Purification of the terminal oxidases of the facultative anerobe, M. denitrificans, was undertaken. The bacterium contains both a cytochrome c oxidase and a cyanide-insensitive DPNH oxidase. A procedure for purification of each of these enzymes is given. These procedures involve fractionation with ammonium sulfate, acetone, and calcium phosphate gel. By the use of the procedures given, cytochrome c oxidase can be purified seventeen-fold and the DPNH oxidase ninty-fold over the original cell-free extract. The cytochrome c oxidase was found to be associated with the particulate material of the cell, had an optimum activity at pH 7.0 to 7.4, and was not affected by aluminum, magnesium, or manganous ion. The enzyme was completely inhibited by cyanide and carbon monoxide but only 65% by azide. The cytochrome c oxidase oxidized reduced mammalian cytochrome c directly. Oxidative phosphorylation was demonstrated during oxidation of reduced mammalian cytochrome c by cell-free extracts of the bacteria. The absorption maxima of the reduced spectrum of the partially purified cytochrome c oxidase were at 420, 522, and 554 mμ., and the maxima of the difference spectrum were at 427, 522, and 551 mμ. The DPNH oxidase appears to be a soluble flavoprotein. An active DPNH oxidase preparation which was inactivated by ammonium sulfate fractionation, could be reactivated by addition of flavin mononucleotide; flavin adenine dinucleotide restored only partial activity. The Michaelis constant with respect to DPNH of the partially purified DPNH oxidase was found to be 3.42 x 10^-6 moles/liter. The activation energy of the DPNH oxidase was determined and found to be 14,900 cals/mole. The oxygen uptake of a cell-free extract of M. denitrificans functioning as a cytochrome c oxidase and as a DPNH oxidase, was 7.54 u liters/minute and 3.52 u liters/minute respectively. The cytochrome c oxidase appears to be the primary terminal oxidase, however, the DPNH oxidase does make a significant contribution to the aerobic respiration of the bacterium. The participation of the bacterial cytochrome c oxidase and DPNH oxidase in the aerobic respiration of the bacterium is discussed.

The problem in this investigation consists of, first, the procurement of several strains of Micrococcus pyogenes var. aureus; second, the comparison of the degree and rate of development of resistance of these organisms to terramycin; and, third, to study the morphological and physiological changes which occur during the development of resistance.

Universiẗat, Diss., 2004--Bielefeld.

The problem in this investigation consisted of, first, the isolation from human sources and identification of thirteen cultures of typical micrococci to be used as test organisms; second, the acquisition of blood plasma from thirty different human beings; and third, the determination of the possibility of individual variation in sensitivity of blood plasma to the micrococci used as test organisms as revealed by the coagulase test.

A hemoglobina (Hb) é uma fonte reconhecida de peptídeos com funções biológicas diversas. O fragmento 33-61 da cadeia α da Hb, isolado do trato gastrointestinal do carrapato Boophilus microplus, foi o primeiro a ser descrito com ação antimicrobiana. O seu análogo sintético amidado, Hb33-61a, mostrou-se ativo contra bactérias Gram-positivas e fungos [Fogaça et al. (1999) J. Biol. Chem. 274, 25330-4]. O estudo de análogos do Hb33-61 nas formas amidada e com carboxila livre revelou que a amidação provoca aumento significativo da atividade frente a Candida albicans. Por apresentar propriedades biológicas e estruturais idênticas às do Hb33-61a, o Hb40-61a pareceu ser a sua porção mÌnima ativa [Sforça et al. (2005) Biochemistry 44, 6440- 51; Machado et al. (2007) Biopolymers 88, 413-26]. Para comprovar tal sugestão, no presente trabalho, sintetizamos, purificamos e caracterizamos novos an·logos do Hb33-61a, bem como os avaliamos quanto às suas atividades frente a C. albicans e Micrococcus luteus. Os resultados confirmaram a sugestão apenas para a ação antifúngica. O análogo Hb40-61a também se mostrou ativo frente a C. albicans resistente a fluconazol. A sua atividade antifúngica se mostrou fortemente dependente da força iônica do meio. A sua baixa atividade hemolítica foi confirmada mesmo em meio de baixa força iônica. O peptídeo Hb40-61a não apresentou sinergismo com o fluconazol frente a C. albicans. A cinética de morte celular mostrou que ele mata a levedura de forma rápida. Portanto, esta hemocidina sintética pode apresentar valor comercial se a via de administração for tópica ou se o seu uso envolver meios de baixa força iônica. Além disso, ela é um modelo valioso para o estudo do mecanismo de ação de peptídeos antimicrobianos com características estruturais similares e pode servir de base para o desenho de novos agentes antibiôticos. / It is well known that hemoglobin (Hb) is a source of biologically active peptides. The fragment 33-61 of bovine hemoglobin α-chain, isolated from the gut contents of the tick Boophilus microplus, was the first identified with antimicrobial activity . Its amidated analogue, Hb33-61a, showed to be active against Gram-positive bacteria and fungi strains [FogaÁa et al. (1999) J. Biol. Chem. 274, 25330-4]. The study of a series of carboxyl-free and amidated synthetic analogues of Hb33-61 revealed that C-terminus amidation enhances the activity against Candida albicans. Since Hb33-61a and Hb40-61a presented identical biological and structural properties, it seemed that Hb40-61a was Hb33-61a minimal active motif [SforÁa et al. (2005) Biochemistry 44, 6440- To test this suggestion, in the present study 51; Machado et al. (2007) Biopolymers 88, 413-26]. we synthesized, purified and characterized Hb40-61a analogues and assayed them against C. albicans and Micrococcus luteus. The results confirmed the suggestion only for the antifungal activity. When tested against fluconazole-resistant C. albicans, Hb40-61a was also active. Its antifungal activity showed to be dependent on the ionic strength of the medium. Its low hemolytic activity was confirmed even under low ionic strength conditions. Hb40-61a had no synergic effect with fluconazole on C. albicans. In vitro time-kill assays demonstrated that Hb40-61a kills the yeast rapidly. Therefore, this synthetic hemocidin may be of commercial interest for topical application or other uses involving low ionic strength medium. Moreover, it can serve as a template for the study of the mechanism of action of structurally related antimicrobial peptides or for the design of novel antibiotic drugs.

A hemoglobina (Hb) é uma fonte reconhecida de peptídeos com funções biológicas diversas. O fragmento 33-61 da cadeia α da Hb, isolado do trato gastrointestinal do carrapato Boophilus microplus, foi o primeiro a ser descrito com ação antimicrobiana. O seu análogo sintético amidado, Hb33-61a, mostrou-se ativo contra bactérias Gram-positivas e fungos [Fogaça et al. (1999) J. Biol. Chem. 274, 25330-4]. O estudo de análogos do Hb33-61 nas formas amidada e com carboxila livre revelou que a amidação provoca aumento significativo da atividade frente a Candida albicans. Por apresentar propriedades biológicas e estruturais idênticas às do Hb33-61a, o Hb40-61a pareceu ser a sua porção mÌnima ativa [Sforça et al. (2005) Biochemistry 44, 6440- 51; Machado et al. (2007) Biopolymers 88, 413-26]. Para comprovar tal sugestão, no presente trabalho, sintetizamos, purificamos e caracterizamos novos an·logos do Hb33-61a, bem como os avaliamos quanto às suas atividades frente a C. albicans e Micrococcus luteus. Os resultados confirmaram a sugestão apenas para a ação antifúngica. O análogo Hb40-61a também se mostrou ativo frente a C. albicans resistente a fluconazol. A sua atividade antifúngica se mostrou fortemente dependente da força iônica do meio. A sua baixa atividade hemolítica foi confirmada mesmo em meio de baixa força iônica. O peptídeo Hb40-61a não apresentou sinergismo com o fluconazol frente a C. albicans. A cinética de morte celular mostrou que ele mata a levedura de forma rápida. Portanto, esta hemocidina sintética pode apresentar valor comercial se a via de administração for tópica ou se o seu uso envolver meios de baixa força iônica. Além disso, ela é um modelo valioso para o estudo do mecanismo de ação de peptídeos antimicrobianos com características estruturais similares e pode servir de base para o desenho de novos agentes antibiôticos. / It is well known that hemoglobin (Hb) is a source of biologically active peptides. The fragment 33-61 of bovine hemoglobin α-chain, isolated from the gut contents of the tick Boophilus microplus, was the first identified with antimicrobial activity . Its amidated analogue, Hb33-61a, showed to be active against Gram-positive bacteria and fungi strains [FogaÁa et al. (1999) J. Biol. Chem. 274, 25330-4]. The study of a series of carboxyl-free and amidated synthetic analogues of Hb33-61 revealed that C-terminus amidation enhances the activity against Candida albicans. Since Hb33-61a and Hb40-61a presented identical biological and structural properties, it seemed that Hb40-61a was Hb33-61a minimal active motif [SforÁa et al. (2005) Biochemistry 44, 6440- To test this suggestion, in the present study 51; Machado et al. (2007) Biopolymers 88, 413-26]. we synthesized, purified and characterized Hb40-61a analogues and assayed them against C. albicans and Micrococcus luteus. The results confirmed the suggestion only for the antifungal activity. When tested against fluconazole-resistant C. albicans, Hb40-61a was also active. Its antifungal activity showed to be dependent on the ionic strength of the medium. Its low hemolytic activity was confirmed even under low ionic strength conditions. Hb40-61a had no synergic effect with fluconazole on C. albicans. In vitro time-kill assays demonstrated that Hb40-61a kills the yeast rapidly. Therefore, this synthetic hemocidin may be of commercial interest for topical application or other uses involving low ionic strength medium. Moreover, it can serve as a template for the study of the mechanism of action of structurally related antimicrobial peptides or for the design of novel antibiotic drugs.

New and rapid techniques for the characterization and identification of bacteria would have an important role in clinical microbiology and in food analysis because of an increasing prevalence of infectious diseases and In this work we carried out two approaches. In the first study the characterization and differentiation of mesophilic and thermophilic bacteria were investigated by using Fourier Transform Infrared (FTIR) Spectroscopic technique. In the second study, we investigated the characterization and identification of 3 Bacillus and Micrococcus species Our results from first approach show that there was a dramatic difference between mesophilic and thermophilic bacteria. The protein concentration was high, lipid concentration, the level of triglycerides and the unsaturated acyl chains decreased in thermophilic bacteria. We found that in thermophilic bacteria PO- 2 groups become hydrogen bounded. In addition, our results suggest that the cellular DNA content was low in thermophilic bacteria. Moreover there were characteristic peaks for both mesophilic and thermophilic bacteria and these peaks can be used for the differentiation of these two bacteria group. There were also some specific peaks that can be used for the differentiation of Escherichia coli and Lactobacillus plantarum at species level. In the second approach, our results show that there were significant spectral differences between Bacillus and Micrococcus species such as the proportion of unsaturated acyl chains in triglycerides were higher in Micrococcus species. Moreover we observed different bands that may be explained by an acetate oxidation via the tricarboxylic acid cycle and an exopolymer formation in Micrococcus species. In addition to that another band similar to glycogen, may be explained by a glycogen-like storage material in Micrococcus species. Also there are characteristic peaks that can be used for identification of Micrococcus spp.

This master thesis is oriented on study antimicrobial effects extracts and macerates from cure Stevia rebaudiana Bertoni. Teoretical part describes basic information about plant Stevia, summary of health significant matters contained in Stevia and posobilities preparing extracts from Stevia. Antimicrobial effects extracts and macerates from cure Stevia were testing on food-borne bacteria (Bacillus cereus and Micrococcus luteus) and yeasts (Geotrichum candidum and Hansenula anomala). Microorganism, extracts and macerates were chosen on basis previous study (Study of antimicrobial effects Stevia Rebaudiana extracts, Eva Rakovská). For screening antimicrobial activity were determined the growth curves by using turbidimetrie for bacteria and direct treetment metod of cells number for yeasts. Antimicrobial effects were confirmed aplication with diffusion pit method on the agar ranges. From the results flow the testing extracts and macerates from stevia analysed antimicrobial effects. The best effect was demostrated on macerates and the most sensitive was bacteria Micrococcus luteus with the best inhibitoring effects.

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:30Z No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:42Z (GMT) No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:53Z (GMT) No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Made available in DSpace on 2015-06-30T13:13:53Z (GMT). No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Os produtos farmacêuticos que requerem a característica de esterilidade devem ser submetidos ao Ensaio de Esterilidade que deve ser realizado em áreas limpas, a fim de evitar resultados falso-positivos. A legislação brasileira recomenda a identificação de microrganismos provenientes dos Ensaios e do ambiente onde estes foram realizados. A dificuldade da identificação de vários gêneros bacterianos por metodologias fenotípicas têm sido relatada em vários estudos e mostram a necessidade da utilização de metodologias moleculares para esta finalidade. Neste estudo foi realizada a caracterização fenotípica (API e VITEK BioMerieux) e genotípica (análise da sequência do gene 16S rRNA) de 58 estirpes de cocos Gram positivos não fermentadores da glicose, provenientes de produtos farmacêuticos estéreis e ambiente controlado. O resultado da caracterização fenotípica realizada utilizando o sistema VITEK demonstrou que 100% das identificações foram equivocadas quanto ao gênero e espécie bacteriana. O sistema API identificou corretamente 69% das estirpes quanto ao gênero bacteriano quando comparado com a análise da sequência do gene 16S rRNA. Vinte e cinco estirpes foram submetidas ao sistema VITEK 2 e 68% dessas foram identificados corretamente quanto ao gênero bacteriano. A análise da sequência do gene 16S rRNA mostrou-se eficiente na determinação do gênero e mostrou a diversidade bacteriana deste grupo de organismos. Entre os cocos Gram positivos não fermentadores da glicose analisados foram identificados os gêneros Micrococcus, Kocuria, Demetria, Macrococcus, Arthrobacter, Dietzia, Janibacter e Brachybacterium. Essa análise também mostrou que 8,6% das estirpes avaliadas podem representar espécies ainda não descritas. Esta metodologia possibilita a diferenciação de quase todas as espécies do gênero encontrado com mais frequência, o Micrococcus, exceto o Micrococcus yunnanesis e Micrococcus luteus. Essas espécies também não puderam ser diferenciadas pela análise da sequência de segmentos de genes conservados (rpoB, gyrB, groEL and recA). Os equívocos das identificações fenotípicas alertam para a necessidade da implementação de metodologias moleculares para concluir a identificação correta de estirpes bacterianas provenientes de testes de esterilidade e ambientes controlados. / Sterile pharmaceutical products must be submitted to sterility testing to be carried out in clean rooms, in order to avoid false positive results. Brazilian law recommends the identification of microorganisms from sterility tests and the environment where these tests were performed. It has been reported in several studies difficulty in identifying various genera using phenotypic methods. This suggests the need of molecular methods which are more suitable for this purpose. In this study we performed phenotypic (API and VITEK systems (BioMerieux)) and genotypic (sequence analysis of 16S rRNA) characterization of 58 strains of Gram positive cocci non-fermenting glucose, from pharmaceuticals sterile and controlled environment. The results of phenotypic characterization performed using the VITEK system showed that 100% of the identifications of bacterial genus and species were misleading. The API system correctly identified the bacterial genus of 69% of the strains compared with the sequence analysis of 16S rRNA. Twenty-five strains were identified with the Vitek 2 system and 68% of the strains were identified with the correct bacterial genus. Sequence analysis of 16S rRNA gene was effective in determining the bacterial genus and also showed bacterial diversity of this group of organisms. Among the glucose non-fermenting Gram-positive cocci analyzed, the identified genera were: Micrococcus, Kocuria, Demetria, Macrococcus, Arthrobacter, Dietzia, Janibacter and, Brachybacterium. This analysis also showed that 8.6% of the strains tested may represent species not yet described. This methodology allowed the differentiation of almost all species of the genus Micrococcus, except Micrococcus yunnanesis and Micrococcus luteus. These species were also not differentiated by sequence analysis of fragments of housekeeping genes (rpoB, gyrB, groEL and recA). The mistake phenotypic identifications highlight the need of the implementation of molecular methods to achieve the correct identification of bacterial strains from sterility testing and controlled environments.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The Organization for Economic Co-operation and Development (OECD) and the Food and Agriculture Organization (FAO) of the United Nations predict that milk production and the dairy sector will remain one of the fastest-growing agricultural subsectors over the coming decade. The global milk production is projected to expand over the 2011-2020 period at an annual rate of 2%. In South Africa alone, approximately 14 – 15 million litres of milk are wasted annually due to microbial spoilage. Therefore, the identification of the spoilage microorganisms in the milk products is necessary. This will contribute towards the design of appropriate measures to prevent wastage due to spoilage and in turn contribute towards sustainability of the sector. Accordingly, one hundred samples of spoiled full cream UHT milk were collected from two plants of each of the two largest milk processors. These samples were examined visually, and the pH was measured. A presumptive identification up to genus level was conducted by examining morphological features and conducting Gram-stain, catalase and oxidase tests. Species-specific identification was done by using the Analytical Profile Index and Biolog system. Molecular profiling was done by sequencing the rDNA genes. The main spoilage organisms identified in the samples were Pseudomonas, Micrococcus, Bacillus, Enterococcus and Lactobacillus. All organisms belonging to the five genera were psychrotrophs, which are commonly found in biofilms in UHT milk processing equipment. Therefore, according to the study, the spoilage bacteria apparently entered into the milk due to inadequate cleaning-in-place (CIP) processes. More importantly, further studies should be conducted in order to identify the spoilage microbes and how CIP processes can be improved.