Orientador: Taís Maria Bauab / Banca: Eliana Aparecida Varanda / Banca: Daisy Nakamura Sato / Resumo: O gênero Maytenus (Celastraceae), atualmente compreende cerca de 80 espécies distribuídas por todo território brasileiro. A espécie Maytenus ilicifolia é conhecida popularmente pelos nomes de espinheira-santa, espinheira-diva, salva-vidas, sombra de touro, cancerosa e coromilho do campo, e utilizada contra hiperacidez e ulcerações do estômago. Com o objetivo de caracterizar o potencial biológico desta planta, foi estudada a atividade antibacteriana e antioxidante do extrato diclorometânico (DCM), frações, subfrações e duas substâncias puras (maitenina e catequina) isoladas das cascas de raízes de M.ilicifolia, obtidas por maceração a frio. Foi utilizada a técnica de diluição em microplacas frente às bactérias Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 19659, Escherichia coli ATCC 25922 e Pseudomonas aeruginosa ATCC 27853. A concentração inibitória mínima (CIM) para o extrato e as substâncias testadas, expresso como a menor concentração capaz de inibir crescimento bacteriano foi avaliada em microplacas contendo meio líquido aos quais foram adicionados extratos, frações, substâncias puras e antibióticos, sendo estes últimos usados como controles positivos. Para a atividade antioxidante foram realizados ensaios espectrofotométricos do 2,2'-Azinobis-3-etilbenzotiazolina-6-acido sulfônico (ABTS.+); do íon hipoclorito (OCl-) e da taurina-cloramina (via oxidação do 3,3',5,5', tetrametilbenzidina), utilizando como padrões quercetina e cisteína. Foi demonstrada forte atividade da maitenina com CIM de 0,48 μg/mL para as bactérias Gram positivas, e a catequina não apresentou atividades contra as bactérias testadas. Na avaliação da atividade antioxidante o extrato DCM e a maitenina não foram tão eficazes quanto a atividade da catequina que... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The genus Maytenus (Celastraceae) currently includes 80 species distributed throughout the Brazilian territory. The species Maytenus ilicifolia popularly known by the names of "espinheira-santa", "espinheira-diva", "salva-vidas", "sombra de touro", "cancerosa" and "coromilho do campo", with activity against hyperacidity and gastric ulcers. In order to characterize the biological potential of this plant, was studied the antibacterial activity of the dichloromethane extract (DCM), fractions, subfractions and two substances (maitenin and catechin) isolated from the roots of M. ilicifolia obtained by cold maceration. To evaluation of the antimicrobial potential was used the microplates dilution technique using the bacteria Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 19659, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Minimum inhibitory concentration (MIC) for the extract and the substances tested, expressed as the lowest concentration able to inhibit bacterial growth, was evaluated in microplates containing liquid medium to which were added extracts, fractions, subfractions, pures substances and antibiotics, the latter being used as positive controls. For the antioxidant activity tests were performed spectrophotometric: 2,2 '-Azinobis-3-ethylbenzothiazoline-6- sulfonic acid (ABTS.+); of OCl- and taurinecloramine (via oxidation of 3,3', 5,5 ' , tetramethylbenzidine), using quercetin and cysteine as standards. It was demonstrated a strong activity to maitenin, with MIC of 0,48 μg/mL for Gram positive bacteria and catechin did not show activity against the bacteria tested. In the evaluation of antioxidant activity the extract DCM and maitenin... (Complete abstract click electronic access below) / Mestre

Helicobacter pylori is a Gram-negative bacterium that is the major cause of many upper gastrointestinal diseases such as gastritis and peptic ulcer disease. It has the unique ability of colonising the human gastric mucosa. Failure in complete eradication of H. pylori in infected patients, mainly due to antibiotic resistance, has necessitated the development of better therapeutics, especially from natural sources. In this study, extract of Maris piper potatoes were obtained and evaluated for antibacterial activity against H. pylori in vitro. Antibacterial activity was carried out against antibiotic-sensitive and clinical antibiotic-resistant H. pylori strains, as well as a range of Gram-negative bacteria including Helicobacter and Campylobacter species, using the viable count method. Result of the antibacterial assays indicated that potato extract is bactericidal against H. pylori lab strain as well as clinical antibiotic-resistant strains, with minimum inhibitory concentration at 15.6 mg/ml. Potato extract also showed minimal antibacterial activity against other Gram- negative bacteria tested, with minimum inhibitory concentration at 250 mg/ml. The effect of the extract on the morphology of H. pylori was also observed by transmission electron microscopy (TEM). TEM analysis of potato extract-treated H. pylori cells showed disruption of the morphology of H. pylori, characterized by separation of the outer membrane from the inner membrane and loss of cell shape. Potato extract also caused hyperpolarisation of H. pylori plasma membrane; however it is unclear whether the membrane active pumping activity is affected. Mutants of H. pylori that are resistant to potato extract were generated as a means to identify the target of potato extract within the H. pylori genome. Genome sequence analysis led to the discovery of a hypothetical protein, encoded by HP0603 gene, which may be involved in inducing resistance to potato extract. The results obtained in this study provide great insights into the anti-H. pylori activity of potato extract. Overall, this study suggests the potential use of potato extract as a source of anti-H. pylori agents; and stimulates further studies into identifying its mechanism of action.

abstract: The prevalence of antibiotic resistant bacterial pathogens has increased since the introduction of penicillin in the 1940s. Insufficient development of novel antibacterial agents is leaving us with a failing arsenal of therapies to combat these pathogenic organisms. We have identified a clay mineral mixture (designated CB) that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens, yet the antibacterial mechanism of action remains unknown. Antibacterial susceptibility testing of four different clay samples collected from the same source revealed that these natural clays had markedly different antibacterial activity. X-ray diffraction analyses of these minerals revealed minor mineralogical differences across the samples; however, ICP analyses demonstrated that the concentrations of many elements, Fe, Co, Cu, Ni, and Zn in particular, vary greatly across the four clay mixture leachates. Supplementation of a non-antibacterial leachate containing lower concentrations of Fe, Co, Ni, Cu, and Zn to final ion concentrations and a pH equivalent to that of the antibacterial leachate resulted in antibacterial activity against E. coli and MRSA, confirming the role of these ions in the in vitro antibacterial clay mixture leachates. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to CB-L. Supplementation of CB-L with ROS scavengers eliminated oxidative damage in E. coli, but did not rescue the cells from killing, indicating that in vitro killing is due to direct metal toxicity and not to indirect oxidative damage. Finally, we ion-exchanged non-antibacterial clays with Fe, Co, Cu, and Zn and established antibacterial activity in these samples. Treatment of MRSA skin infections with both natural and ion-exchanged clays significantly decreased the bacterial load after 7 days of treatment. We conclude that 1) in vitro clay-mediated killing is due to toxicity associated directly with released metal ions and not to indirect oxidative damage and 2) that in vivo killing is due to the physical properties of the clays rather than metal ion toxicity. / Dissertation/Thesis / Ph.D. Microbiology 2014

No description available.

In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides and aptamers, as therapeutic and diagnostic entities. Therapeutic Bio-Oligomers; A series of peptoid analogs have been designed and synthesised using solid phase synthesis. These peptoids have been subjected to biological evaluation to determine structure-activity relationships that define their antimicrobial activity. In total 13 peptoids were synthesised. Out of 13 different peptoids, only one peptoid called Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal activity. TOP kills bacteria under non-dividing and dividing conditions. The Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM. The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the 10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for TOP as 100. This TI is well above previously reported peptidomimetics TI of around 20. TOP demonstrates selective bacterial killing in co-culture systems and intracellular bacterial killing activity. Diagnostic Bio-Oligomers; In the second part of my thesis, I investigated aptamer and peptide-based molecular probes to detect MRSA. As well as screening aptamers and peptide probes against whole MRSA, I over-expressed and purified PBP2A protein. This purified protein was used as a target for aptamer and peptide probes to detect MRSA. Two different aptamer libraries were initially screened for utility. In-vitro conditions for SELEX were optimised. Biopanning with a phage derived peptides was also performed. Target sequences for both methods were identified and chemically synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and confocal imaging showed no specificity for MRSA detection with either method. The Bio-Oligomers and the in-vitro selection methodology require further refinement to improve diagnostic utility.

Potassium ions are the most abundant cation and potassium transport is essential in maintaining cellular homeostasis through the regulation of cell turgor and cytoplasmic pH. It allows bacteria to grow and survive, therefore, the potassium pool needs to be strictly controlled, which is mainly performed by transport systems that contain a KTN domain. The potassium efflux system, Kef, is such a KTN-bearing system and it is widespread among Gram negative bacteria. The system provides protection against harmful electrophiles through cytoplasmic acidification. Kef is a glutathione-regulated protein: it is inhibited by glutathione (GSH), but it becomes activated by binding glutathione-S-conjugates (GSX), that are formed in the presence of electrophiles. GSH or GSX are bound in the same pocket that is located in a cytosolic regulatory domain which controls the K+ flux. Previous studies already showed that bacterial growth is inhibited when the gating of Kef is manipulated, which makes Kef a potential target for developing novel antibacterial drugs. Structure-Function studies have already lead to a better understanding of the regulation of potassium efflux activity, but no quantitative analyses had been performed until now. A simplified model Kef system (SdKef) is presented and a novel assay was developed that provided new insights into the structural components necessary for the gating of Kef. This assay makes the search for modulators of Kef, and therefore potential novel antibacterial drugs, more easily accessible. Another objective was to identify the nucleotide(s) bound and to determine its role in controlling the Kef system. This nucleotide was identified as AMP which is essential for stability of the Kef system.

Self-etching dentine bonding agents are a recent addition to the choice of bonding agents which a clinician has available to bond resin restorations to tooth structure. The so-called ‘traditional’, total-etch fourth and fifth generation dentine bonding agents have proven their clinical abilities and the question now remains whether these ‘new’ self-etching dentine bonding agents will clinically perform as well as the ‘proven’ total-etch dentine bonding agents. For the purpose of this dissertation the author completed three research projects which were performed to evaluate the efficacy of a selection of dentine bonding agents and then used the results to compare some properties (shear bond strength, microleakage, and anti-bacterial properties) of total-etch dentine bonding agents with some self-etching dentine bonding agents. All discussions will focus on the three dentine bonding agent properties evaluated by the three research projects performed. The three specific aims of this study were: <ul> <li> To compare the dentine shear bond strength of a selection of self-etching dentine bonding agents with that of a total-etch dentine bonding agent control.</li> <li> To compare dentine and enamel microleakage values of a selection of self-etching bonding agents with that of a total-etch dentine bonding agent control.</li> <li> To evaluate the possible anti-bacterial properties of a selection of dentine bonding agents, with focus placed on the self-etching dentine bonding agent ABFb (Clearfil Protect Bond).</li></ul> The studies performed by the author achieved comparative/similar results to some studies described in the literature but it is clear from the literature that some studies provide conflicting results, especially leakage of enamel margins when using self-etching bonding agents. Taking into consideration the limitations of the three studies performed, it can be concluded that as far as the three evaluated properties of self-etching dentine bonding agents are concerned, they should prove to be acceptable clinical alternatives for use in place of total-etch dentine bonding agents. Copyright / Dissertation (MSc(Odont))--University of Pretoria, 2010. / Odontology / unrestricted

Cyclic dipeptides (CDPs) are amino acid-based compounds, some of which possess antibacterial activity. The encapsulation of certain drugs into liposomes has been found to improve their activity in terms of bioavailability and duration of action. Liposomes are small vesicles that are under investigation as drug carriers for the delivery of therapeutic agents. A number of liposome formulations are currently under clinical trial review, whilst some have already been approved for clinical use. The aim of this study was to optimize a liposomal cyclo(Tyr-Pro) formulation and to assess its antibacterial activity against various Gram-positive and Gram-negative bacteria. Response surface methodology (RSM) using the central composite design (CCD) model was used to optimize liposomal formulations of cyclo(Tyr-Pro) for each of the four bacteria, namely Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Percent drug encapsulated and bacterial inhibition were investigated with respect to two independent variables, i.e. lipid composition and cholesterol content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each bacterium. The model selected by the software failed to adequately correlate the predicted models to the experimental data. The in vitro experiments showed that the antibacterial activity of liposome-encapsulated cyclo(Tyr-Pro) was superior to that of its free counterpart. Binding maximum or Bmax for the encapsulated compound at concentrations as low as 0.412 mg/ml, was significantly higher than that obtained for free cyclo(Tyr-Pro) which was tested at a concentration of 20 mg/ml. Furthermore, encapsulation of cyclo(Tyr-Pro) into a liposome formulation enhanced its potency. This was evident in the lower IC50 values for the liposomal compound when compared to free cyclo(Tyr-Pro).

At the author’s request, the abstract has been removed due to the confidential nature of the thesis. It will be added once the embargo period has passed.

The in vitro activity of twelve cephalosporins (first generation: Cephalothin, Cefazolin; second generation: Cefoxitin, Cefamandole, Cefuroxime, Cedonicid; third generation: Ceftazidime, Ceftizoxime, Cefotaxime, Cefoperazone, Ceftriaxone, Moxalactam) were studied against 146 strains of Gram negative bacilli belonging to the following families: Enterobacteriaceae Proteus vulgarius (2), P. mirabilis (5), Providencia stuartti (6), P. alkalifaciens (5), Morganella morganii (16), Serratia marcescens (14), Enterobacter cloacae (17), E. aerogenes (9), Kluyvera ascorbata (3), Citrobacter freundii (14), C. diversus (3), C. amalonaticus (1), Yersinia intermedia (1), Y. enterocolitica (2); Pseudomonadaceae: Pseudomonas aeruginosa (31), P. fluorescens (3); Neisseriacaee: Acinetobacter anitratus (3), Acinetobacter lwoffi (1); and Vibrionaceae: Aeromonas hydrophilia (4), Plesiomonas shigelloides (1), Campylobacter jejuni (5)). This investigation, which studied the activity of all the mentioned cephalosporins against each strain, suggests that resistance to the third generation cephalosporins has already emerged in such species as S. marcescens, E. cloacae, E. aerogenes, C. freundii, P. aeruginosa, P. fluorescens, A. anitratus, A. lwoffi, and Campylobacter jejuni. This resistance is most pronounced in Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa. The second generation cephalosporins, particularly Cefoxitin and perhaps Cefuroxime, are chief inducers of resistance in Enterobacter and Serratia. In the pseudomonads difference mechanisms seem to operate than those taking place in Enterobacter-Serratia. The study also shows that resistance is not a generic characteristic in Proteus, Providencia, or Citrobacter but rather specific. Some aspects of mechanisms of resistance to cephalosporins are discussed. Concern is here indicated that at least in certain groups of bacteria, the use of the second generation cephalosporins may lead to emergence of resistance to the third generation group.