatment replicating gastrointestinal conditions, showing high levels of auto and co-aggregation with L. monocytogenes and diverse levels of hydrophobicity, demonstrating that E. hirae ST57ACC and P. pentosaceus ST65ACC might prevent the establishment of infections caused by this pathogen. Interference of 33 commercial drugs from different groups on growth of E. hirae ST57ACC and P. pentosaceus ST65ACC was tested by agar-spot method, revealing that only anti-inflammatories and drugs containing loratadine and propranolol hydrochloride influenced the growth of bacteriocinogenic strains. Phenotypical tests employed to determine antibiotic susceptibility have shown that E. hirae ST57ACC and P. pentosaceus ST65ACC were resistant to vancomycin, oxacillin and sulfa/trimethoprim out of 11 antibiotics tested by disk-diffusion test, nonetheless low number of antibiotic resistance genes was observed by PCR analysis. None of the isolates amplified biogenic amines encoding genes neither presented phenotypical evidence of their production. Expression of different ABC transporters linked to bacteriocin export and sugar metabolism was detected, for both isolates. Changes in inoculum size did not influenced the growth of E. hirae ST57ACC and P. pentosaceus ST65ACC; however, bacteriocin production was affected, and bacteriocins were detected only after 9 h with inoculation at 5% and 10% of bacteriocinogenic strains. Additionally, it was observed that cell density of both bacteriocinogenic strains was linked to bacteriocin production in traditional and pH at 5.5 and agitation controlled fermentation continuous. E. hirae ST57ACC and P. pentosaceus ST65ACC were capable to grow and produce bacteriocins in the presence of xylo-oligossacharides after 6 h of incubation, but in lower levels than those obtained with cultivation in MRS broth. Finally, E. hirae ST57ACC and P. pentosaceus ST65ACC were purified from different methods. The bacteriocin produced by P. pentosaceus ST65ACC was purified in two-steps, with final yield of 101.33, recognized as a 3.5 to 8.5 kDa peptide, determined by Tricine-SDS-PAGE. In contrast, a three-step-protocol was used to purify the bacteriocin produced by E. hirae ST57ACC, with final yield of 3.05. Moreover, a semi-purified fraction of E. hirae ST57ACC bacteriocin was tested in HT-29 cell-line, demonstrating no-cytotoxic effects in human cells, which means the bacteriocin can be considered safe in this aspect. Obtained data from this study indicate that E. hirae ST57ACC and P. pentosaceus ST57ACC may be considered as important biotechnological tools for bacteriocin production to control L. monocytogenes and as biopreservatives in food.

Bacillus thuringiensis NEB17 is a plant growth promoting rhizobacterium that produces a compound that directly increases plant growth. The compound is a bacteriocin and we propose the name thuricin 17. Thuricin 17 is a novel peptide inhibiting the growth of Bacillus species/strains, displaying both bactericidal and static effects. Its molecular weight, estimated via SDS-PAGE and verified by MALDI-QTOF mass spectroscopy, is 3162 Da. The partial amino acid sequence was determined and is N-term---WTCWSCLVCAACSVELL, C-term-CAS. Heat and pH stability, production and susceptibility to proteolysis were conducted. Thuricin 17 is active in pH 1.00-9.25, stable above 60°C and produced in the late exponential growth phase. This is the first bacteriocin from a Bacillus PGPR and the first reported to increase plant growth. This work presents an original discovery regarding PGPR mechanisms.

Thesis (PhD)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The possible use of the bacterially produced antimicrobial peptides, and in particular class IIa bacteriocins as food preservatives is a motivating factor in studies on resistance to them by food-borne pathogens like Listeria monocytogenes. The high frequencies of resistance to class Ha bacteriocins have however sparked concern regarding their adequacy as potential biopreservatives. Activity of these cationic peptides was reported to occur by membrane permeabilisation due to pore formation, which results in the leakage of the intracellular contents followed by cell death. The cell envelope (cell wall and cell membrane) is therefore envisaged as a key site of modification in suscepti bility of bacteria to class Ha bacteriocins. Mutants of the L. monocytogenes 873 isolate, resistant to the class IIa bacteriocin, leucocin A, were generated at the start of the study to complement the existing array of L. monocytagenes wild-type and resistant isolates obtained from other sources. The fifty percent inhibitory concentrations using a highly sensitive and reproducible bioassay were determined. This allowed categorisation of the mutants into intermediate and highly resistant phenotypes. Analysis of the growth patterns of all these strains showed decreased growth rates and higher growth yields for all the resistant strains in general. This provided evidence for possible effects of membrane adaptation and metabolic changes in the resistant strains and prompted further investigation. The major focus of the study on the class Ha resistant mutants were: (1) analysis of membrane compositional changes and factors influencing cell surface charge; (2) assessment of physical changes in the membrane and bacteriocin itself using circular dichroism and fourier transform infrared spectroscopy; (3) and, determination of changes in glucose metabolism. Electrospray mass spectrometry analysis of the major listerial phospholipid, phosphatidylglycerol, revealed that membranes of resistant strains had increased levels of unsaturated and short-acyl-chain phosphatidylglycerol molecular species, indicating more fluid membranes. In addition, treatment with a desaturase inhibitor resulted in increased sensitivity of only the intermediate resistant strains to the class na bacteriocin, leucocin A. This indicated the influence of membrane adaptation in only lower levels of resistance. It is conceivable that more fluid membranes could also impact on decreased stability of pore formation by the bacteriocin. Complementary biophysical studies using fourier transform infrared spectroscopy indicated the possible occurrence of greater membrane fluidity of resistant cells, by the notable shift in the anti symmetric CH2 stretching vibration from 2921 cm-I to 2922 cm-I. Additionally, circular dichroism revealed a decreased a-helical and increased random structure of leucocin A in the presence of listerial liposomes derived from highly resistant cell membrane extracts. It is possible that this may result in reduced activity of the bacteriocin in resistant cell membranes as a-helical stucture is a critical feature for membrane insertion of cationic antimicrobial peptides. Cell surface charge was determined by quantification of alanine and lysine esterification of the anionic cell surface polymer, teichoic acid, and membrane phospholipids respectively. Increased D-alanine, which causes neutralisation of the cell surface, was observed in all resistant cells. A tendency for greater lysine content in membrane phospholipids, which also impacts on neutralisation of the anionic phospholipid of listerial membranes, was observed in highly resistant strains only. This neutralisation of the negative charge of the cell surface may interfere with initial electrostatic interaction of bacteriocin with the cell, and subsequent interactions required for permeabilisation of the cell membrane. These differences in alanine and lysine esterification were not the result of increased expression of certain associated genes (d/tA and /mo1695) and may be the result of post-transcriptional regulation. It was, however, found that all resistant L. monocytogenes strains, including the intermediate resistant strains, exhibited decreased expression of a putative docking molecule, the mannose-specific phosphotransferase system EIIAB subunit (EIlABMan).A clear correlation existed between the levels of resistance and EIIABMandown-regulation. Finally, analysis of the glucose metabolism in highly resistant and wild-type strains, indicated a more efficient metabolism with regards to higher growth yields and ATP yield, in contrast to a lower specific growth rate in a spontaneous and genetically defined (EIlABMan inactivated) highly resistant mutant. The switch in metabolic end-product observed, was attributed to the loss of the glucose transporter, EIlABMan,and may cast doubts on the feasibility of the use of class Ha bacteriocins as food preservatives in light of a stable and efficient resistant phenotype.
AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Orientador: Reginaldo Bruno Gonçalves
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Esta tese, apresentada na forma de 3 artigos, teve por objetivos: (1) analisar a freqüência dos genes de produção de mutacinas I, II, III e IV, em genótipos de S. mutans isolados de indivíduos cárie-ativos e livres de cárie, (2) analisar a freqüência dos genes de produção das mutacinas I, II, III, IV, N, B-Ny 266, 1140 e genes homólogos às bacteriocinas, identificadas em outras espécies bacterianas, em cepas de S. mutans isolados de crianças, bem como detectar a expressão diferencial dos genes identificados, em células de S. mutans crescidas na condição planctônica e séssil, (3) analisar a expressão dos genes de produção das mutacinas I, II e proteínas kinases CiaH, Dgk e ComD, em diferentes fases do crescimento planctônico e séssil. O rastreamento e a freqüência dos genes estruturais de diferentes mutacinas em isolados de S. mutans, foram realizados pela técnica de PCR e a análise da expressão gênica, pela técnica de RT-PCR semi-quantitativa. Os estudos, apresentados nesta tese, demonstraram o papel das mutacinas como um fator de virulência, altamente diversificado entre a espécie S. mutans, e relacionado com o risco de cárie. Este fator de virulência, pode ser regulado por mecanismos quorum-sensing, sendo assim, dependente da condição de crescimento planctônica ou séssil. A regulação da produção de mutacinas, por mecanismos quorum-sensing, pode representar uma vantagem seletiva à espécie produtora, principalmente em ambiente complexo, como o biofilme dental e lesões de cárie. Futuramente, mais estudos serão necessários para identificar novos determinantes genéticos, necessários para a síntese de substâncias semelhantes às mutacinas, bem como, identificar os mecanismos e componentes, que modulam a expressão deste importante fatorde virulência em S. mutans
Abstract: This thesis, comprised of 3 manuscripts was designed (1) to analyse the frequency of biosynthesis genes of the mutacins types I, II, III and IV, in S. mutans isolated from caries-affected and caries-free individuals, (2) to analyse the frequency of biosynthesis genes of the mutacins types I, II, III, IV, N, B-Ny 266, 1140 and genes homologues to bacteriocins identified in other bacterial species, in S. mutans isolated from children, in addition, to detect the differential expression of these genes, in S. mutans cells grown in planktonic and sessil conditions, (3) and to analyse the expression of the mutacins I and II production genes and kinase proteins genes (ciaH, dgk e comD), in different phases of the planktonic and sessile growth. The screening and frequency of the mutacins structural genes in S. mutans isolates were realized by PCR technique and the analysis of genetic expression, by RT-PCR semiquantitative method. The studies, presented in this thesis, demonstrate the role of mutacins as a virulence factor, highly diverse among S. mutans, and related to risk of dental caries. The mutacins production may be regulated by quorum-sensing mechanisms and is dependent on planktonic and sessile conditions. The modulation by quorum-sensing mechanisms may represent a selective advantage for producer S. mutans strain, mainly in complex environments as the dental biofilm and caries. Hereafter, more studies will be necessary to identify new genetic determinants for synthesis of mutacin-like substances and elucidate the mechanisms and components that modulate the genetic expression of this important virulence factor in S. mutans
Doutorado
Microbiologia e Imunologia
Doutor em Biologia Buco-Dental

Além das características tecnológicas das bactérias lácticas para o processamento de alimentos fermentados, novas propriedades são requeridas, como a produção de bacteriocinas. Vários desses compostos protéicos possuem atividade antimicrobiana direcionada à patógenos e apresentam potencial de uso como bioconservadores naturais de alimentos. O objetivo desse trabalho foi avaliar a viabilidade de aplicação de cinco culturas lácticas produtoras de bacteriocinas como bioconservantes em alimentos. Avaliaram-se as propriedades de segurança e probióticas dessas culturas e determinaram-se os parâmetros de produção e purificação parcial das bacteriocinas, bem como os efeitos de fatores limitantes de crescimento (cloreto de sódio e nitrito). Os resultados demonstraram que essas linhagens não possuem atividade de enzimas indicadoras de patogenicidade (termonuclease, -hemolisinas e gelatinases). A tolerância às condições ácidas variou dependendo da linhagem. Lactobacillus plantarum CTC 368 e Enterococcus avium CTC 469 apresentaram maior tolerância a valores de pH 2,0 e 3,0. Lactococcus lactis subsp. cremoris CTC 204 e Enterococcus avium CTC 483 também apresentaram 100% de sobrevivência a pH 3,0 e, mesmo com uma diminuição da tolerância a pH 2,0, a sobrevivência foi cerca de 80%. Com relação à susceptibilidade aos sais de bile, observou-se que a viabilidade variou entre as linhagens em função da concentração e tempo de incubação. Enterococcus avium CTC 469 e Lactococcus lactis subsp. hordinae CTC 484 foram as mais tolerantes e apresentaram sobrevivência de 57% e 58%, respectivamente. As linhagens mostraram excelente sobrevivência tanto a baixos valores de pH, quanto às concentrações de sais biliares indicando potencial probiótico. As características de crescimento e de produção de bacteriocinas foram semelhantes para todas as linhagens e meios avaliados. A viabilidade aumentou durante o período de incubação e a produção de bacteriocinas mostrou uma cinética de metabólito primário. A suplementação do meio MRS com 1,0% de NaCl e 0,1% de NaNO2, não afetou o crescimento de Lc. lactis subsp. cremoris CTC 204, mas promoveu a redução da atividade da bacteriocina de 42,5% e 17,5%, respectivamente. O crescimento de Lactococcus lactis subsp. hordinae CTC 484 foi afetado com a adição de NaCl e NaNO2 ao meio APT, mas ainda reteve 50% da atividade bacteriocigênica. O espectro de atividade das bacteriocinas variou de acordo com a susceptibilidade da linhagem indicadora, a concentração ou atividade da bacteriocina, a viabilidade e/ou presença das células produtoras e os procedimentos de extração das bacteriocinas. Os resultados revelaram que as bacteriocinas de Lactococcus lactis subsp. cremoris CTC 204 e Lactococcus. lactis subsp. hordinae CTC 484 promoveram um efeito bactericida contra Staphylococcus aureus. Concluiu-se que essas duas culturas produtoras de bacteriocinas apresentam propriedades para utilização como bioconservantes em alimentos e que estudos mais aprofundados, relacionados com a purificação da bacteriocina e avaliação de seu uso em alimentos, devem ser realizados.
In addition to the technological characteristics of lactic bacteria for the processing of fermented foods, other properties need to be developed and explored, such as bacteriocin production. Several of these protein-based compounds have antimicrobial activities targeted towards pathogens and have a significant potential for use as natural biopreservatives in processed foods. The objective of this study was to evaluate the viability of using five bacteriocin-producing lactic cultures as biopreservatives in foods. The safety and probiotic properties of these cultures were evaluated, followed by the determination of bacteriocin production and purification parameters and the effect of growth-limiting factors (sodium chloride and nitrite). The results demonstrated that these strains do not show activity of marker enzymes of pathogenicity (thermonuclease, - hemolysins and gelatinases). Tolerance to acidic conditions varied depending on the specific strain. Lactobacillus plantarum CTC 368 and Enterococcus avium CTC 469 exhibited greater tolerance to pH values 2,0 and 3,0. Lactococcus lactis subsp. cremoris CTC 204 and En. avium CTC 483 also presented 100% survival at pH 3,0, and even with reduced tolerance at pH 2,0, the survival rate was 80%. Variation in susceptibility to bile salts was observed between the strains as a function of concentration and incubation time. En. avium CTC 469 and Lactococcus lactis subsp. hordinae CTC 484 were found to be the most tolerant, exhibiting survival rates of 57% and 58%, respectively. The strains demonstrated excellent survival not only at low pH values, but also at high bile salt concentrations, thereby indicating probiotic potential. The growth and bacteriocin-producing characteristics were similar for all the strains and culture media evaluated. Bacterial multiplication increased during the incubation period, while bacteriocin production displayed primary metabolite kinetics. The activity spectrum of the bacteriocins varied with the susceptibility of the indicator strain, the concentration or activity of the bacteriocin, the viability and/or presence of bacteriocinproducing cells and the bacteriocin extraction procedures. The results reveal that the bacteriocins produced by Lc. lactis subsp. cremoris CTC 204 and Lc. lactis subsp. hordinae CTC 484 exerted a bactericidal effect against Staphylococcus aureus. Supplementation of the MRS medium with 1,0% NaCl and 0,1% NaNO2, did not affect the growth of Lc. lactis subsp. cremoris CTC 204, but did reduce by 42,5% and 17,5% the activity of the bacteriocin, respectively. Growth of Lc. lactis subsp. hordinae CTC 484 was affected by the addition of NaCl and NaNO2 to the APT medium, but even so still exerted 50% of its bacteriocinogenic activity. It was concluded that Lc. lactis subsp. cremoris CTC 204 and Lc. lactis subsp. hordinae CTC 484 exhibited adequate properties for use as biopreservatives in foods and that more extensive studies related to the purification of bacteriocins and assessment of their use in foods should be conducted.

Xylella fastidiosa é o agente causal de uma série de doenças que ocorrem em plantas economicamente importantes como laranjeiras, videiras e cafeeiros, causando no Estado de São Paulo prejuízos relevantes à indústria citrícola. Esta bactéria Gram-negativa é restrita ao xilema das plantas e à porção anterior do trato digestório dos insetos vetores das famílias Cicadellidae e Cercopidae, conhecidos como cigarrinhas. Tentativas de elucidar os mecanismos de virulência e patogenicidade adotados por esta bactéria apontam a formação do biofilme como etapa fundamental para o estabelecimento da infecção e o consequente desenvolvimento da doença na planta, mas fatores adicionais parecem contribuir, tais como a produção de toxinas. As bacteriocinas são proteínas com atividade antibiótica contra cepas próximas à espécie produtora e que já foram associadas à virulência e patogenicidade de outras bactérias. Uma varredura in silico no genoma de X. fastidiosa 9a5c revelou 13 sequências codificadoras de microcinas putativas no cromossomo. Transcritos de todos esses genes foram detectados por RT-qPCR em culturas de X. fastidiosa 9a5c, e análises comparativas com genomas públicos (cepas Temecula1, Dixon, Ann-1, M23, M12, EB92-1 e GB514) e recém sequenciados por nosso grupo de pesquisa (cepas U24d, J1a12, 3124, Hib4, Pr8x e Fb7) revelaram que cada cepa possui seu próprio arsenal de bacteriocinas. Diferenças encontradas in silico entre os loci de bacteriocinas nas cepas foram demonstradas experimentalmente. Nossos resultados comprovam a variabilidade predita nos quatro clusters de bacteriocinas que identificamos, o que é esperado para genes relacionados à adaptação e patogenicidade. Destes loci, três foram detectados por RT-PCR como transcritos policistrônicos. Nossa tentativa de detectar essas proteínas em culturas de X. fastidiosa (através de sequenciamento de polipeptídeos por HPLC-MS/MS) foi capaz de identificar uma das bacteriocinas putativas e, portanto, o conjunto de nossas observações apóia a continuidade dos estudos para elucidar o papel das bacteriocinas na fisiopatologia de X. fastidiosa.
Xylella fastidiosa is the causal agent of diseases that affect several economically important crops such as sweet orange trees, grapevines and coffee trees, causing in the State of São Paulo considerable losses mainly to the citrus industry. This Gram-negative bacterium is restricted to the plant xylem and to the upper gastrointestinal tract of its insect vectors, the sharpshooters from the Cicadellidae and Cercopidae families. Attempts to elucidate the virulence and pathogenicity pathways employed by this bacterium point the biofilm formation as a fundamental step for the establishment of the infection and the consequent development of the plant disease, but additional factors seem to contribute to these processes, such as the production of toxins. Bacteriocins are proteinaceous antibiotics that act against closely-related species and have been previously associated with virulence and pathogenicity in other bacteria. An in silico screening of the X. fastidiosa 9a5c genome revealed 13 coding sequences as putative microcins in the chromosome. Transcripts from all those genes were detected through RT-qPCR in X. fastidiosa 9a5c cultures, and comparative analyses on the public genomes (Temecula1, Dixon, Ann-1, M23, M12, EB92-1 and GB514) plus the ones recently sequenced by our group (U24d, J1a12, 3124, Hib4, Pr8x and Fb7) revealed that each strain possesses its own arsenal of bacteriocins. Differences found in silico among the loci in all strains were experimentally confirmed. Our results demonstrated the predicted variability in the four bacteriocins clusters as expected for adaptation and pathogenicity-related genes. Three out of the four bacteriocins loci were detected by RT-PCR as polycistronic transcripts. Our attempt to detect these proteins in X. fastidiosa cultures (using HPLC-MS/MS polypeptide sequencing) identified one of the putative bacteriocins, and therefore our observations warrant further efforts to elucidate the role of bacteriocins in the X. fastidiosa physiopathology.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008.
Lactobacillus plantarum JW3BZ and Lactobacillus fermentum JW15BZ isolated from boza, a Bulgarian cereal based fermented beverage, produce bacteriocins JW3BZ and JW15BZ active against a wide range of food spoilage and pathogenic bacteria. Strains JW3BZ and JW15BZ are resistant to low pH (pH 2.0–4.0). Both strains grow well in MRS broth with an initial pH ranging from 5.0 to 10.0. Strain JW3BZ displayed intrinsic resistance to bile salts. Strain JW15BZ, on the other hand, is sensitive to bile salts exceeding concentrations of 0.3% (w/v). Both strains are weakly hydrophobic and are resistant to a wide range of antibiotics, antiinflammatory drugs and painkillers. Strains JW3BZ and JW15BZ adhered at 4% to Caco-2 cells and they did not compete with Listeria monocytogenes Scott A for adhesion. A homologue of MapA, a gene known to play a role in adhesion, was detected in L. plantarum JW3BZ. Both strains have high auto- and co-aggregation properties. Bacteriocin JW15BZ was partially purified with ammonium sulfate, followed by separation on Sep-Pak C18 and reverse phase High Pressure Liquid Chromatography (HPLC). Two separate peaks with antimicrobial activity were recorded for bacteriocin JW15BZ, suggesting that it consists of at least two antimicrobial peptides. Lactobacillus plantarum JW3BZ contains genes homologous to plnE, plnF and plnI of the plnEFI operon that encode for two small cationic bacteriocin-like peptides with double-glycine-type leader peptides and its respective immunity proteins. The antimicrobial activity displayed by strain JW3BZ may thus be ascribed to the production of plantaricins E and F. Bacteriocin JW3BZ and JW15BZ displayed activity against herpes simplex virus (HSV-1) (EC50=200 μg/ml). Both strains were identified in boza after 7 days at storage at 4 oC and repressed the growth of Lactobacillus sakei DSM 20017, indicating that the bacteriocins are produced in situ. The sensory attributes of boza prepared with different starter cultures did not vary considerably, although statistical differences were observed for acidity and yeasty aroma. Encapsulation of strain JW3BZ and JW15BZ in 2% sodium alginate protected the cells from low pH (1.6) and 2.0% (w/v) bile. The rate at which cells were released from the matrix varied, depending on the conditions. Better survival of strains JW3BZ and JW15BZ encapsulated in 2% (w/v) alginate was observed during 9 h in a gastro-intestinal model. Highest release of cells was observed at conditions simulating colonic pH (pH 7.4), starting from 56-65% during the first 30 min, followed by 87%. Complete (100%) release was recorded after 2.5 h at these conditions. Strains JW3BZ and JW15BZ could be used as starter cultures in boza. The broad spectrum of antimicrobial activity of bacteriocins JW3BZ and JW15BZ is an added advantage, rendering the cells additional probiotic properties. Encapsulation of the cells in alginate gel increased their resistance to harsh environmental conditions and may be the ideal method to deliver viable cells in vivo.

Orientadores: Arnaldo Yoshiteru Kuaye, Izildinha Moreno
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A biopreservação é uma técnica utilizada para estender a vida útil e aumentar a segurança dos alimentos por meio do emprego de microbiota protetora e/ou seus peptídeos antimicrobianos. O objetivo deste trabalho foi avaliar a atividade antimicrobiana de culturas produtoras de bacteriocinas sobre alguns patógenos Gram-positivos de ocorrência comum em produtos lácteos. As culturas bacteriocinogênicas Lactococcus lactis subsp. Lactis ATCC 11454, Lactobacillus plantarum ALC 01 e Enterococcus faecium FAIR-E 198 apresentaram comportamento distinto em relação à produção de bacteriocinas quando inoculadas em caldo MRS e em leite desnatado reconstituído (LDR) a 10%. Na avaliação do espectro de ação antimicrobiano pelo método de difusão em ágar por inoculação em poços, as bacteriocinas produzidas por Lb. plantarum ALC 01 e E. faecium FAIR-E 198 apresentaram atividade inibitória apenas sobre as linhagens de Listeria monocytogenes, já a nisina produzida por Lc. lactis subsp. lactis ATCC 11454 demonstrou espectro de ação mais amplo, porém com menor atividade que as demais culturas bacteriocinogênicas. Na avaliação da compatibilidade de desenvolvimento associativo com fermentos lácticos comerciais, somente Lc. lactis subsp. lactis ATCC 11454 apresentou atividade (halo de 6mm) sobre as linhagens constituintes de ambos os fermentos lácticos avaliados. A determinação da atividade acidificante foi realizada em LDR 10% após 8 e 24h de incubação a 35ºC; a adição de 0,1% e de 0,5% das culturas bacteriocinogênicas não afetou significativamente a capacidade de acidificação dos fermentos lácticos. Além disso, observou-se que o desenvolvimento associativo dos fermentos lácticos com Lb. Plantarum ALC 01 e E. faecium FAIR-E 198, em ambas as concentrações, proporcionou significativo aumento da atividade das bacteriocinas destas culturas, enquanto que a atividade da nisina de Lc. lactis subsp. lactis ATCC 11454 foi suprimida. A atividade de aminopeptidases foi determinada após desenvolvimento das culturas lácticas em caldo MRS, Lb. Plantarum ALC 01 apresentou as maiores atividades. Também foi analisado o comportamento de patógenos Gram-positivos durante desenvolvimento associativo com as culturas produtoras de bacteriocinas e fermento láctico em LDR 10% a 35ºC por 48h. Lb. plantarum ALC 01 e E. faecium FAIR-E 198 não influenciaram significativamente o desenvolvimento de Bacillus cereus K1-B041 e de Staphylococcus aureus ATCC 27154 durante as primeiras 24h de incubação a 35ºC, contudo apresentaram forte ação inibitória sobre L monocytogenes Scott A. Já Lc. lactis subsp. lactis ATCC 11454 afetou o desenvolvimento de todos os patógenos apenas durante as primeiras 12h de incubação. O fermento láctico demonstrou significativa ação inibitória sobre B. cereus K1-B041 (>7,37 log UFC/ml) e L monocytogenes Scott A (>6,26 log UFC/ml). Em queijo Minas Frescal, não foi observada diferença significativa entre a ação das culturas bacteriocinogênicas e o fermento láctico sobre L. monocytogenes Scott A e S. aureus ATCC 27154. B. cereus K1-B041 demonstrou susceptibilidade a Lb. plantarum ALC 01 e E. faecium FAIR-E 198 após 7 dias. Pelo método de diluição crítica não foi detectada atividade de bacteriocina nos queijos durante os 21 dias de estocagem a 8 ± 1ºC. A adição das culturas produtoras de bacteriocinas como adjuntas ao queijo Minas Frescal não promoveu alteração no pH e na acidez, contudo Lb. plantarum ALC 01 e E. faecium FAIR-E 198 promoveram aumento da proteólise primária e secundária. Embora os resultados obtidos demonstrem que as culturas bacteriocinogênicas avaliadas não possam ser empregadas como único método de conservação, estas podem atuar em sinergia com outros fatores para aumentar a eficiência no controle de patógenos Gram-positivos, especialmente L. monocytogenes, em produtos lácteos fermentados
Abstract: The biopreservation system, such as bacteriocinogenic lactic bacteria cultures and/or their bacteriocins, have received increasing attention and new approaches to control pathogenic and spoilage microorganisms have been developed. The purpose of this study was to evaluate the action of bacteriocin-producing cultures (Lactococcus lactis subsp. lactis ATCC 11454, Lactobacillus plantarum ALC 01 and Enterococcus faecium FAIR-E 198) on some Gram-positive pathogens in different culture media and dairy products. The bacteriocin production was influenced by the media. The antimicrobial activity of these cultures was evaluated by agar-well diffusion assay. The bacteriocins produced by Lb. plantarum ALC 01 and E. faecium FAIR-E 198 presented inhibitory activity on Listeria monocytogenes alone, on the other hand, the nisin produced by Lc. lactis subsp. Lactis ATCC 11454 demonstrated wide action spectrum, albeit with lower activity. In compatibility of associative development evaluation with the commercial starter culture, only Lc. lactis subsp. lactis ATCC 11454 presented activity on the starter culture. The acidifier activity determination was carried out in skimmed milk after 8h and 24h of incubation at 35ºC. The addition of 0.1% and 0.5% of the bacteriocinogenic cultures did not affect the production of lactic acid by the starter culture. The associative development of the starter culture with Lb. plantarum ALC 01 and E. faecium FAIR-E 198 provided significant increase in bacteriocin activity of these cultures, while the activity of Lc. Lactis subsp. lactis ATCC 11454 nisin was suppressed. The aminopeptidase activity was determined after cellular lise of the lactic cultures previously grown in MRS broth. Lb plantarum ALC 01 presented the largest activity. Moreover, the behavior of Gram-positive pathogens was analyzed during co-culture with the bacteriocin-producing bacteria and with the starter culture in skimmed milk. Lb. plantarum ALC 01 and E. faecium FAIR-E 198 did not influence the development of Bacillus cereus K1-B041 and of Staphylococcus aureus ATCC 27154 during the first 24h of incubation at 35ºC. They presented strong inhibition on L. monocytogenes Scott A. Lc. lactis subsp. lactis ATCC 11454 affected the development of the pathogens studied during the first 12h of incubation. The starter culture demonstrated good inhibition of B. cereus K1-B041 (>7.37 log UFC/ml) and L monocytogenes Scott A (>6.26 log UFC/ml). In Minas Frescal cheese, significant difference was not observed between the action of the bacteriocinogenic cultures and the starter culture on L. monocytogenes Scott A and S. aureus ATCC 27154. B. cereus K1- B041 demonstrated susceptibility to Lb. plantarum ALC 01 and E. faecium FAIR-E 198 after 7 days. Bacteriocin activity was not detected in the cheeses during 21 days at 8 ± 1ºC. The addition of the bacteriocin-producing bacteria as an adjunct culture to the Minas Frescal cheese did not promote alteration in the pH and in the acidity, however Lb. plantarum ALC 01 and E. faecium FAIR-E 198 promoted an increase of the cheese proteolysis. Although the obtained results demonstrated that bacteriocinogenic cultures cannot be used as only method of conservation, these can be used as an additional barrier to optimize the control of Gram-positive pathogens, especially L. monocytogenes, in dairy products
Doutorado
Doutor em Tecnologia de Alimentos

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida.
Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Skin is the largest organ in the human body and serves as a barrier that protects the underlying tissue of the host from infection. Injury, however, destroys this protective barrier and provides a perfect opportunity for microorganisms to invade the host and cause infection, thereby affecting the normal wound healing processes. Furthermore, the ability of microbial pathogens to rapidly develop resistance towards a variety of antimicrobial compounds hampers the effective treatment and control of infections. Antimicrobial-resistant pathogens are increasingly being isolated from patients, placing a huge burden on the health care sector. The search for new and novel antimicrobial agents and treatments is thus of utmost importance and will continue to play an integral role in medical research. Antimicrobial peptides (AMPs) may serve as possible alternatives to antibiotics, or may be used in combination with antibiotics to reduce the risk of antimicrobial resistance. AMPs play a role in innate defence and are produced by a variety of mammals, plants, reptiles, amphibians, birds, fish and insects. The AMPs of bacteria (bacteriocins), especially those of lactic acid bacteria (LAB), are receiving increased attention as antimicrobial agents to treat bacterial infections. Electrospun nanofibers have characteristics that make them suitable as wound dressings, i.e. high oxygen permeability, variable pore size, high surface area to volume ratio and nanofibers are morphologically similar to the extracellular matrix. The ability to incorporate of a variety of biologically active compounds into nanofibers increases their potential as wound dressings. A novel approach would be to incorporate bacteriocins from LAB into nanofiber scaffolds to generate antimicrobial wound dressings. In this study, the feasibility of co-electrospinning bacteriocins from LAB into nanofibers was investigated. Plantaricin 423, produced by Lactobacillus plantarum 423, was successfully co-electrospun into poly(ethylene oxide) (PEO) nanofibers. Plantaricin 423 retained activity after the electrospinning process and continued to inhibit the growth of Lactobacillus sakei DSM 20017T and Enterococcus faecium HKLHS. Viable cells of L. plantarum 423 were also successfully co-electrospun into PEO nanofibers, albeit with a slight reduction in viability. A nanofiber drug delivery system was developed for plantaricin 423 and bacteriocin ST4SA, produced by Enterococcus mundtii ST4SA, by blending PEO and poly(D,L-lactide) (PDLLA) in a suitable solvent before electrospinning. Nanofibers were produced that released the bacteriocins over an extended time period. The PEO:PDLLA (50:50) nanofiber scaffold retained its structure the best upon incubation at 37 °C and released active plantaricin 423 and bacteriocin ST4SA. Nisin A was also successfully co-electrospun into a PEO:PDLLA (50:50) nanofiber scaffold and nisin A, released from the nanofibers, inhibited the growth of Staphylococcus aureus in vitro. Nisin A-containing nanofiber scaffolds significantly reduced viable S. aureus cells in infected skin wounds and promoted wound healing in non-infected wounds. As far as we could determine we are the first to show that bacteriocin-eluting nanofiber scaffolds can be used to treat skin infections and influence wound healing.
AFRIKAANSE OPSOMMING: Vel is die grootse orgaan in die menslike liggaam en dien as buitelaag wat die gasheer se onderliggende weefsel teen infeksie beskerm. Beskadigde vel verloor egter hierdie beskermende eienskap en gee mikroörganismes die geleentheid om die liggaam binne te dring, infeksie te veroorsaak en die normale prosesse geassosieer met wondgenesing te beïnvloed. Die suksesvolle behandeling en beheer van infeksies word gedemp deur die vermoë van mikroörganismes om vinnig weerstand teen antimikrobiese middels te ontwikkel. Mikroörganismes met antimikrobiese weerstand word geredelik van pasiënte geïsoleer en dit plaas enorme druk op die gesondheidssektor. Die soeke na nuwe antimikrobiese middels en behandelings is dus van uiterste belang en sal altyd ‘n integrale rol in geneeskunde navorsing speel. Antimikrobiese peptiede (AMPe) kan moontlik as alternatief tot antibiotika dien, of kan in kombinasie daarmee gebruik word om die ontwikkeling van antimikrobiese- weerstandbiedenheid te verhoed. AMPe speel ‘n rol in ingebore beskerming en word deur soogdiere, plante, reptiele, voëls, visse en insekte geproduseer. AMPe van bakterieë (bakteriosiene), veral die van melksuurbakterieë (MSB), wek toenemende belangstelling as antimikrobiese middels vir die behandeling van bakteriële infeksies. Nanovesels, wat deur middel van ‘n elektrospin proses geproduseer word, het eienskappe wat hul aanloklik maak as wondbedekking, naamlik hoë suurstof deurlaatbaarheid, verskeie porie grottes, ‘n hoë oppervlakte tot volume verhouding, sowel as ‘n morfologiese struktuur wat die ekstrasellulêre matriks naboots. Die vermoë om ‘n verskeidenheid biologies aktiewe komponente in nanovesels te inkorporeer verhoog hul potensiaal as wondbedekkingsmateriaal. ‘n Unieke benadering is die inkorporasie van bakteriosiene van MSB in nanovesels om ‘n antimikrobiese wondbedekking te ontwikkel. In hierdie studie is die vermoë om bakteriosiene van MSB in nanovesels te inkorporeer, deur middel van ‘n mede-elektrospin proses, ondersoek. Plantarisien 423, geproduseer deur Lactobacillus plantarum 423, was suksesvol deur die mede-elektrospin proses in poliëtileen oksied (PEO) nanovesels geinkorporeer. Plantarisien 423 het na die elektrospin proses steeds sy antimikrobiese aktiwiteit behou en het die groei van Lactobacillus sakei DSM 20017T en Enterococcus faecium HKLHS geïnhibeer. Lewende selle van L. plantarum 423 was ook suksesvol deur die mede-elektrospin proses in PEO nanovesels geinkorporeer, alhoewel die lewensvatbaarheid van die selle effens afgeneem het. ‘n Nanovesel matriks is ontwikkel om die vrystelling van plantarisien 423 en bakteriosien ST4SA, geproduseer deur Enterococcus mundtii ST4SA, te beheer deur PEO en poli(D,L-melksuur) (PDLMS) in ‘n geskikte oplosmiddel te vermeng voor die elektrospin proses. Nanovesels is geproduseer wat die bakteriosiene oor ‘n verlengde tydperk kon vrystel. ‘n PEO:PDLMS (50:50) nanovesel matriks het sy stuktuur die beste behou tydens inkubasie by 37 °C en het aktiewe plantarisien 423 en bakteriosien ST4SA vrygestel. Nisien A was met dieselfde tegniek in PEO:PDLMS (50:50) geinkorporeer en nisien A, wat deur die nanovesels vrygestel was, het die groei van Staphylococcus aureus in vitro geïnhibeer. Die nisien A-bevattende nanovesel matriks het die aantal lewende selle van S. aureus noemenswaardig verminder in geïnfekteerde wonde en kon die genesing van wonde, wat nie geïnfekteer was, stimuleer. Sover ons kon vastel is hierdie die eerste gepubliseerde navorsing wat toon dat bakteriosiene, geinkorporeer in nanovesels, gebruik kan word om vel infeksies te beheer en wondgenesing te stimuleer.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Because of the diversity of its indigenous microbiota, goat milk is considered a good source of new strains of lactic acid bacteria (LAB) with potential for exploitation as alternatives to food biopreservation. The increasing consumer demand for these alternatives justifies studies to investigate the antimicrobial potential of these microorganisms. The present study aimed to evaluate the genetic diversity and the antimicrobial action range of autochthonous strains of BAL isolated from goat milk that can be potentially used as biopreservatives in food. The bacteriocinogenic activity against Listeria monocytogenes and stability in different environmental conditions were also the target of this study. Fifty-seven isolates of BAL (33 isolates of Enterococcus spp., and 24 isolates of Lactococcus spp.), previously characterized as bacteriocinogenic by genotypic and phenotypic methods, were subjected to macrorestriction with SmaI and PFGE and their profiles were compared with the results of bacteriocinogenic genes previously searched. High genetic diversity was observed for both genders. Although PFGE showed sufficient discriminatory power, isolates with different results for bacteriocinogenic genes were grouped as identical in both genders. Twelve isolates from Lactococcus spp. and eighteen isolates of Enterococcus spp., representing different pulsotypes, were selected for evaluation of the antimicrobial activity range against 46 target microorganisms (including BAL, pathogens and spoilage microorganisms). Lactococcus strains showed a wide spectrum against the targets, especially against L. monocytogenes and Clostridium spp.. Enterococcus spp., similarly, was able to inhibit various targets, acting mainly against Listeria spp. Gram- negative microorganisms also showed sensitivity to isolates of both genders. Six isolates (four Enterococcus spp., and two Lactococcus spp.) were evaluated for bacteriocinogenic potential against L. monocytogenes strains from different serotypes and all of them were inhibited by bacteriocins produced by these LAB. Additionally, bacteriocins produced by these isolates showed a wide range of stability at different pH values and temperatures. The data showed that goat milk can contain a diverse microbiota able to inhibit microorganisms of interest to the food industry and can be potentially employed in biopreservation of foods produced at different processing conditions.
O leite de cabra, devido a diversidade de sua microbiota autóctone, é considerado uma boa fonte de novas cepas de Bactérias Ácido Láticas (BAL) com potencial para exploração como alternativas à biopreservação de alimentos. A crescente demanda dos consumidores por essas alternativas justifica estudos que investiguem o potencial antimicrobiano desses micro-organismos. O presente estudo teve como objetivo avaliar a diversidade genética e o espectro de ação de cepas de BAL antagonistas autóctones de leite de cabra que podem ser potencialmente utilizadas como biopreservantes em alimentos. A atividade bacteriocinogênica contra Listeria monocytogenes e estabilidade das bacteriocinas em diferentes condições ambientais também foram alvo deste trabalho. Cinquenta e sete isolados de BAL (33 isolados de Enterococcus spp. e 24 isolados de Lactococcus spp.), previamente caracterizados como bacteriocinogênicos por metodologias genotípicas e fenotípicas, foram submetidos à macrorrestrição com a enzima SmaI e PFGE e seus perfis foram comparados aos resultados de genes bacteriocinogênicos previamente pesquisados. Alta variabilidade genética foi observada para ambos os gêneros. Embora o PFGE tenha apresentado poder discriminatório suficiente, isolados com diferentes resultados para genes de bacteriocinas foram agrupados como idênticos em ambos os gêneros. Doze isolados de Lactococcus spp. e dezoito isolados de Enterococcus spp., representativos de diferentes pulsotipos, foram selecionados para avaliação do espectro de ação antimicrobiana contra 46 micro- organismos indicadores (incluindo BAL, micro-organismos deteriorantes e patógenos). Os isolados de Lactococcus spp. demonstraram amplo espectro de ação sobre os micro-organismos alvo, com destaque para a atividade contra os patógenos L. monocytogenes e Clostridium spp.. Enterococcus spp., de modo similar, foi capaz de inibir diversos micro-organismos alvo, apresentando atividade principalmente contra Listeria spp. Cepas Gram-negativas também apresentaram sensibilidade aos isolados de ambos os gêneros. Seis isolados (quatro Enterococcus spp. e dois Lactococcus spp.) foram avaliados quanto ao potencial bacteriocinogênico contra cepas de L. monocytogenes de diferentes sorotipos e todos os sorotipos foram inibidos pelas bacteriocinas produzidas pelas BAL. Adicionalmente, as bacteriocinas produzidas por estes isolados apresentaram ampla faixa de estabilidade em diferentes valores de pH e temperaturas. Os dados obtidos demonstraram que o leite de cabra pode conter uma microbiota bacteriocinogênica diversificada, capaz de inibir micro-organismos de interesse à indústria de alimentos, podendo ser potencialmente empregadas na bioconservação de alimentos produzidos em diferentes condições de processamento.