A própolis é uma substância resinosa coletada pelas abelhas de diversas partes das plantas, à qual têm sido atribuídas propriedades antimicrobiana, antioxidante, antinflamatória, antiviral, entre outras. Sua composição química depende de vários fatores, como a localização geográfica. Um novo tipo de própolis brasileira, denominada de própolis vermelha, por sua coloração intensa característica, foi identificada e coletada em região de mangue do Estado de Alagoas. O objetivo deste trabalho foi fracionar e isolar compostos com atividade antibacteriana dessa nova variedade de própolis. O extrato etanólico da própolis vermelha foi fracionado pela técnica de extração líquido-líquido, originando as frações hexânica (fr-Hex) e clorofórmica (fr-Clo). A fr-Clo apresentou alta atividade antibacteriana pelos testes de Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM), contra as bactérias Staphylococcus aureus, Streptococcus mutans e Actinomyces naeslundii. Dessa forma, a fr-Clo foi refracionada por coluna seca gerando sete sub-frações. Após a avaliação da atividade antibacteriana das sub-frações pelas técnicas de CIM e CBM, a sub-fração 3, a mais bioativa, foi purificada em coluna de Sephadex LH-20. Dessa purificação foram obtidas três sub-frações bioativas, as quais foram submetidas ao isolamento dos compostos pela técnica de Cromatografia Líquida de Alta Eficiência (CLAE) preparativa. Dois compostos foram isolados e denominados de composto 1 e composto 2. O composto 2 foi o mais potente para as atividades inibitória e bactericida e sua CIM para os três tipos de bactéria testados variou entre 15,6 e 31,2 µg/mL, enquanto para o composto 1, este parâmetro variou de 31,2 a 62,5 µg/mL. As CBM do composto 1 e do composto 2 variaram entre 125 e 250 µg/mL e entre 31,2 e 62,5 µg/mL, respectivamente. Por meio da técnica Ressonância Magnética Nuclear (RMN) foi possível identificar o composto 1 como pertencente à classe das isoflavanas e o composto 2 como uma chalcona (isoliquiritigenina). A forte atividade antibacteriana apresentada pelos compostos isolados da própolis vermelha torna este produto uma importante fonte de compostos antibacterianos naturais. / Propolis is a resinous material, collected by honeybees from several parts of plants, known for its antimicrobial, antioxidant, anti-inflammatory, antiviral properties, among others. Its composition varies according to several factors, such as the geographical location. A novel type of Brazilian propolis, named red propolis due to its intense characteristic color, was collected in a mangrove area in the State of Alagoas. This research aimed to fractionate and isolate the compounds in this new type of propolis that present antibacterial activity. The ethanolic extract of red propolis (EEP) was fractionated using the liquid-liquid extraction technique, yielding the hexanic (Hex-fr) and the chloroformic fractions (Chlo-fr). Chlo-fr showed high antibacterial activity determined by the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests against Staphylococcus aureus Streptococcus mutans, and Actinomyces naeslundii. Thus, Chlo-fr was refractionated by Dry-Column Chromatography, yielding seven subfractions. After submitted to MIC and MBC techniques to assess their antibacterial activity, and subfraction 3, the most bioactive of them, was purified in Sephadex LH-20 column. After this purification, three bioactive subfractions were obtained, submitted to isolation of compounds using preparative High Performance Liquid Chromatography (HPLC). Two compounds were isolated and named compound 1 and compound 2. Compound 2 presented the highest inhibitory and bactericidal activities and its MIC for the three types of bacteria tested ranged from 15.6 to 31.2 µg/mL, whereas for compound 1, this parameter ranged from 31.2 to 62.5 µg/mL. MBC of compound 1 and compound 2 ranged from 125 to 250 µg/mL and from 31.2 to 62.5 µg/mL, respectively. Through Nuclear Magnetic Resonance tecnique (NMR), it was possible to identify the compound 1 as belonging to class of isoflavans and the compound 2 as a chalcone (isoliquiritigenin). Due to the strong antibacterial activity presented by the compounds isolated from red propolis, it can be concluded that this product is an important source of natural antibacterial compounds.

As b-defensinas são pequenos peptídeos catiônicos com estrutura rica em folhas b pregueadas e seis cisteínas conservadas. São bastante estudadas em mamíferos, mas pouco em serpentes. Utilizando a PCR foram descritos 13 genes b-defensina-símiles em serpentes do gênero Bothrops e Lachesis . Eles se organizam em três éxons e dois íntrons; há alta similaridade no éxon1, íntron 1 e 2, mas os éxons 2 e 3 estão sob evolução acelerada. A análise filogenética por máxima parcimônia revelou que o gene ancestral de b-defensina-símile pode ter três éxons em vertebrados e sua evolução ocorreu de acordo com o modelo de nascimento-e-morte. Peptídeos b-defensina-símiles reduzidos, testados por ensaio de inibição de crescimento em meio líquido, apresentaram atividade inibitória contra Escherichia coli, Citrobacter freundii, Micrococcus luteus e Staphylococcus aureus. A crotamina reduzida foi mais ativa que a forma nativa. Os resultados indicam que a carga líquida era a característica bioquímica mais importante na atividade antibiótica dos peptídeos b-defensina-símiles. / b-defensins are small basic cationic peptides with b-sheet-rich fold plus six conserved cysteines. They are fully studied in mammals, but scarce in snakes. Using a PCR approach, we described 13 b-defensin-like sequences in Bothrops and Lachesis snakes. They are organized in three exons and two introns; they show high similarities in exon 1, intron 1 and intron 2, but exons 2 and 3 have undergone accelerated evolution. Phylogenetic analysis was done using maximum parsimony indicate that the ancestral b-defensin-like gene may have three exons in vertebrates and that their evolution occurred according to a birth-and-death model. Reduced b-defensin-like peptides were tested by microbroth dilution assay, showed inhibitory activity against Escherichia coli, Citrobacter freundii, Micrococcus luteus e Staphylococcus aureus. The reduced form of crotamine was more active than native. The results indicate that the positive net charge is the most important biochemical characteristic of b-defensin-like peptides to antibiotic activity.

Os tipos de cassete cromossômico estafilocócico mec (SCCmec) encontrados em cepas de Staphylococcus aureus resistente a oxacilina (ORSA) isoladas em ambiente hospitalar são denominados I, II e III. Um novo SCCmec, tipo IV, foi identificado em cepas isoladas de pacientes que não tinham conexão aparente com hospitais. Essas cepas, denominadas ORSA adquiridas na comunidade ou CA-ORSA, não costumam conter genes de resistência a antimicrobianos além do gene mecA, porém, são mais virulentas. Com a finalidade de conhecer a diversidade estrutural do SCCmec de cepas ORSA brasileiras, foram estudadas 50 cepas ORSA, entre elas algumas S. aureus com resistência intermediária a vancomicina (VISA) e S. aureus com resistência heterogênea intermediária a vancomicina (HVISA). As cepas foram isoladas entre 1995 e 2000, provenientes de hospitais de 13 Estados brasileiros, pertencentes a diversos clones de PFGE. As cepas foram analisadas quanto ao perfil de sensibilidade, RFLP do gene da coagulase, fagotipagem e tipagem do SCCmec. A maioria das cepas é pertencente ao clone endêmico brasileiro, que carrega o SCCmec tipo III, também presente em outros clones de PFGE. O SCCmec tipo IV, encontrado em 3 cepas (clones J, L e D), mostrou suscetibilidade a um maior número de antimicrobianos pertencentes a diferentes classes, em comparação aos outros tipos de SCCmec. O SCCmec tipo IV está presente entre os ORSA brasileiros e pode estar disseminando na comunidade e hospitais do país. / The types of SCCmec found in nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains can be designated type I, II or III. A novel type of SCCmec, designated type IV, was identified in strains isolated in outpatients. These strains, called community-acquired MRSA or CA-MRSA, do not contain antimicrobial resistance genes other than mecA, however, they are more virulent. In order to characterise the structural diversity of SCCmec in Brazilian MRSA strains, 50 MRSA strains were studied, including some vancomycin intermediate resistant S. aureus (VISA) and heterogeneous vancomycin intermediate S. aureus (HVISA) strains. All strains were isolated between 1995-2000, in hospitaIs in 13 Brazilian States, and belonged to different PFGE clones. Strains were analysed as to their susceptibility profile, RFLP of the coagulase gene, phagetype and SCCmec type. The majority of strains belonged to the Brazilian Endemic Clone, which carries an type IH SCCmec, which in turn, was also found in other PFGE clones. The type IV SCCmec was found in 3 strains (belonging to the J, L and D clones) and presented a susceptibility profile to a number of drugs of different antimicrobial classes. The type IV SCCmec is present among Brazilian MRSA strains and can be disseminated in the community and in hospitaIs throughout the country.

Partículas e filmes híbridos de polímeros e compostos de amônio quaternário (CAQ) foram caracterizados quanto a propriedades físicas e atividade contra cepas de Pseudomonas aeruginosa ATCC 27853 e Staphylococcus aureus ATCC 25923. Partículas híbridas foram obtidas a partir de fragmentos de bicamada (BF) de brometo de dioctadecildimetilamônio (DODAB), adicionados consecutivamente de soluções de carboximetilcelulose (CMC) e cloreto de poli (dialildimetilamônio) (PDDA) e, a seguir, caracterizadas por espalhamento de luz dinâmico para determinação de distribuições de tamanho, diâmetro médio (Dz) e potencial-zeta (ζ). Filmes híbridos foram obtidos por revestimento rotacional de lamínulas de vidro a partir de uma solução clorofórmica de poli (metil metacrilato) (PMMA) e CAQ onde [PMMA] = 10 mg/mL e [CAQs] = 0,03 4 mM de DODAB, brometo de cetiltrimetilamônio (CTAB) ou brometo de tetrapropilamônio (TPAB). Molhabilidade dos filmes e difusibilidade dos CAQs nos filmes imersos em água foram avaliadas por determinação de ângulo de contato e de tensão superficial na interface ar-água, respectivamente. Atividade antimicrobiana foi avaliada por plaqueamento e contagem de viáveis, tanto para os filmes quanto para as partículas. Para os filmes, também foram determinados halos de inibição. Partículas de DODAB BF/ CMC/ PDDA exibiram Dz e ζ dependentes da concentração dos componentes. A 0,1 mM de DODAB, 0,1 mg/mL de CMC e 0,1 mg/mL de PDDA, foram obtidas partículas pequenas (Dz = 100 nm; ζ = 30 mV). Com 0,5 mM de DODAB, 0,5 mg/mL de CMC e 0,5 mg/mL de PDDA foram obtidas partículas grandes (Dz = 470 nm; ζ = 50 mV). Contra 107 UFC/mL, 1 - 2 µg/mL de PDDA, sozinho em solução ou em forma de partículas, matou 99 % das células de P. aeruginosa. O mesmo ocorreu contra S. aureus (107 UFC/mL) em 10 µg/mL de PDDA. A ação antimicrobiana mostrou-se dependente da quantidade de cargas positivas nas partículas e independente do tamanho da partícula. Filmes de PMMA/ CAQ apresentaram maior molhabilidade do que aqueles de PMMA puro. Filmes de PMMA/ DODAB e PMMA/ TPAB submersos em água não causaram alterações de tensão superficial na interface ar-água, indicando baixa difusibilidade destes CAQs a partir dos filmes. Filmes de PMMA/ CTAB submersos em água reduziram a tensão superficial até aproximadamente 60 mN/m, mostrando a difusibilidade do CTAB no filme e sua organização na interface ar-água. O efeito antimicrobiano dos filmes PMMA/ DODAB e PMMA/ CTAB foi dependente da concentração do CAQ utilizada no preparo do filme. Viabilidade celular de 0% contra 107 UFC/mL de ambas as bactérias ocorreu com 4 mM de DODAB ou, com 2 ou 0,2 mM de CTAB contra P. aeruginosa ou S. aureus, respectivamente. Filmes de PMMA/ TPAB não apresentaram atividade antimicrobiana. Com a emergência de micro-organismos resistentes aos antibióticos, os novos nanomateriais antimicrobianos híbridos de polímeros e compostos de amônio quaternário podem representar uma alternativa de fácil obtenção e baixo custo. / Hybrid particles and films from polymers and quaternary ammonium compounds (QAC) were characterized regarding its physical properties and antimicrobial activity against strains of Pseudomonas aeruginosa ATCC 27853 or Staphylococcus aureus ATCC 25923. Hybrid particles were obtained from dioctadecyldimethylammonium bromide (DODAB) bilayer fragments (BF), consecutively added of carboximethylcellulose (CMC) and poly (diallyldimethylammonium) chloride (PDDA) solutions, and then, characterized by dynamic light-scattering for determination of size distributions, z-average diameter (Dz) and zeta-potential (ζ). Hybrid films were obtained by spin-coating of a chloroformic solution of poly (methylmethacrylate) (PMMA) and QAC on glass coverslips at [PMMA] = 10 mg/mL and [QACs] = 0.03 -4 mM where QACs were DODAB, cetyltrimethylammonium bromide (CTAB) or tetrapropylammonium bromide (TPAB). Films wettability and QACs diffusibility in the films immersed in water were evaluated by determinations of contact angle and surface tension at air-water interface, respectively. Antimicrobial activity was evaluated by plating and CFU counting both for particles and films. For the hybrid films, width of inhibition zone was also determined. DODAB BF/ CMC/ PDDA particles exhibited Dz and ζ dependent on the concentrations of the components. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles were obtained (Dz = 100 nm; ζ = 30 mV). At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles were obtained (Dz = 470 nm; ζ = 50 mV). At 107 CFU/mL, cell viability of 1 % for P. aeruginosa was obtained for PDDA in solution or covering particles at 1 or 2 µg/mL PDDA, respectively. Against S. aureus (107 CFU/mL) at 10 µg/mL PDDA, a similar result was obtained. The antimicrobial effect was dependent on the amount of positive charge on particles and independent on particle size. The hybrid films of PMMA/ QAC showed higher wettability than those of pure PMMA. PMMA/ DODAB and PMMA/ TPAB hybrid films, submerged in water, did not cause changes in surface tension at the air-water interface, indicating low diffusibility for both DODAB and TPAB in hybrid films. Films of PMMA/ CTAB, submerged in water, reduced the surface tension to about 60 mN/m, showing that CTAB could diffuse from the film to the air-water interface and change its surface tension. The antimicrobial effect of PMMA/ DODAB and PMMA/ CTAB hybrid films was clearly dependent on the QAC concentration used to prepare the films. Cell viability of 0% for P. aeruginosa and S. aureus (107 CFU/mL) occurred at 4 mM DODAB, or 2 and 0.2 mM CTAB, respectively. Films of PMMA/ TPAB showed no antimicrobial activity. With the emergence of microorganisms resistant to antibiotics, the novel hybrid antimicrobial nanomaterials may become important since they are inexpensive and easy to obtain.

<p>U skladu sa postavljenim ciljevima rada formirana je kolekcija&nbsp;<br />fenotipski i genotipski okarakterisanih multiplo rezistentnih&nbsp;<br />kultura genomske vrste <em>Acinetobacter baumannii</em>. Za sojeve iz&nbsp;<br />kolekcije kultura utvrđen je anti-<em>A. baumannii</em> efekat etarskih&nbsp;<br />ulja, biljnih ekstrakata i njihovih bioaktivnih komponenti, kao&nbsp;<br />nekonvencionalnih antimikrobnih agensa. Takođe, osim njihovog&nbsp;<br />pojedinačnog efekta utvrđen je i kombinovani efekat&nbsp;<br />konvencionalnih i nekonvencionalnih antimikrobnih agenasa, koji&nbsp;<br />je dovedo do redukcije minimalnih ihnibitronih koncentracija oba&nbsp;<br />agensa u kombinaciji. Kombinacije etarskih ulja i njihovih&nbsp;<br />bioaktivnih komponenti sa trenutno dostupnim konvencionalnim&nbsp;<br />antibioticima, čija je efikasnost smanjena kada se primenjuju&nbsp;<br />pojedinačno, predstavljaju novu perspektivnu strategiju u terapiji&nbsp;<br />infekcija multiplo rezistentnim sojevima vrste <em>A. baumannii</em>.</p> / <p>In accordance to the aims of the work the collection of phenotypic and<br />genotypic characterized multidrug-resistant cultures of genomic species<br /><em>Acinetobacter baumannii</em> was formed. For the strains from the culture<br />collection an anti-<em>A. baumannii </em>effect of the essential oils, plant extracts<br />and their bioactive components, as non-conventional antimicrobial<br />agents, was determined. Also, in addition to their individual effect the<br />combined effect of conventional and non-conventional antimicrobial<br />agents was determined, which lead to a reduction of the minimal<br />inhibitroy cocnentrations of both agents in combination. Combinations<br />of the essential oils and their bioactive components with the currently<br />available conventional antibiotics, whose efficiency reduced when<br />administered individually, represent a promising new strategy for the<br />treatment of infections caused by multidrug-resistant <em>A. baumannii.</em></p>

Dehydroquinate synthase (DHQS) catalyses the five-step transformation of the seven carbon sugar 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) to the carbacycle dehydroquinate (DHQ). Multiple studies have described in detail the mechanism of most of the steps carried out by DHQS with the exception of the final cyclisation step. In this study, (3S)-3-fluoro-DAH7P and (3R)-3-fluoro-DAH7P (fluorinated analogues of DAH7P) were produced and assayed across three phylogenetically distinct sources of DHQS in order to determine the role of the enzyme during the cyclisation step of the reaction. Incubation of (3S)-3-fluoro-DAH7P with DHQS from Escherichia coli, Pyrococcus furiosus, and Kiwifruit resulted in the production of different ratios of (6S)-6-fluoro-DHQ and 1-epi-(6S)-6-fluoro-DHQ for each enzyme. In addition, enzyme catalysis showed a slowing of reaction rates when (3S)-3-fluoro-DAH7P was used, suggesting that the fluorine at C-3 is stabilising the enol pyranose. An increase in the stabilisation of the fluoro-enol pyranose would allow release of this substrate intermediate from the enzyme to compete with the on-going on-enzyme reaction. The differences in the ratio of products formed suggest that the cyclisation occurs in part on the enzyme and that the epimeric product arises only by an abortive reaction pathway where the (3S)-3-fluoro-enol pyranose is prematurely released and allowed to cyclise free in solution. Once in solution, the (3S)-3-fluoro-enol pyranose could undergo a conformational change in the ring leading to the formation of the epimeric product. Furthermore, it is suspected that the position of fluorine influences the likely transition-state in carbacycle formation leading to the production of the epimeric product. This research has illuminated the role of the enzyme in guiding the correct stereochemistry of the product and illustrates the important molecular interplay between the enzyme and substrate.

Made available in DSpace on 2016-08-17T18:39:51Z (GMT). No. of bitstreams: 1 6095.pdf: 2718900 bytes, checksum: c4d734a7b2927b9a3c31ecfb042e26bd (MD5) Previous issue date: 2014-04-16 / Many efforts have been dedicated in the searching of natural compounds which are suitable to be applied in food conservation. Among them, the chitosan has presented promising results concerning the antimicrobial activity, structural preservation and delay of fruits senescence. However, the chitosan, as found commercially, is soluble only in acidic medium solutions what is considered a hindrance for fully applications as food conservative. In this sense, chitosan derivations which are able to be solubilized in neutral pH have been developed. The N,N,N Trimethylchitosan (TMC) is one of them, characterized as a hydrosoluble derivative of chitosan, which has been considered as an efficient antimicrobial agent with positive action as fresh food conservative. The aim of the present was the evaluation of the antimicrobial activity of the TMC against Gram-positive and Gram-negative bacteria and filament fungi. The TMC antimicrobial activity was assessed by comparing with the Commercial Chitosan (CC), by means of the determination of the Minimum Inhibitory Concentration (MIC), following the Clinical and Laboratory Standards Institute (CLSI) according to M7-A6 (NCCLS, 2003) and M38-A (NCCLS, 2002) standards, both recognized by the Agência Nacional de Vigilância Sanitária (ANVISA). Additionally, the TMC and CC antifungal activity was carried out by recoding the visual fungi growth and estimated from the percentage of infected samples by the fungy on cut faces of grape-tomatoes (Solanum lycopersicum variety cerasiforme), in coated and non-coated conditions, along seven days of incubation. The results concerning antibacterial activity points to a greater action of the TMC when comparing to some analysis performed on CC coating against four of the six strains analyzed: Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Salmonella enterica. Nevetheless the CC antifungal activity was considerable superior to that observed to TMC for three different fungi tested: Penicillium sp., Aspergillus flavus standard and Aspergillus flavus wild. However, both polymers showed high antibacterial and antifungal activity and are therefore considered as natural agents with relevant antimicrobial potential for the fresh food conservation. / Muitos têm sido os esforços pela procura de substâncias naturais que possam ser aplicadas à conservação de alimentos. Dentre as substâncias em estudo, a quitosana tem apresentado resultados promissores tanto com relação à atividade antimicrobiana quanto na manutenção estrutural de alimentos e redução da senescência em frutos. No entanto, a quitosana, em sua forma comercial, é solúvel apenas em soluções ácidas diluídas, o que dificulta sua plena utilização como conservante alimentício. Dessa forma, derivados de quitosana que apresentem capacidade de solubilização em pH neutro têm sido desenvolvidos. Dentre esses derivados a N,N,N-trimetilquitosana (TMQ), um polímero hidrossolúvel derivado da quitosana, tem sido avaliado como promissor agente antimicrobiano e conservador de alimentos frescos. Assim, o presente projeto teve como objetivo avaliar a atividade antimicrobiana da N,N,N-trimetilquitosana sobre o crescimento de bactérias Gram-positivas e Gram-negativas e de fungos filamentosos. A atividade antimicrobiana da TMQ foi avaliada mediante comparação com a Quitosana Comercial (QC) pela determinação das Concentrações Inibitórias Mínimas (CIM) a partir de testes padronizados pela Clinical and Laboratory Standards Institute (CLSI), segundo suas normas M7-A6 (NCCLS, 2003) e M38-A (NCCLS, 2002), ambas reconhecidas pela Agência Nacional de Vigilância Sanitária (ANVISA). Além disso, a avaliação da atividade antifúngica das substâncias foi realizada mediante leitura visual e estimada a partir do percentual de amostras infectadas pelos fungos sobre fatias de tomatecereja (Solanum lycopersicum variedade cerasiforme) revestidas e não revestidas, ao longo de sete dias de incubação. Os resultados indicaram uma maior atividade antibacteriana da TMQ em relação à QC para quatro das seis cepas bacterianas analisadas: Enterococcus faecalis, Staphylococcus aureus, Escherichia coli e Salmonella enterica. No entanto, a atividade antifúngica da QC superou a da TMQ para os três fungos testados: Penicillium sp., Aspergillus flavus padrão e Aspergillus flavus selvagem. Entretanto, ambos os polímeros avaliados apresentaram atividade antibacteriana e antifúngica satisfatória sendo, portanto, considerados como agentes naturais com potencial antimicrobiano relevante para a conservação de alimentos frescos.

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Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2015. / The increase in antibiotic resistant bacterial and fungal infections and the prevalence of oxidative stress-related conditions including cancers, cardiovascular diseases and diabetes has led to a consensus among pharmaceutical companies, clinicians and researchers that novel antimicrobial and antioxidant approaches are needed. These should be ideally efficacious, non toxic, easily accessible and affordable. There has been an increased interest in the identification of medicinal plants that possess both these bioactivities in an intrinsically related manner, allowing the simultaneous prevention of these ailments. Two South African herbal teas, rooibos and honeybush have been associated with a long history of medicinal use, hence their consideration for the current study. Numerous studies have been performed to evaluate the antioxidant activities of these South African herbal teas, however limited information about their antimicrobial activity currently exists. / National Research Foundation