Yes / Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are opportunistic pathogens that can be transmitted via drinking water, when tiny droplets containing the bacteria are aerosolized and inhaled during activities such as showering. The resulting respiratory illnesses, Legionnaires' Disease and NTM lung disease, are among the leading sources of drinking water associated disease in the United States and other parts of the world. Lp and NTM are both difficult to control, because they establish as part of natural biofilms that form within the interiors of pipes and fixtures that deliver drinking water to the point of use. These pathogens are especially problematic within premise (i.e., building) plumbing, where intermittent use throughout the day leads to long periods of stagnation, increased water age, warmer temperatures, and depleted disinfectant residuals that exacerbate bacterial growth. The recent advent of high throughput DNA sequencing has led to the discovery that drinking water microbiomes are diverse, complex, and largely comprised of non-pathogenic microbes. This has further led researchers to hypothesize that the microbial ecology of this diverse microbiome could be harnessed as a natural means to control Lp and NTM, i.e., a "probiotic" approach, but such an approach has not yet been demonstrated. The objective of this study was to assess this hypothesis by utilizing biologically active granular activated carbon (GAC) filters, which are already a widely used drinking water treatment both at the municipal and household scale, as a means to naturally shape the microbial ecology of downstream premise plumbing and inhibit Lp and NTM proliferation. GAC has an extremely high surface area that aids removal of organic carbon via adsorption but also provides an ideal habitat for establishment of biofilms, which removes organic carbon from the water via biodegradation. Convectively-mixed pipe reactors (CMPRs) were used for replicable simulation of premise plumbing distal taps. The CMPRs consisted of four-foot-long closed polyvinyl chloride (PVC) pipe segments with the sealed bottom portion resting in a ~48 °C water bath and with the top portion plugged and exposed to the cooler, ambient atmosphere (25 °C in this study), inducing convective mixing and resulting in an internal water temperature of 37 °C. PVC was chosen because it is common in premise plumbing and generally leaches the least organic carbon among the different types of plastic pipe. Four different influent water conditions were implemented in the experimental design: 1) Untreated, dechlorinated municipal tap water with high organic carbon and low biomass; 2) GAC-treated tap water with low organic carbon and elevated, viable biomass; 3) GAC-treated + 0.22-m pore size membrane-filtered tap water to remove both nutrients and biomass; 4) GAC-treated tap water pasteurized at 70 °C with low nutrients and elevated, killed biomass. The 0.22-m pore size membrane filter simulated the use of a building scale particle filter, while pasteurization simulated water passing through a hot water heater at an elevated temperature recommended for pathogen thermal disinfection. To understand the influence of these experimental conditions on older pipes containing mature biofilms versus new pipes that leach more organics and are being freshly colonized, a set of older pipes colonized with mature ~4-year-old biofilms were compared to newly purchased pipes. Each set of pipes was tested in triplicate for the four different experimental conditions with the full volume replaced three times a week for eight months, simulating infrequently used taps containing warm, continuously mixing water thought to create conditions at a very high risk for opportunistic pathogen growth. In the aged CMPR bulk water effluents, droplet-digital-polymerase-chain-reaction measurements showed a one-log reduction of Lp and NTM when receiving GAC-treated or GAC-treated + particle-filtered influent water versus receiving dechlorinated municipal tap water or GAC-treated + pasteurized water. These findings suggest that decreased biodegradable dissolved organic carbon achieved by GAC filtration acted to suppress Lp and NTM growth, while the additional step of biomass removal by particle filtration provided a more modest benefit. In the CMPRs consisting of new pipes, concentrations of Lp and NTMs in the effluent bulk water were similar among the experimental conditions, except that the CMPRs receiving the GAC-treated + particle-filtered influent water experienced a two-log reduction in NTMs. These results demonstrate that the colonization and proliferation of NTM within premise plumbing can be significantly controlled by limiting nutrients and biomass in the influent water. This work demonstrates the potential of harnessing GAC-treatment as a means to Control Lp and NTM in premise plumbing via nutrient removal. In scenarios where chemical disinfectants have been depleted, off-the-shelf GAC-treatment used as point-of-entry treatment to large buildings with recirculating plumbing could provide benefits that have previously been unrecognized. Alternatively, pasteurization in very hot water heaters could provide a short-term disinfection benefit, but eventually the nutrients embodied in the dead biomass undermine the positive influence of the nutrient removal provided by the GAC-treatment. Improved mechanistic understanding of probiotic strategies to opportunistic pathogen control would be needed to overcome inherent limitations to the approaches examined herein, if more effective control is desired in the absence of thermal or chemical disinfection. / Master of Science / Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are bacterial pathogens that are the leading source of drinking water-associated disease in the US. Unfortunately, they are not effectively controlled by protections put in place by the US Safe Drinking Water Act (SDWA). Firstly, they cause respiratory infections, which are spread when tiny droplets of water are inhaled during activities such as showering, whereas the SDWA is specifically designed to protect against ingested pathogens. Secondly, unlike fecal-derived organisms (e.g. E. coli) that are the focus of the SDWA, Lp and NTM grow naturally in drinking water distribution systems, especially in premise (i.e., building) plumbing, where water is warmer and more stagnant. Therefore, even if water leaving the treatment plant is devoid of Lp or NTM, this does not guarantee that the consumer's tap water will be Lp- or NTM-free. Also, even though chlorine or other chemical disinfectant is required by the SDWA to be added to the water leaving the treatment plant to control downstream microbial growth, the disinfectant can be depleted or absent within the premise plumbing itself. Additionally, both Lp and NTM tend to more naturally resist chemical disinfectants than fecal-derived organisms. This research is aimed at overcoming these challenges, opening the door to new approaches to controlling Lp and NTM in premise plumbing. Historically, any microbial growth occurring in drinking water has been viewed as problematic, as it usually indicates the chemical disinfectant is inadequately protecting consumers. However, this work explores whether having an abundant community of beneficial bacteria could improve microbial water quality by competing against pathogens for limited space for attachment and nutrients. Such an approach would be analogous to the use of probiotics in humans, to establish a beneficial gut flora that is less susceptible to pathogen invasion. Granular activated carbon (GAC) filters are often used at drinking water treatment plants and by consumers as a point-of-use (e.g., installed on the kitchen tap or in a refrigerator) or whole-house treatment to remove any contaminants of concern and improve the taste and odor of tap water. The granules within GAC filters have a high surface area that helps remove contaminants, but also provides an environment where microbes can live and thrive. As water enters the filter, beneficial microbes can break down any remaining nutrients in the water (e.g., organic carbon and nitrogen). Additionally, the water leaving the filter carries high levels of microbes that grow on the GAC filter that are shed as water passes through. The resulting water with reduced nutrients and higher concentrations of potentially beneficial microbes could create a competitive environment that alters growth of harmful bacteria, like Lp and NTM, in downstream portions of plumbing. The incoming cold water is also warmed by the building envelope, which increases bacterial growth rates. Thus, the underlying hypothesis of this research is that GAC treatment could provide a combination of reduced nutrients and competitive microbes as water enters downstream premise plumbing and reduce the growth of Lp and NTM. However, GAC-treated water within a building can be further altered by other treatments, like a very hot water heater, which would heat and kill the microbes flowing through it, or a particle filter, which could remove the microbes in the water. This work also seeks to understand how these additional treatments might improve or interfere the nutrient reduction and addition of competitive microbes provided by GAC treatment. This research explores how all these different scenarios affect the growth of Lp and NTM using a lab-scale simulated premise plumbing system constructed out of polyvinyl chloride (PVC) pipe that is a common plumbing material used in homes. Water that was added to the pipes was prepared in four different ways to test the probiotic control hypothesis across distinct experimental conditions that replicate the different influent water scenarios. The four conditions were implemented over the course of eight months with regular chemical and biological analyses conducted to understand the effects of the different influent waters on Lp and NTM. It was discovered that premise plumbing with mature biofilms receiving GAC-treated water or GAC-treated + particle-filtered water contained ~90% less Lp and NTM than premise plumbing receiving non-filtered municipal tap water. However, if the GAC-treated water passes through a water heater, the capacity to limit Lp or NTM growth was lost. While GAC filters are currently thought of as an instantaneous treatment that removes contaminants from water, this work demonstrates how GAC treatment might provide prolonged benefits to water, after it has passed through the filter on its journey to a shower head or faucet. Increased understanding of the exact mechanisms of limited pathogen growth gained by this research can lead to new and effective approaches to protect people from contracting diseases caused by Lp and NTM.

Molecular typing is increasingly essential to tuberculosis (TB) control programmes, providing public health practitioners with a tool to characterize transmission patterns, track the emergence and spread of strains of M. tuberculosis complex (MTC) in populations. While molecular typing is already used extensively as a tool for TB control in many developed settings across the globe, its use in resource-poor settings is still limited. Moreover, information on the role, contribution and burden of nontuberculous mycobacteria (NTM) and other pathogens in aetiology of TB-like syndromes is also lacking in such settings. The broad objective of this dissertation was to determine the genetic diversity of MTC and their drug resistance profiles as well as the prevalence of NTM and other potentially pathogenic bacteria among TB suspects in Northeastern, Tanzania in order to generate insights that may inform the design of a rational TB control programmes. A total of 18 distinct spoligotypes were identified in this study area, with CAS1-KILI and EAI8 being the most predominant families. Major lineages prediction by conformal Bayesian network (CBN) revealed that 70% of TB infections in this area is due to modern lineages, whereas 30% of TB infections is due to the ancestral lineages mainly of Indo-oceanic lineage. The study also revealed that the overall proportions of any drug resistance and MDR-TB were 12.7% and 6.3% respectively. With the prevalence of any drug resistance and MDR-TB among new cases being 11.4% and 4.3% respectively, among previously, treated cases were 22.2%. The prevalence of NTM was found to be 9.7 %, with HIV being a significant predictor of NTM detection (P < 0.001). Four out of 30 patients with NTM diagnosed by culture received 1st line anti-TB treatment suggesting that a proportion of patients diagnosed by smear microscopy (4/65, 6.2%) were mistreated as TB patients. Our findings further showed that 17 (4.6%) out of 372 TB suspects were due to pulmonary nocardiosis. Overall this dissertation has revealed that TB is still a major problem in Tanga and is characterized by a diverse array of MTB strains. Additionally, modern MTB strains contribute significantly to TB infections in this area. High proportions of anti-TB drug resistance among new treated cases observed suggest that more efforts need to be done to identify individual cases at facility level for improved TB control programmes. Inefficient screening of TB patients and a prevalent increase of NTM may contribute to both unrealistic and mismanagement of TB cases. A diverse array of pathogenic Nocardia species among TB suspects further indicates that they are likely cause of human disease in this population. Therefore, need to integrate NTM and pathogens causing TB-like syndromes in diagnosis and management of TB is urgent. Results of these investigations contribute to the understanding of the dynamics of TB transmission in resource poor settings of Tanzania and highlight key factors that should be considered in the development of rational approaches to design effective TB prevention and control programmes in the country.

nÃo hà / As infecÃÃes por micobactÃrias nÃo tuberculosas (MNT) estÃo se tornando mais frequentes em todo o mundo, inclusive no Brasil. A capacidade das MNT em produzir doenÃa està claramente documentada na literatura e sua importÃncia clÃnica vem aumentando progressivamente. Embora a micobactÃria possa comprometer qualquer ÃrgÃo ou sÃtio, a forma mais frequente de comprometimento à a doenÃa pulmonar. Esse estudo se propÃs a avaliar aspectos clÃnicos e epidemiolÃgicos de pacientes com doenÃa pulmonar por MNT e a diversidade das espÃcies isoladas. Foram estudados retrospectivamente, 59 pacientes inscritos para tratamento no ambulatÃrio de referÃncia do Hospital de Messejana, perÃodo de 1998 a 2008. As espÃcies de MNT isoladas em meio de cultura a partir de amostras de escarro foram identificadas atravÃs da metodologia PRA no LaboratÃrio de Microbiologia da Faculdade de Medicina, UFC e classificadas conforme a patogenicidade em potencialmente ou raramente patogÃnicas. As variÃveis foram: ano diagnÃstico, idade, procedÃncia, sexo, profissÃo e risco biolÃgico de adoecimento, grau de escolaridade, renda familiar, comorbidades, histÃria prÃvia de tuberculose (TB), sorologia HIV, teste tuberculÃnico, histÃria de tabagismo e etilismo, sintomas clÃnicos, achados radiolÃgicos e desfecho. As variÃveis categÃricas e quantitativas foram analisadas atravÃs de proporÃÃes e frequÃncias com cÃlculo das medidas de localizaÃÃo central e de dispersÃo para cada variÃvel isoladamente. As anÃlises de associaÃÃes foram feitas utilizando o teste nÃo-paramÃtrico de Pearson e o teste paramÃtrico da razÃo de verossimilhanÃas, com IC=95% e p = 0,05. Resultados: As espÃcies identificadas mais frequentes foram: M. abscessus, 28,5% (17/59); M. lentiflavum 18,6% (11/59); M. avium, 16,6% (10/59). A mÃdia de idade  dp do grupo foi de 50,4  16,1 anos; 62,7% (37/59) do sexo masculino e 62, 7% tinham no mÃximo o ensino fundamental; 72,9% (43/59) renda atà um salÃrio mÃnimo; 49,2% (29/59) uso de Ãlcool e 40,6% (24/59) tabagismo; 62,7% (37/59) profissÃo com risco biolÃgico; 86% (51/59) residiam em Fortaleza; A tosse foi observada em 91,5% (54/59) e expectoraÃÃo em 72,9% (43/59); Fibrose e cavidade foram observadas em 71,2% (42/59) e 45,8 (27/59), respectivamente. Sorologia HIV foi positiva em 7% (4/59); teste tuberculÃnico realizado em 22% (13/59), desses, 92,3% (12/13) positivos; 54% (32/59) tiveram alta por cura e 15,3% (9/59) Ãbito. As associaÃÃes entre profissÃo com risco biolÃgico e sexo feminino, profissÃo com risco biolÃgico e comorbidade, e profissÃo e renda familiar mostraram-se significantes. A anÃlise de associaÃÃo da patogenicidade da espÃcie de MNT isolada com as outras variÃveis do estudo nÃo mostrou significÃncia, ou seja, nenhuma foi discriminativa. Este estudo demonstra a importÃncia para o reconhecimento da doenÃa pulmonar por MNT, cujas espÃcies podem ser identificadas em amostras de escarro. / Infections caused by nontuberculous mycobacteria (NTM) are becoming more frequent around the world and also in Brazil. The ability of produce disease is clearly documented in the literature and its importance has been increasing steadily. Although mycobacteria can affect any human organ, the most common form of involvement is the lung disease. This study aimed to evaluate clinical and epidemiological characteristics of patients with NTM lung disease and the diversity of species isolated. We studied retrospectively 59 patients enrolled for treatment at the outpatient clinic reference Messejana Hospital, from 1998 to 2008. The species of NTM isolated from sputum were identified using PRA method in the Microbiology Laboratory, Faculty of Medicine, Federal University of CearÃ. The variables studied were: year of diagnosis, age, origin, gender, profession and biological risk of illness, education level, family income, clinical symptoms, comorbidity, previous history of tuberculosis (TB), radiological findings, anti-HIV serology, tuberculin test, history of smoking and alcoholism and outcome. The quantitative and categorical variables were analysed by calculating frequencies and proportions of the measures of central location and dispersion for each variable separately. The analyzes of associations were performed using the nonparametric Pearsonâs test and parametric test of the likelihood ratio (based on product binomial distributions / multinomial) using CI = 95% and p < 0,05. The PRA method identified all 59 species, with predominance of the following: M. abscessus, 28.5% (17/59); M. lentiflavum 18.6% (11/59); M. avium, 16.6% (10/59); M. fortuitum and M. smegmatis, 10.2% (06/59) each; M. kansasii, 6.8% (4/59). The mean age  SD of the group was 50.4  16.1 years; 62.7% (37/59) were male and 62, 7% had at most basic education, 72.9% (43/59) with income below the poverty level, 49.2% (29/59) reported using alcohol and 40.6% (24/59) smoking, 59.3% (35/59) were involved with biological risk, 86% (51%) lived in Fortaleza. Cough was observed in 91.5% (54/59) and expectoration in 72.9% (43/59). Fibrosis and cavity were in 71.2% (42/59) and 45.8% (27/59) of the patients, respectively. HIV serology was positive in 7% (4/59), tuberculin skin test was performed in only 22% (13/59), of which 92.3% (12/13) were positive, 54% (32/59) had cured and 15.3% (9/59) had died. The associations between the variable profession with biological risk and gender (60% female), and between profession with biological risk and comorbidity (75% had associated disease), and profession with biological risk and family income proved to be significant. The association analysis of the groups of species of NTM isolated, if potentially pathogenic or rarely pathogenic with the others different study variables were not significant, thus did not discriminate to differentiate the groups. This study demonstrated the importance for the recognition of NTM lung disease whose species can be identified in sputum samples and for the epidemiological importance and the need for differential diagnosis in cases of tuberculosis.

Molecular typing is increasingly essential to tuberculosis (TB) control programmes, providing public health practitioners with a tool to characterize transmission patterns, track the emergence and spread of strains of M. tuberculosis complex (MTC) in populations. While molecular typing is already used extensively as a tool for TB control in many developed settings across the globe, its use in resource-poor settings is still limited. Moreover, information on the role, contribution and burden of nontuberculous mycobacteria (NTM) and other pathogens in aetiology of TB-like syndromes is also lacking in such settings. The broad objective of this dissertation was to determine the genetic diversity of MTC and their drug resistance profiles as well as the prevalence of NTM and other potentially pathogenic bacteria among TB suspects in Northeastern, Tanzania in order to generate insights that may inform the design of a rational TB control programmes. A total of 18 distinct spoligotypes were identified in this study area, with CAS1-KILI and EAI8 being the most predominant families. Major lineages prediction by conformal Bayesian network (CBN) revealed that 70% of TB infections in this area is due to modern lineages, whereas 30% of TB infections is due to the ancestral lineages mainly of Indo-oceanic lineage. The study also revealed that the overall proportions of any drug resistance and MDR-TB were 12.7% and 6.3% respectively. With the prevalence of any drug resistance and MDR-TB among new cases being 11.4% and 4.3% respectively, among previously, treated cases were 22.2%. The prevalence of NTM was found to be 9.7 %, with HIV being a significant predictor of NTM detection (P < 0.001). Four out of 30 patients with NTM diagnosed by culture received 1st line anti-TB treatment suggesting that a proportion of patients diagnosed by smear microscopy (4/65, 6.2%) were mistreated as TB patients. Our findings further showed that 17 (4.6%) out of 372 TB suspects were due to pulmonary nocardiosis. Overall this dissertation has revealed that TB is still a major problem in Tanga and is characterized by a diverse array of MTB strains. Additionally, modern MTB strains contribute significantly to TB infections in this area. High proportions of anti-TB drug resistance among new treated cases observed suggest that more efforts need to be done to identify individual cases at facility level for improved TB control programmes. Inefficient screening of TB patients and a prevalent increase of NTM may contribute to both unrealistic and mismanagement of TB cases. A diverse array of pathogenic Nocardia species among TB suspects further indicates that they are likely cause of human disease in this population. Therefore, need to integrate NTM and pathogens causing TB-like syndromes in diagnosis and management of TB is urgent. Results of these investigations contribute to the understanding of the dynamics of TB transmission in resource poor settings of Tanzania and highlight key factors that should be considered in the development of rational approaches to design effective TB prevention and control programmes in the country.

L’émergence de formes multirésistantes de tuberculose et la résistance intrinsèque de Mycobacterium abscessus à de nombreux anti-infectieux imposent l’identification de nouveaux antibiotiques et de nouvelles stratégies thérapeutiques. Les mycobactéries sont naturellement peu sensibles aux β-lactamines par production d’une β-lactamase et de cibles atypiques de faible affinité, les L,D-transpeptidases, qui sont efficacement inactivées par une seule classe de β-lactamines, les carbapénèmes. L’objectif de la thèse est d’étudier le mode d’action des β-lactamines afin de proposer des stratégies permettant d’optimiser ces antibiotiques. Pour comprendre la spécificité des L,D-transpeptidases vis-à-vis des carbapénèmes, nous avons étudié la cinétique et le mécanisme de la réaction d’inactivation de ces enzymes par différentes méthodes de spectroscopie en flux arrêté. Nos résultats indiquent que l’efficacité des carbapénèmes est due à leur capacité à former rapidement un intermédiaire tétrahédrique et à la stabilité de l’acylenzyme. La spécificité des L,D-transpeptidases pour les carbapénèmes ne dépend pas de leurs chaînes latérales, qui pourraient être modifiées pour améliorer les propriétés pharmacologiques de ces antibiotiques. Chez M. abscessus, nous avons identifié un inhibiteur de la β-lactamase, l’avibactam, qui augmente l’activité de certaines β-lactamines in vitro, en intracellulaire et dans un modèle d’infection du poisson zèbre. Nos résultats montrent que les β-lactamines peuvent être optimisées pour le traitement des infections dues aux mycobactéries multirésistantes par l’amélioration de l’inactivation des cibles ou l’inhibition des β-lactamases. / The emergence of multidrug-resistant tuberculosis and the intrinsic resistance of Mycobacterium abscessus to most antibiotics require the identification of new drugs and new therapeutic strategies. Mycobacteria are naturally poorly susceptible to β-lactam antibiotics due to production of a β-lactamase and of atypical low-affinity targets, the L,D-transpeptidases, which are effectively inactivated by a single class of β-lactams, the carbapenems. The aim of the thesis is to study the mode of action of β-lactams to propose strategies for the optimization of these antibiotics. To understand the specificity of L,D-transpeptidase for carbapenems, we have studied the kinetics and mechanism of inactivation of these enzymes using various stopped-flow spectroscopic methods. Our results indicate that the efficacy of carbapenems is due to their ability to rapidly form a tetrahedral intermediate and to the stability of the acylenzyme. The specificity of the L,D-transpeptidases for carbapenems does not depend upon the side chains of the drugs, which may be modified to improve their pharmacological properties. In M. abscessus, we have shown that the β-lactamase inhibitor avibactam increases the activity of various β-lactams in vitro, intracellularly, and in zebrafish model. Our results show that β-lactams can be optimized for the treatment of infections due to multidrug-resistant mycobacteria by improving inactivation of the targets and by inhibiting the β-lactamases.

Orientador: Paulo José Fortes Villas Bôas / Coorientador: Adriana Polachini do Valle / Resumo: Estima-se que um em cada quatro brasileiros esteja infectado pelo bacilo de Koch, e apenas em 2013, 80 mil casos de tuberculose foram notificados ao Ministério da Saúde, dos quais 6,5 mil foram a óbito. O exame laboratorial para o diagnóstico da tuberculose é feito pelos seguintes métodos tradicionais: Baciloscopia (BAAR) e Cultura, considerada o padrão ouro, porém com um tempo de realização demorado (em torno de oito semanas). A Reação em Cadeia da Polimerase (PCR) é um método de amplificação de DNA que pode ser útil no diagnóstico diferencial da tuberculose com outras infecções pulmonares. Além disso, a PCR pode confirmar a identificação da micobactéria em materiais clínicos que apresentem uma difícil positividade. Objetivos - Avaliar a positividade de Mycobacterium spp. em diferentes materiais biológicos utilizando a técnica de PCR para os alvos Internal Transcribed Spacer (ITS) 16S-23S rDNA e gene hsp 65. Material e métodos - Cento e trinta materiais biológicos de pacientes com suspeita de tuberculose pulmonar ou extrapulmonar foram investigados para pesquisa de Mycobacterium spp. por ITS-PCR e pesquisa de fragmento do gene hsp65, no período de março de 2012 a abril de 2013. Para identificação das espécies de micobactérias foi utilizada a pesquisa do gene IS6110, e técnica Restriction Enzyme Pattern Analysis (PCR-PRA). Para algumas amostras positivas pela PCR foi realizado o sequenciamento para identificação da espécie. Foram avaliados dados demográficos e condições ass... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is estimated that one in every four Brazilians is infected with Koch's bacillus, and just in 2013, eighty thousand cases of tuberculosis were reported to the Department of Health, of which 6.5 million died. The laboratory test for the diagnosis of tuberculosis is made by the following traditional methods: baciloscopy and culture, considered the gold standard, but with a long holding time (around 8 weeks). Polymerase chain reaction (PCR) is a DNA amplification method that can be useful in the differential diagnosis of pulmonary tuberculosis with other infections. In addition, PCR can confirm the identification of mycobacteria in clinical materials that exhibit a difficult positivity. Objectives - To evaluate the positivity of Mycobacterium spp. in different biological materials using the PCR technique for the Internal Transcribed Spacer (ITS) 16S-23S rDNA gene and hsp 65 targets. Materials and Methods - One hundred and thirty biological materials of patients with suspected pulmonary or extrapulmonary tuberculosis were investigated for Mycobacterium tuberculosis spp. by ITS-PCR research and gene fragment hsp65 research, from March 2012 to April 2013. To identify the species of mycobacteria was used the IS6110 gene research and PCR-PRA technique). For some positive samples was performed by PCR sequencing to species identification. They evaluated demographics and associated conditions. The comparison between the techniques was checked by χ² test. The samples surveyed in sput... (Complete abstract click electronic access below) / Doutor

No description available.

Bien que présentes dans tous les écosystèmes lacustres, la composition ainsi que l'abondance des communautés bactériennes peut varier à l'échelle régionale mais également à l'échelle locale. Une meilleure compréhension des facteurs responsables de ces patrons biogéographiques permettrait d'améliorer la connaissance des fonctionnements de ces systèmes aquatiques et donc de leur réponse potentielle face aux pressions anthropiques. Dans ce contexte, cette thèse a visé à étudier la biogéographie du compartiment bactérien dans un ensemble de lacs peu profonds. Les objectifs principaux de cette étude ont été d'évaluer aux échelles régionale et locale les facteurs responsables des patrons biogéographiques pour (i) l'ensemble de la communauté bactérienne et (ii) pour un groupe bactérien spécifique, i.e. les mycobactéries non-tuberculeuses. Pour atteindre ces objectifs deux approches complémentaires ont été entreprises. Tout d'abord, la variabilité spatiale à l'échelle régionale a été évaluée par l'échantillonnage en Ile-de-France de 49 lacs pendant trois étés. Couplé à cette approche, une étude plus fine a été entreprise pour caractériser la dynamique spatiotemporelle du compartiment bactérien par le suivi mensuel pendant deux ans et de six évènements pluvieux importants au sein du lac de Créteil (Val de Marne).À l'échelle régionale, la variabilité spatiale de la structure de la communauté bactérienne pour les trois années de suivi (caractérisé par T-RFLP) a été prédite à hauteur de 76% (r carré moyen) par les processus stochastiques et moins de 14% par les facteurs déterministes incluant les paramètres environnementaux (statut trophique) et les processus de dispersion (connexion du lac à une rivière et les axes PCNM). L'analyse de la composition de la communauté bactérienne par séquençage à haut débit (MiSeq Illumina) a mis en évidence des résultats similaires à ceux acquis par T-RFLP. Cependant, cette analyse a révélé que l'importance des processus impliqués dans les patrons biogéographiques pouvait évoluer en fonction des phylums ou des classes bactériens considérés. La variabilité spatiale des densités de mycobactéries (PCR en temps réel), était quant à elle expliquée à hauteur de 50% par des facteurs déterministes (pH de l'eau, concentration en fer labile et connexion à une rivière).À l'échelle locale, le suivi du lac de Créteil n'a révélé aucune variation spatiale significative (le long des transects horizontal et vertical) de la structure de la communauté bactérienne ainsi que des densités de mycobactéries. Par contre une étude spécifique sur deux lacs a révélé des variations significatives de densité et la diversité des mycobactéries au sein de différents compartiments des lacs. À l'inverse, d'importantes variations temporelles de la structure des communautés bactériennes ont été observées au cours des deux années de suivi, principalement associées aux variations de température de l'eau. Par ailleurs, bien que relativement stable au cours des deux années de suivi, les variations de densités des mycobactéries ont seulement été prédites par les processus stochastiques à hauteur de 35% / Although bacteria are widespread in lacustrine environments, their composition and abundance vary at the regional and also at the local scale. A better understanding of the factors responsible for these biogeographic patterns would improve our knowledge of these aquatic systems and thus their potential response to anthropogenic pressures. In this context, this thesis studied the biogeography of the bacterial compartment in a set of shallow lakes located in the Paris area. The main objectives of this study were to assess at the regional and local scale the factors responsible for the biogeographical patterns on (i) the entire bacterial community, and (ii) a specific bacterial group, i.e. the nontuberculous mycobacteria. To achieve these objectives two complementary approaches were undertaken. First, at the regional scale, the spatial variability was assessed by sampling 49 lakes during three consecutive summers. A finer study was also performed to characterize the spatiotemporal dynamics of the bacterial compartment over a two-year monthly monitoring and during six important rain events within the Créteil Lake (Val de Marne).At the regional scale, the spatial variability of the bacterial community structure for the three summers (assessed by T-RFLP) was predicted for 76% (mean r-squared) by stochastic processes and less than 14% by deterministic factors including environmental parameters (trophic status) and dispersal-related process (connection to a river and PCNM axes). The analysis of the bacterial composition by high-throughput sequencing (Illumina MiSeq) showed similar tendencies to those acquired by T-RFLP. However, this analysis revealed that the importance of the processes involved in biogeographical patterns could vary according to the bacterial phyla or classes considered. Spatial variability of mycobacterial densities (real time PCR) was explained up to 50% by deterministic factors (water pH, amount of labile iron and connection to a river).At the local scale, the monitoring of Creteil Lake revealed no significant spatial variation (along the horizontal and vertical transect) on the structure of the bacterial community and mycobacterial densities. However, a specific study of two lakes showed that mycobacterial density and diversity significantly varied among the different compartments of the lakes. Inversely, significant temporal variations on the bacterial community structure were observed over the two-year of monitoring, mainly related to water temperature changes. Although mycobacterial densities were relatively stable over the Créteil Lake monitoring, their variations were only predicted by stochastic processes up to 35%

Introdução: No ano de 2007, foram diagnosticados 190 casos de infecção hospitalar por Micobactéria de Crescimento Rápido (MCR) em pacientes submetidos a cirurgias videoassistidas em hospitais da região metropolitana de Vitória/ES. Os pacientes foram acompanhados na unidade de referência do Hospital Universitário Cassiano Antonio de Moraes da Universidade Federal do Espírito Santo (HUCAM/UFES), onde foi instituído tratamento específico e estabelecida investigação por métodos de imagens, sobretudo Tomografia Computadorizada (TC), para identificação do número, extensão e localização das lesões. Objetivo: Identificar a distribuição e a frequência dos achados de TC em pacientes com diagnóstico clínico e epidemiológico de infecção hospitalar por MCR após cirurgias laparoscópicas. Métodos: Foi conduzido um estudo descritivo utilizando os dados de prontuários da unidade de referência do HUCAM/UFES e as imagens das tomografias computadorizadas realizadas. Os exames foram analisados, em consenso, por dois radiologistas, que identificaram, separadamente, o comprometimento de pele/subcutâneo, de planos musculofasciais da parede abdominal e do intraperitoneal. Os padrões de comprometimento tabulados foram: densificações, coleções, nódulos maiores ou iguais a 1,0 cm (nódulos), nódulos menores que 1,0 cm (nódulos pequenos), nódulos com pseudocavitação e nódulos pequenos com pseudocavitação. Resultados: 26 pacientes atendiam aos critérios preestabelecidos. As infecções foram relacionadas a: nove cirurgias bariátricas, uma cirurgia bariátrica com colecistectomia, sete colecistectomias, uma colecistectomia somada à correção de hérnia inguinal com colocação de tela, três cirurgias para correção de refluxo gastroesofágico (CRGE), três laparoscopias diagnósticas, uma salpingectomia e uma apendicectomia.O menor intervalo de tempo entre a cirurgia e o exame de tomografia computadorizada foi de 8 dias, o maior, 351 dias, com média de 112 dias e mediana de 83. Todos os pacientes apresentaram algum achado de imagem no subcutâneo, sendo que seis pacientes tiveram comprometimento exclusivo na pele/subcutâneo, e os demais apresentaram comprometimento concomitante de planos musculofasciais e/ou intraperitoneal. Os achados no subcutâneo foram: densificação (88,4%), nódulo pequeno (61,5%), nódulo pequeno pseudocavitado (23,0%), nódulo (38,4%), nódulo pseudocavitado (15,3%) e coleção (26,9%); os achados nos planos musculofasciais foram: densificação (61,5%), nódulo pseudocavitado (3,8%) e coleção (15,3%); e os achados intraperitoneais foram: densificação (46,1%), nódulo pequeno (42,3%), nódulo (15,3%) e coleção (11,5%). Nenhum dos cinco pacientes que realizaram a biópsia subcutânea anteriormente à TC apresentou nódulo subcutâneo maior do que 1,0 cm; comparativamente, dentre aqueles que realizaram a biópsia após a TC, constatou-se que 10 pacientes (47,6%) apresentaram nódulos subcutâneos. A comparação entre os 16 pacientes com intervalo de tempo entre cirurgia e TC menor do que 3 meses e os 10 pacientes com intervalo maior do que 3 meses demonstrou que: os pacientes com menor intervalo de tempo apresentavam maior porcentagem de densificações no subcutâneo (100%), nos planos musculofasciais (81,2%) e intraperitoneais (70,0%); já no grupo com maior intervalo, as porcentagens foram de 66,6%, 30,0% e 10,0%, respectivamente. Conclusão: os achados tomográficos, em ordem decrescente de frequência, foram: a) no subcutâneo: densificação, nódulo pequeno, nódulo, nódulo pequeno pseudocavitado, nódulo pseudocavitado e coleção; b) nos planos musculofasciais: densificação, coleção e nódulo pseudocavitado; e c) intraperitoneal: densificação, nódulo pequeno, nódulo e coleção / Introduction: In 2007, 190 cases of hospital-acquired infection due to rapidly growing mycobacterial (RGM) were diagnosed in patients undergoing video-assisted surgery in the hospitals of the metropolitan region of Vitória, ES (Brazil). The patients were followed at the referral unit of the University Hospital Cassiano Antonio de Moraes of the Federal University of Espírito Santo (HUCAM), where specific treatment was instituted and research by imaging methods - particularly computed tomography (CT) - was initiated to identify the number, extent, and location of the lesions. Objective: To identify the distribution and frequency of CT findings in patients with clinical and epidemiological diagnosis of hospital-acquired RGM infection after laparoscopic surgery. Method: A descriptive study was conducted using medical records data from the referral unit of the HUCAM and the computed tomography (CT) images. The scans were analyzed by two radiologists, in consensus, who individually identified compromised skin/subcutaneous areas, muscle-fascial planes of the abdominal wall and intraperitoneal regions. The involvement patterns were tabulated as: densification, collections, nodules >= 1.0 cm (nodules), nodules < 1.0 cm (small nodules), pseudocavitated nodules, and small pseudocavitated nodules. Results: Twenty-six patients met the established criteria. The infections were related to 9 bariatric surgeries, 1 bariatric surgery with cholecystectomy, 7 cholecystectomies, 1 cholecystectomy along with inguinal hernia correction with screen placement, 3 surgeries for correction of gastroesophageal reflux, 3 diagnostic laparoscopies, 1 salpingectomy, and 1 appendectomy. The shortest time interval between surgery and CT examination was 8 days and the longest interval was 351 days, with a mean of 112 days and a median of 83 days. All patients presented subcutaneous involvement on the CT image; 6 patients had exclusive impairment in the skin/subcutaneous tissue whereas the others had concomitant impairment in musculo-fascial and/or intraperitoneal planes. The subcutaneous findings were: densification (88.4%), small nodules (61.5%), small pseudocavitated nodules (23.0%), nodules (38.4%), pseudocavitated nodules (15.3%), and collections (26.9%). The findings in the musculo-fascial planes were: densification (61.5%), pseudocavitated nodules (3.8%), and collections (15.3%). The intraperitoneal findings were: densification (46.1%), small nodules (42.3%), nodules (15.3%) and collections (11,5%). None of the 5 patients who underwent biopsy before CT showed subcutaneous nodules larger than 1.0 cm; in contrast, of those who underwent biopsy after CT, 10 patients (47.6%) had subcutaneous nodules. A comparison between the 16 patients with a time interval of less than 3 months between surgery and CT, and the 10 patients with an interval of more than 3 months showed that patients with a shorter time interval had a higher percentage of subcutaneous densification (100%), musculo-fascial plane densification (81.2%), and intraperitoneal densification (70.0%); for those with a longer interval, the percentages were 66.6%, 30.0%, and 10.0%, respectively. Conclusion: the subcutaneous CT findings in descending order of frequency were: densification, small nodules, nodules, small pseudocavitated nodules, pseudocavitated nodules, and collections. The musculo-fascial plane CT findings were: densification, collections, and pseudocavitated nodules. The intraperitoneal CT findings were: densification, small nodule, nodules and collections