L'eau et le sol sont considérés comme des sources potentielles de mycobactéries non-tuberculeuses (MNT). Parmi les infections humaines causées par les MNT d'origine environnementale, les infections pulmonaires et cutanées sont souvent décrites. Le manque de connaissances sur leur cycle de vie dans l'environnement requiert des outils analytiques, qui ne sont actuellement pas adaptés à ce type d'échantillons. Cette thèse vise donc premièrement à proposer des méthodes de quantification en bactériologie et en biologie moléculaire dans le but de déterminer les sources des MNT dans les bassins versants. Ainsi, la comparaison des méthodes d'isolement de MNT a montré que le traitement au chlorure de cetylpyridininium de l'eau suivi d'une culture en milieu riche supplémenté par un mélange d'antibiotiques (polymyxine B, amphotéricine, acide nalidixique, triméthoprime, carboxy-pénicilline) limitait la croissance des microorganismes interférents et éliminait moins de MNT que les autres méthodes comparées (Radomski et al. 2010, doi: 10.1128/AEM.00942-10). Bien que des espèces de MNT potentiellement pathogènes aient été isolées de l'eau de surface de la Seine en utilisant ces outils bactériologiques, la quantification des MNT ne s'est pas avérée reproductible. En conséquence, une méthode de quantification par polymérisation en chaîne en temps réel (qPCR) a été développée pour énumérer le genre Mycobacterium dans l'eau (Radomski et al. 2010, doi: 10.1128/AEM.02659-09). La nouvelle méthode développée, ciblant l'ARNr 16S, était plus spécifique que les autres méthodes qPCR publiées, ciblant un autre locus de l'ARNr 16S et le gène hsp65 (respectivement 100 % versus 44 % et 91 %). La comparaison des méthodes d'extraction d'ADN mycobactérien a montré que la lyse enzymatique combinée au bromure d'hexadécyltriméthylammonium était la procédure la plus efficace pour énumérer par qPCR les MNT dans des échantillons environnementaux. Ainsi, ces méthodes d'extraction d'ADN et de qPCR ont été utilisées pour étudier des sources de MNT dans des bassins versants. Dans un second temps, nous avons étudié trois sources potentielles de MNT : une ponctuelle et deux diffuses. Plus précisément, une station d'épuration (STEP) a été choisie comme source ponctuelle de MNT et a été étudiée en temps sec en fonction d'indicateurs de contamination fécale et des paramètres globaux habituellement contrôlés. Les MNT ont atteint 5,52×105±3,97×105 copies/L dans l'eau en entrée de STEP (84 % d'échantillons positifs), n'ont pas été détectées dans l'eau en sortie de STEP après décantation physico-chimique et biofiltration et ont été estimées à 1,04×106 ±1,75×106 copies/g dans les boues de STEP (50 % d'échantillons positifs). La plupart des MNT (98±2 %, correspondant à 2,45±0,78 log10) ont été éliminées par décantation physico-chimique et les MNT restantes (0,74×104 ±1,40×104 copies/L) ont été éliminées par biofiltration (53 % d'échantillons positifs). Ces résultats ont montré également que Mycobacterium, Escherichia coli et les entérocoques intestinaux possèdent des comportements significativement différents conduisant respectivement à trois modèles : hydrophobe, hydrophile et intermédiaire. Concernant les sources diffuses, la densité de MNT a été mesurée dans divers sols ruraux et urbains qui ont été caractérisés par différents paramètres physico-chimiques. Les densités de MNT les plus importantes ont été mesurées dans des sols de forêts tourbeuses (9,27×104±5,00×104 copies/g sec) et dans des sols faiblement urbanisés proches de marécages côtiers (1,71×106±2,85×106 copies/g sec) alors qu'aucune MNT n'a été détectée dans les autres types de sols étudiés. De plus, la densité de MNT a été significativement associée à des sols proches de zones acides et des teneurs fortes des sols en eau, matière organique et fer. Ces résultats suggéreraient que les MNT sont dépendantes de leur production intra et extracellulaire de chélateurs de fer et indiqueraient que les zones faiblement urbanisées pourraient être impactées par la proximité de marais acides. Afin d'étudier une autre source diffuse, les MNT et d'autres paramètres ont été mesurés lors d'événements pluvieux dans l'eau de surface de la Marne et de ses principaux affluents. Les densités de MNT ont été estimées à 2,16×105±2,36×105 copies/L dans environ 20 % des échantillons d'eau collectés, et elles ne différaient pas entre les zones péri-urbaines et rurales échantillonnées. Nos résultats ont montré que la pluviométrie et la durée de l'évènement expliquaient la diminution du nombre de MNT détectées dans l'eau de surface au cours de l'événement pluvieux de faible intensité (6,6 mm/h de pluviométrie cumulées en 5,5 h). Ces résultats ont souligné que certains affluents de la Marne pouvaient apporter des MNT en temps sec, mais qu'au cours de l'évènement pluvieux suivi les densités de MNT diminuaient.En guise d'amélioration à ces études appliquées, des réflexions sur les défis relatifs à la surveillance des microorganismes pathogènes dans l'environnement ont été explorées. En nous focalisant sur la MNT la plus pathogène, M. avium, nous avons discuté des défis de la détection et de l'énumération et proposé un guide d'adaptation des méthodes médicales aux échantillons environnementaux (Radomski et al. 2011, ed. A. Méndez-Vilas, Vol. 2). Ce guide se présente sous la forme d'un arbre de décision permettant de choisir les outils analytiques les plus appropriés pour surveiller les microorganismes pathogènes dans l'environnement. De plus, une stratégie in silico de comparaison de génomes bactériens totalement séquencés a été développée dans le but de décrire des nouvelles cibles de détection. L'analyse in silico des génomes totalement séquencés a permis de détecter 11 protéines présentant entre 80 % et 100 % de similarité dans les génomes mycobactériens et moins de 50 % de similarité dans les génomes non-mycobactériens des genres Corynebacterium, Nocardia et Rhodococcus. Sur la base d'alignements des séquences d'ADN de ces cibles potentielles, il a été possible de dessiner des amorces PCR et une sonde pour détecter le gène codant la sous-unité C de la synthase de l'adénosine triphosphate qui semble exclusivement conservée dans le génome mycobactérien. Le développement d'outils analytiques, en particulier la qPCR, a permis de montrer qu'une STEP éliminait efficacement les MNT et que le traitement des eaux usées est nécessaire pour préserver l'eau de surface de cette source ponctuelle de MNT. Il a été mis en évidence que les événements pluvieux diminuent la densité de MNT dans l'eau de surface et que les sols acides sont des sources naturelles majeures de MNT qui pourraient impacter des zones faiblement urbanisées en temps de pluie via le ruissellement. Concernant les réflexions sur la surveillance des microorganismes pathogènes dans l'environnement, l'arbre de décision des outils analytiques appropriés et la nouvelle stratégie in silico de détection de cibles moléculaires pourraient être appliqués pour l'étude d'autres microorganismes de l'environnement / Water and soil are considered as potential sources of nontuberculous mycobacteria (NTM) infections. Among human infections caused by environmental NTM, pulmonary infections and cutaneous infections are often described. However, lack of knowledge about their life cycle in the environment requires analytical tools, which are not currently adapted to these kinds of samples. The aim of this thesis is to propose bacteriological and molecular quantitative methods, in order to determine the sources of NTM in watersheds. Comparison of NTM isolation methods showed that treatment with cetylpyridinium chloride of water, followed by culture on a rich medium supplemented with antibiotic cocktail (polymyxin B, amphotericin, nalidixic acid, de trimethoprim, azlocillin) decreased the growth of nontarget microorganisms, while inhibiting less NTM than the other compared methods (Radomski et al. 2010 doi: 10.1128/AEM.00942-10). Although potentially pathogenic NTM species were isolated from surface water of the Seine River using these bacteriological tools, enumeration of NTM was not reproducible. Consequently, a quantitative real-time polymerase chain reaction (qPCR) method was developed in order to enumerate Mycobacterium spp. in water (Radomski et al. 2010 doi: 10.1128/AEM.02659-09). This newly developed method, targeting 16S rRNA, was more specific than the two previously published qPCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively). Comparison of DNA extraction methods showed that the enzymatic lysis and hexadecyltrimethylammonium bromide procedure was the most effective combination for mycobacterial DNA extraction with the aim to enumerate NTM in environmental samples by qPCR. Thus, these extraction and qPCR methods were used in order to study NTM sources in watersheds.Secondly, we studied three potential sources of NTM : one point source and two nonpoint sources. More precisely, a wastewater treatment plant (WWTP) was chosen as a potential point source of NTM and was studied according to indicators of fecal contamination and usually monitored parameters. NTM reached 5.52×105±3.97×105 copies/L in the influent of WWTP (84% of positive samples). They were not detected in the effluent after physico-chemical decantation and biofiltration, and were estimated at 1.04×106 ±1.75×106 copies/g in sludge (50% of positive samples). Most NTM (98±2%, i.e. 2.45±0.78 log10) were removed by the physical-chemical decantation, and the remaining NTM (0.74×104 ±1.40×104 copies/L) were removed by biofiltration (53% of positive samples). These results showed also that Mycobacterium, Escherichia coli and intestinal enterococci follow significantly different behaviors as hydrophobic, hydrophilic and intermediate models, respectively. Concerning the nonpoint sources, NTM were enumerated in a variety of rural and urban soils which were characterized by different physico-chemical parameters. The highest NTM densities were measured in peat forest soils (9.27×104±5.00×104 copies/g dw) and in lightly urbanized soils near a costal swamp (1.71×106±2.85×106 copies/g dw), whereas they were not detected in the other monitored soils. NTM density was significantly associated with soils near acidic areas and high moisture, organic matter, and iron content in soils. These results emphasized that NTM are dependent upon the production of surface and extracellular iron-binding compounds, and may mean that lightly urbanized area could be impacted by the proximity of the acidic swamp. In order to study another nonpoint source, NTM and other parameters were measured during wet events in surface water of Marne River and their main effluents. NTM density was estimated at 2.16×105±2.36×105 copies/L in about 20% of surface water samples, and NTM densites did not differ among rural and peri-urban sampling areas. Our results showed that the pluviometry and rain duration explained the decrease of detected NTM abundances in surface water during a slightly intense wet event (6.6 mm/h of cumulated rain during 5.5 h). These results emphasized that some tributaries of the Marne River may constitute a source of NTM, however their influence on NTM density in surface water of the Marne River decreased during the slightly intense wet event.In order to improve these applied studies, challenges dealing with pathogenic microorganism monitoring in environment were explored. Focusing on the most pathogenic NTM, M. avium, we discussed the challenges for detection and enumeration and proposed a guidance for the adaptation of clinical methods to environmental samples (Radomski et al. 2010 ed. A. Méndez-Vilas, Vol. 2). This guidance was proposed as a decision tree allowing to choose the most suitable analytical tools in order to monitor pathogenic microorganisms in environment. Moreover, an in silico strategy of whole sequenced bacterial genome comparison was developed in order to describe new targets for NTM detection. In silico analysis of whole sequenced genomes allowed to detect 11 proteins showing between 80% and 100% of similarity with mycobacterial genomes, and less than 50% of similarity with closely related genomes of Corynebacterium, Nocardia and Rhodococcus genera. Based on the DNA sequence alignments of these potential targets, it was possible to design a primer pair and a probe in order to detect by PCR the gene coding for adenosine-5'-triphosphate synthase subunits C which seems exclusively conserved in mycobacterial genome.Using the developed analytical tools, especially the qPCR, we showed that a WWTP removed efficiently NTM from the influent, and that waste water treatment is necessary in order to preserve surface water against this NTM point source. It was shown that storm events decrease NTM densities in surface water and in contrast that acidic soils are major NTM natural sources which may impact lightly urbanized areas during wet weather when runoff water suspends soil matter. Concerning challenges dealing with pathogenic microorganism monitoring in environment, the decision tree of suitable analytical tools and the new in silico strategy of molecular target detection might be also useful for the study of other environmental microorganisms

Because of the potential health hazards associated with the use of sludge for agricultural purposes, Dudley et al (1980) published a scheme for the routine analysis of bacteria in municipal sewage sludge. In this study, the Dudley et al scheme (1980) was modified by updating some of the procedures. Aerobically digested sludge generated by the Bowling Green Wastewater Treatment Plant, Bowling Green, Kentucky, was analyzed using the modified scheme. Sludge samples were collected once every two months over a one-year period from October 1989 to August 1990. Egg yolk-free tryptose sulfite cycloserine agar in conjunction with the revewrse CAMP test was used to assay for Clostridium perfringens. This procedure improved the one proposed by Dudley et al. (1980) by achieving a higher confirmation rate, reducing testing time, allowing for easier interpretation of results, and increasing accuracy. Selective and differential media by Rippey and Cabelli (1979) were added to the scheme to isolate Aeromonas, Aeroomonas hydrophila and Aeromonas caviae were successfully isolated wand were identified using the system by Cunliffe and Adcock (1989) for speciating aeromonads. Baird-Parker medium was compared to mannitol salt agar for effectiveness in isolating Staphylococcus from sludge. Statistical analysis showed Baird-Parker medium to be significantly more effective than mannitol salt agar. However, neither agar reduced background flora to acceptable levels. Staphylococcus isolates were subject to species identification by the API Staph Ident system (Analytab Products, Plainview, New York). Staphylococcus xylosus, Staphylococcus haemolyticus, and Staphylococcus epidermidis were found to be present in the sludge. A procedure by Ottolenghi and Hamparian (1987) was employed to isolate Salmonella in sludge. No salmonellae were isolated over the one year period. Over the year-long study, bacterial numbers, with the exception of Clostridium perfringens and the total aerobic count, fluctuated with variations in the aerobic digester temperature. Numbers decreased as temperature increased. Clostridium perfringens counts were the most consistent throughout the year and exceeded fecal coliform and fecal streptococci counts in five of the six samplings.

Este trabalho teve por objetivo contribuir para a implantação de uma Rede de Laboratórios de Bacteriologia da Tuberculose no Estado de São Paulo, visando a obter uma modificação efetiva da situação epidemiológica da tuberculose em nosso meio. O autor justifica inicialmente a bacteriologia como medida diagnóstica prioritária nos atuais programas de luta contra a doença. Apresenta, a seguir, um estudo preliminar sobre a implantação de Uma Rede de Laboratórios em uma das Divisões Regionais de Saúde do Estado de São Paulo, mostrando a sua viabilidade e exequibilidade. / The objective of this paper was to contribute to the implementation of a network of Tuberculosis Bacteriological Laboratories throughout the State of São Paulo (Brazil), designed to change effectively the epidemiological situation of tuberculosis among us. The author justifies bacteriology as the priority diagnostic procedure in the current programmes aiming the control of the disease, and presents a preliminary study regarding the implementation of a network of laboratories in a Regional Health Division of the State of São Paulo, showing its viability and operational possibility.

Este trabalho procurou definir a existência de espécies emergentes de micoplasmas (Mycoplasma genitalium, M. fermentans e M. penetrans) em indivíduos HIV-positivos e indivíduos com queixa clínica de retrite, por não haver dados a esse respeito no Brasil. A pesquisa das espécies mais conhecidas (M. hominis e Ureaplasma urealyticum), cuja metodologia para sua detecção já está bem estabelecida, proporcionou uma visão global da participação das espécies de importância clínica conhecidas até o momento. Foram avaliadas amostras de raspado uretral e primeiro jato urinário de 106 indivíduos HIV-positivos e 110 indivíduos HIV-negativos com DST, no total de 212 e 220 amostras do primeiro e do segundo grupo, respectivamente. Deste modo, foram submetidas a análise 432 amostras para pesquisa de micoplasmas por cultura e PCR. Para pesquisa de anticorpos séricos anti- M. penetrans, foram obtidas amostras de soro de cada indivíduo dos dois grupos estudados, bem como de 86 doadores de sangue saudáveis para cálculo do limite de reatividade do ELISA. As taxas de infecção obtidas em nosso estudo, considerando os resultados do cultivo e da PCR, nos indivíduos HIV-positivos, foram: 2,8% para a espécie M. genitalium, 5,7% para M. fermentans, e 6,6% para M. penetrans. Nos indivíduos com queixa de DST, os valores encontrados foram: 9,1% para M. genitalium e 0,9% para M. fermentans, não tendo sido observado nenhum caso positivo para M. penetrans. Estes últimos resultados revelam que, apesar da pouca importância dada até então às três espécies, estas responderam, no total, por taxas de infecção de 15,1% e 10,0%, respectivamente, nos grupos HIV-positivo e DST. Estas taxas são bastante significativas quando comparadas com as observadas para as espécies M. hominis e U. urealyticum, ou seja, 26,4% e 14,5% em cada grupo, respectivamente. Nossos dados demonstraram uma freqüência significativa de anticorpos anti- M. penetrans: 25,5% no grupo de indivíduos HIV-positivos, 17,3% no grupo de DST, e 2,3% no grupo controle para IgG; 3,8%, 9,1% e 5,8% para IgM, e 15,1%,17,3% e 1,2% para IgA, respectivamente. Estes dados, aliados aos resultados de PCR, confirmam a presença de M. penetrans em nosso meio, infectando principalmente os indivíduos HIV-positivos. A presença desta espécie e também de M. fermentans, detectadas por PCR, denotam que muito pouco se conhece sobre o envolvimento destes micoplasmas com o HIV e que ainda não lhes é dada a devida importância na rotina médica. A associação definitiva de sua infecção com a progressão da AIDS, ainda a ser alcançada, e a demonstração em nossa população da infecção por M. genitalium em indivíduos com queixa de uretrite não-gonocócica, abre novos horizontes para o estudo da participação dos micoplasmas na etiologia das doenças humanas no país. / The aim of this work was to define the existence of emergent mycoplasma species, such as Mycoplasma genitalium, M. fermentans and M. penetrans, in HIV-1-infected and HIV-negative individuals with Sexually Trasmitted Diseases (STD), because there are no data concerning this subject in Brazil. The research about well-known species, such as Ureaplasma urealyticum and M. hominis, of wich detection techniques are well established, provided a comprehensive view of the participation of Mollicutes in human disease. We evaluated urethral swabs and urine samples from 106 HIV-1-infected and 110 HIV-negative individuals with clinical symptoms of non-gonococal urethritis, summing up 212 samples from the first group, and 220 from the second. Therefore, we tested a total of 432 samples to mycoplasma detection by culture and PCR. To detection of serum antibodies to M. penetrans, we obtained blood samples of each patient from the groups studied, as well as from 86 healthy blood donors to calculate the ELISA (Enzime Linked Immuno Sorbent Assay) cut-off value. The rates of infection obtained in our study, considering the culture and PCR results, in the HIV-infected individuals were: 2.8% for M. genitalium, 5.7% for M. fermentans, and 6.6% for M. penetrans. In the STD group, the rates were: 9.1% for M. genitalium and 0.9% for M. fermentans. We did not observed any positive case for M. penetrans in this group, and no positive case for M. pneumoniae in both groups, in spite of the existence of some reports of its detection in the genitourinary tract. This results reveal that they account, in the total, for infection rates of 15.1% and 10%, respectively, in the HIV-infected and the STD groups. These rates are very significant when compared to the ones observed for the species M. hominis and U. urealyticum, i.e., 26.4% and 14.5% in each group, respectively. Our results also show a significant antibody frequency to M. penetrans: 25.5% in the HIV-infected group, 17.3% in the STD group, and 2.3% in the control group for IgG; 3.8%, 9.1% and 5.8% for IgM, and 15.1 %, 17.3% and 1.2% for IgA, respectively. These data, together with the PCR results, confirm the presence of M. penetrans in our population, mainly infecting HIV- positive patients. The presence of this species, and also of M. fermentans, detected by PCR, demonstrate that very little is known about the role of this mycoplasma species in HIV infection, and they are not given the appropriate importance in clinical routine in Brazil. The definitive association between mycoplasma infection and the progression of AIDS is still to be established. The demonstration of M. genitalium infection in individuals with clinical symptoms of non-gonococal urethritis in our population, may open new insights in the study of the role of mycoplamas in the ethiology of human diseases in this country.

Thesis (Master of Applied Sciences in Chemistry)--Cape Peninsula University of Technology, 2018. / Maintenance of optimum wound chemistry is important to ensure timely healing of a wound. Bacterial infections impair the process of wound healing by producing toxins that alter the chemical environment in and around the wound. The imbalance in the wound chemistry prolongs healing and opens doors to opportunistic infections. Bacteria have developed resistance to conventional bactericides hence, there is need for search of new bactericides that can control bacteria in and around the wound. Therefore, new chemical or biochemical bactericides, which are not resisted by the bacteria, can be explored to control bacterial life around the wound in a bid to maintain optimum wound healing chemistry. Materials such as chitosan, zinc oxide, copper oxide and silver have showed remarkable potential as both bactericidal and wound healing agents. In this work silver, zinc oxide, and copper oxide nanoparticles (NPs) and their chitosan composites (CH-NPs) were synthesized using the chemical reduction method and simple chelation respectively to produce nanoparticles of Ag, ZnO, and CuO as well as composites of CH-ZnO, CH-Ag, CH-CuO, and CH-ZnO-Ag-CuO. Formation of the NPs was confirmed by the exhibition of characteristic peaks in UV-Visible and Fourier Transform Infrared Resonance (FTIR) spectroscopy as well as X-ray diffraction. The nanoparticles (NPs) had optical and electronic band gaps in the range 1 to 5eV indicating their semi-conductive nature. X-ray diffraction (XRD) investigations depicted the crystalline structures of the NPs to be base-centred, face-centred, and hexagonal for Ag, CuO, and ZnO respectively. Transmission electron microscopy (TEM) studies exhibited spherical, hexagonal, and rod-shaped shapes for silver, copper oxide, zinc oxide NPs respectively. Electrochemical investigations of the pure NPs indicated the existence of both the adsorption and the diffusion controlled electron transfer processes at electrode surfaces as well as fast electron transfer rate as depicted by the charge transfer coefficient and standard rate constant parameter values. FTIR spectra of CH-NPs composites depicted new excitation bands absent in spectra of both chitosan and the NPs. The spectra also indicated the deformation and absence of the amine (-NH2) and hydroxyl bands (-OH) within the CH-NPs composites. UV-Visible spectroscopy investigations of the CH-NPs composites exhibited blue-shifts of the λmax with respect to the NPs. The FTIR and UV-Visible spectra confirmed the existence of bonding between the chitosan and the NPs. The optical band gap energies of all the CH-NPs composites fell within the range of 2.0 to 4.5 eV indicating that the CH-NPs fell in the category of the semi-conducting materials after chelating with the chitosan.

Este trabalho procurou definir a existência de espécies emergentes de micoplasmas (Mycoplasma genitalium, M. fermentans e M. penetrans) em indivíduos HIV-positivos e indivíduos com queixa clínica de retrite, por não haver dados a esse respeito no Brasil. A pesquisa das espécies mais conhecidas (M. hominis e Ureaplasma urealyticum), cuja metodologia para sua detecção já está bem estabelecida, proporcionou uma visão global da participação das espécies de importância clínica conhecidas até o momento. Foram avaliadas amostras de raspado uretral e primeiro jato urinário de 106 indivíduos HIV-positivos e 110 indivíduos HIV-negativos com DST, no total de 212 e 220 amostras do primeiro e do segundo grupo, respectivamente. Deste modo, foram submetidas a análise 432 amostras para pesquisa de micoplasmas por cultura e PCR. Para pesquisa de anticorpos séricos anti- M. penetrans, foram obtidas amostras de soro de cada indivíduo dos dois grupos estudados, bem como de 86 doadores de sangue saudáveis para cálculo do limite de reatividade do ELISA. As taxas de infecção obtidas em nosso estudo, considerando os resultados do cultivo e da PCR, nos indivíduos HIV-positivos, foram: 2,8% para a espécie M. genitalium, 5,7% para M. fermentans, e 6,6% para M. penetrans. Nos indivíduos com queixa de DST, os valores encontrados foram: 9,1% para M. genitalium e 0,9% para M. fermentans, não tendo sido observado nenhum caso positivo para M. penetrans. Estes últimos resultados revelam que, apesar da pouca importância dada até então às três espécies, estas responderam, no total, por taxas de infecção de 15,1% e 10,0%, respectivamente, nos grupos HIV-positivo e DST. Estas taxas são bastante significativas quando comparadas com as observadas para as espécies M. hominis e U. urealyticum, ou seja, 26,4% e 14,5% em cada grupo, respectivamente. Nossos dados demonstraram uma freqüência significativa de anticorpos anti- M. penetrans: 25,5% no grupo de indivíduos HIV-positivos, 17,3% no grupo de DST, e 2,3% no grupo controle para IgG; 3,8%, 9,1% e 5,8% para IgM, e 15,1%,17,3% e 1,2% para IgA, respectivamente. Estes dados, aliados aos resultados de PCR, confirmam a presença de M. penetrans em nosso meio, infectando principalmente os indivíduos HIV-positivos. A presença desta espécie e também de M. fermentans, detectadas por PCR, denotam que muito pouco se conhece sobre o envolvimento destes micoplasmas com o HIV e que ainda não lhes é dada a devida importância na rotina médica. A associação definitiva de sua infecção com a progressão da AIDS, ainda a ser alcançada, e a demonstração em nossa população da infecção por M. genitalium em indivíduos com queixa de uretrite não-gonocócica, abre novos horizontes para o estudo da participação dos micoplasmas na etiologia das doenças humanas no país. / The aim of this work was to define the existence of emergent mycoplasma species, such as Mycoplasma genitalium, M. fermentans and M. penetrans, in HIV-1-infected and HIV-negative individuals with Sexually Trasmitted Diseases (STD), because there are no data concerning this subject in Brazil. The research about well-known species, such as Ureaplasma urealyticum and M. hominis, of wich detection techniques are well established, provided a comprehensive view of the participation of Mollicutes in human disease. We evaluated urethral swabs and urine samples from 106 HIV-1-infected and 110 HIV-negative individuals with clinical symptoms of non-gonococal urethritis, summing up 212 samples from the first group, and 220 from the second. Therefore, we tested a total of 432 samples to mycoplasma detection by culture and PCR. To detection of serum antibodies to M. penetrans, we obtained blood samples of each patient from the groups studied, as well as from 86 healthy blood donors to calculate the ELISA (Enzime Linked Immuno Sorbent Assay) cut-off value. The rates of infection obtained in our study, considering the culture and PCR results, in the HIV-infected individuals were: 2.8% for M. genitalium, 5.7% for M. fermentans, and 6.6% for M. penetrans. In the STD group, the rates were: 9.1% for M. genitalium and 0.9% for M. fermentans. We did not observed any positive case for M. penetrans in this group, and no positive case for M. pneumoniae in both groups, in spite of the existence of some reports of its detection in the genitourinary tract. This results reveal that they account, in the total, for infection rates of 15.1% and 10%, respectively, in the HIV-infected and the STD groups. These rates are very significant when compared to the ones observed for the species M. hominis and U. urealyticum, i.e., 26.4% and 14.5% in each group, respectively. Our results also show a significant antibody frequency to M. penetrans: 25.5% in the HIV-infected group, 17.3% in the STD group, and 2.3% in the control group for IgG; 3.8%, 9.1% and 5.8% for IgM, and 15.1 %, 17.3% and 1.2% for IgA, respectively. These data, together with the PCR results, confirm the presence of M. penetrans in our population, mainly infecting HIV- positive patients. The presence of this species, and also of M. fermentans, detected by PCR, demonstrate that very little is known about the role of this mycoplasma species in HIV infection, and they are not given the appropriate importance in clinical routine in Brazil. The definitive association between mycoplasma infection and the progression of AIDS is still to be established. The demonstration of M. genitalium infection in individuals with clinical symptoms of non-gonococal urethritis in our population, may open new insights in the study of the role of mycoplamas in the ethiology of human diseases in this country.

Este trabalho teve por objetivo contribuir para a implantação de uma Rede de Laboratórios de Bacteriologia da Tuberculose no Estado de São Paulo, visando a obter uma modificação efetiva da situação epidemiológica da tuberculose em nosso meio. O autor justifica inicialmente a bacteriologia como medida diagnóstica prioritária nos atuais programas de luta contra a doença. Apresenta, a seguir, um estudo preliminar sobre a implantação de Uma Rede de Laboratórios em uma das Divisões Regionais de Saúde do Estado de São Paulo, mostrando a sua viabilidade e exequibilidade. / The objective of this paper was to contribute to the implementation of a network of Tuberculosis Bacteriological Laboratories throughout the State of São Paulo (Brazil), designed to change effectively the epidemiological situation of tuberculosis among us. The author justifies bacteriology as the priority diagnostic procedure in the current programmes aiming the control of the disease, and presents a preliminary study regarding the implementation of a network of laboratories in a Regional Health Division of the State of São Paulo, showing its viability and operational possibility.

Rhodococci are notable for their ability to degrade a variety of natural and xenobiotic compounds. Recently, interest in Rhodococcus has increased due to the discovery of a large number of genes for secondary metabolism. Only a few secondary metabolites have been characterized from the rhodococci (including 3 recently described antibiotics). Twenty-four new Rhodococcus strains were isolated from soils in East Tennessee using acetonitrile enrichment culturing and identified using 16S rRNA analysis. Forty-seven Rhodococcus strains were screened for antibiotic production using a growth inhibition assay. One strain, MTM3W5.2, had 90% similarity to the Rhodococcus opacus 16S rRNA gene sequence and produced a large zone of inhibition against R. erythropolis and a large number of closely related species. The antimicrobial compound produced by MTM3W5.2 had a large MW of 911.5452 Da and acts much like a bacteriocin but no amino acids were detected in this molecule based on TLC analysis.

Binding of inorganic phosphate (Pi) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding Pi must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in Pi binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required for transition state stabilization and Pi binding whereas αGly-351 is only indirectly involved in Pi binding and most likely maintains structural integrity of the catalytic site. Results from preliminary experiments on αThr-349 mutants suggest that the residue may be involved in Pi binding; however, further investigation is required to fully test this hypothesis.

Rhizobium leguminosarum is a Gram negative nitrogen-fixing soil bacterium. Due to the limited bioavailability of iron, bacteria utilize siderophores that scavenge and bind available iron. The transport of iron-siderophore complexes is achieved by the TonB-ExbB-ExbD complex. We have previously shown that a functional TonB protein is necessary for iron transport by creating ΔtonB mutants and assessing their growth and 55Fe-siderophore transport ability. We attempted to identify and characterize the roles of putative exbB and exbD genes using a similar approach. Growth curves and sequence analyses suggest putative exbB and exbD may be the tolpal-associated genes tolQ and tolR. Phenotypic and sensitivity assays showed mutants do not exhibit the characteristic tol phenotype and are not sensitive to detergents or changes in ionic strength of the growth medium. We also expressed and purified the 120 amino acid fragment of the TonB C-terminus for further physical and chemical characterization.