Identifica los linajes de Mycobacterium tuberculosis (MTB) en la región Callao mediante el uso de la metodología MIRU-VNTR. El estudio comprende 133 muestras de DNA obtenidas a partir de aislamientos de MTB en medio sólido Löwenstein - Jensen (LJ). Utiliza 24 MIRU-VNTR para la genotipificación de MTB. Los resultados reportan un alto poder discriminatorio de este método con un HGDI de 0.995. El linaje LAM es el más prevalente (51.1 %), seguido por Haarlem (18.8 %), el linaje asiático Beijing (8.3 %), el linaje Uganda se identificó en el 6% y los linajes T y S se observaron con un 2.3 % y 0.8 %, respectivamente. Identifica como Orphans (huérfanos) un 8.3 % de las cepas analizadas. La región Callao muestra una gran diversidad de linajes; así como, la presencia de patrones huérfanos endémicos. Esta información permite reconocer los linajes que se encuentran circulando en toda la región, así mismo su posible relación con multidrogorresistencia, lo cual será de gran utilidad al sector salud, para establecer programas de control y prevención oportuna de casos de tuberculosis. / Tesis

INTRODUÇÃO: A hanseníase é uma doença infectocontagiosa granulomatosa crônica, causada por Mycobacterium leprae e representa ainda um problema de saúde pública no Brasil. Em média 30-40% dos doentes tem risco de desenvolver reações hansênicas, que são considerados estados de exacerbação da resposta imunológica do hospedeiro a antígenos M. leprae. Está bem descrita a importância de células T reguladoras (Tregs) em infecções bacterianas crônicas. Estudos anteriores do nosso grupo demonstraram aumento da proporção de Tregs in situ e em sangue periférico de pacientes multibacilares (mas não em paucibacilares), assim como diminuição da proporção de Tregs na reação tipo 2 (porém não na reação tipo 1), sugerindo a participação deste subtipo celular na imunopatogênese da hanseníase. Entretanto, a baixa frequência de Tregs tem dificultado o estudo da sua capacidade funcional. OBJETIVO: Estabelecer protocolo de expansão de Tregs funcionais em pacientes com hanseníase e, posteriormente, avaliar a capacidade funcional das Tregs expandidas. METODOLOGIA: Células mononucleares do sangue periférico (PBMCs) foram separadas e cultivadas, com anti-CD3 e anti-CD28, acrescidos de IL-2 e rapamicina, por 21 dias. Para avaliar a capacidade funcional das Tregs expandidas, realizou-se um ensaio de co-cultivo das Tregs expandidas e PBMCs autólogas (marcadas com CFSE). Utilizou-se diferentes proporções de Tregs:PBMCs (1:1, 1:2 e 1:5). A casuística foi composta por 29 indivíduos diagnosticados com hanseníase, divididos em quatro grupos, de acordo com a classificação de Ridley e Jopling. Além disso, coletou-se material de 6 indivíduos saudáveis, com a finalidade de caracterizar o pool celular do protocolo de expansão. Para isso, culturas de expansão de indivíduos saudáveis foram submetidas a dois protocolos distintos de purificação celular (beads magnéticas e cell sorting), além de imunofenotipagem das moléculas CD39, PD-1 e LAG3. RESULTADOS: Tregs dos pacientes multibacilar sem reação, nas proporções 1:2 e 1:5, e no grupo reacional tipo 2, na proporção 1:5, mostraram maior capacidade supressora, quando comparados aos grupos pacubacilar sem reação e reacional tipo 2, respectivamente. No grupo de indivíduos saudáveis não houve diferenças na capacidade supressora das Tregs obtidas por separação por beads magnéticas e Tregs obtidas por cell sorting, e não houve expressão significativa de células PD-1+ e LAG-3+ nas culturas de expansão. CONCLUSÃO: Em todos os indivíduos testados, independente da classificação clínica da hanseníase, o protocolo proposto resultou em uma expansão exponencial de uma linhagem de Tregs com atividade supressora, porém com sugestão preliminar de maior eficiência supressora das Tregs na forma MB (com ou sem reação), corroborando a correlação entre atividade supressora de Tregs e maior carga bacilar. Além disso, o protocolo com beads magnéticas se mostrou tão eficaz quanto o protocolo de purificação por cell sorting. A imunofenotipagem das Tregs expandidas de indivíduos saudáveis indicou que não havia número significativo de células exaustas ao fim de nosso protocolo, assim como não houve crescimento concomitante de células T reguladoras do tipo 1 na cultura de expansão / INTRODUCTION: Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae and a public health problem in Brazil. On average, 30-40% of patients are at risk of developing leprosy reactions, which are considered as states of exacerbation of the host\'s immune response to M. leprae antigens. The importance of regulatory T cells (Tregs) in chronic bacterial infections has been well described, but their role in leprosy remains unclear. Previous studies from our group have shown increased proportions of Tregs in situ and in peripheral blood of multibacillary patients (but not in paucibacillary patients), and decrease in the proportion of Tregs in type 2 reaction (but not type 1 reaction), reinforcing the role played by these cells in the immunopathogenesis of leprosy. However, the low numbers of Tregs preclude further studies on their functional capabilities. OBJECTIVE: To establish a protocol for expansion of functional Tregs in patients with leprosy and evaluate the functional capacity of the expanded Tregs. METHOD: Peripheral blood mononuclear cells (PBMCs) were separated and cultured under anti-CD3 and anti-CD28 stimulation, plus IL-2 and rapamycin for 21 days. To evaluate the functional capacity of the expanded Tregs, a co-culture assay of the expanded Tregs and autologous PBMCs (labeled with CFSE) was performed. Different ratios of Tregs: PBMCs (1: 1, 1: 2 and 1: 5) were used. The sample consisted of 29 individuals diagnosed with leprosy, divided into four groups, according to the Ridley and Jopling classification. In addition, samples from 6 healthy individuals were collected to characterize better the cellular characteristics of the expansion protocol. For this, cultures of healthy individuals PBMC were submitted to two distinct cell purification protocols (magnetic beads and cell sorting), as well as immunophenotyping of the molecules CD39, PD-1 and LAG3. RESULTS: Expanded Tregs from the multibacillary patients without reaction, at 1:2 and 1:5 ratios, and with type 2 reaction, at 1: 5 ratio, showed greater suppressive capacity that the respective paucibacillary groups. Tregs from healthy individuals obtained through magnetic beads separation and through cell sorting did not show differences in suppressor capacity, and there was no significant expression of PD-1 + and LAG-3 + on cells of the expansion cultures. CONCLUSION: In all individuals tested, irrespective of the leprosy classification, the proposed protocol resulted in an exponential expansion of Tregs with suppressive capacity; however, preliminary evidence indicated stronger suppressor activity of the Tregs from multibacillary patients (with or without reaction), suggesting a direct correlation between Tregs activity and bacillary load in leprosy. In addition, our protocol with magnetic beads proved to be as effective as the cell sorting protocol. Immunophenotyping of expanded Tregs from healthy subjects revealed that there was no significant number of exhausted cells at the end of our protocol, nor there was the concomitant outgrowth of type 1 regulatory T cells in the expansion protocol

PhD, Faculty of Health Sciences, University of the Witwatersrand / The South African gold-mining workforce has an unusually high incidence of Mycobacterium kansasii disease, yet little is known about the possible sources of M. kansasii infection, genetic diversity and the basis for this organism’s pathogenicity. The purpose of this study was to investigate these issues in a gold-mining environment. Five M. kansasii isolates and 10 other potentially pathogenic mycobacteria were cultured mainly from showerhead biofilms. PCR-restriction analysis (PRA) of the hsp65 gene on 191 clinical and on the 5 environmental M. kansasii isolates revealed 160 subtype I (157 clinical and 3 environmental), 8 subtype II (clinical) and 6 subtype IV (5 clinical and 1 environmental) strains. Twenty-two isolates (21 clinical and 1 environmental) did not show the typical M. kansasii PRA patterns. After confirmation by DNA sequencing as belonging to the M. kansasii species, the results suggested that these isolates were probably new subtypes of M. kansasii. In contrast to the clonal population structure found amongst the subtype I isolates from studies in other countries, DNA fingerprinting of 114 subtype I clinical and environmental isolates showed genetic diversity amongst the isolates. One of the 2 environmental isolates showed 100% identity with a clinical isolate, suggesting that water distribution systems are the possible sources of M. kansasii infection for the miners. An investigation into the genetic differences between clinical (subtype I) and environmental (III, IV and V) isolates, using Hybridisation Monitored Differential Analysis (HMDA), identified 45 open reading frames (ORFs) encoding predominantly membrane-associated proteins that include six potential virulence factors, two family members of transcription regulators for drug and xenobiotic metabolism, three family members of multidrug efflux systems, a number of proteins associated with lipid and carbohydrate metabolism and transport, and a number of hypothetical proteins with unknown function. Immunological analysis of M. kansasii isolates, using the Lymphocyte Transformation and Cytometric Bead Array assays, showed that M. kansasii modulates immune responses through suppression of lymphocyte blastogenesis and by altering the expression of Th1/Th2/Th17 cytokines by human lymphocytes in vivo for its own survival. This study demonstrated for the first time that water distribution systems in South Africa are possible sources of M. kansasii infection, and showed that subtype I strains of M. kansasii from the study region display genetic diversity and have unique or divergent genes not found in other subtypes. It also demonstrated that immunosuppression is one of the pathogenic mechanisms employed by M. kansasii.

Dissertation Submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in the fulfillment of the requirement for the degree of Master of Science in Medicine by Research. Johannesburg, 2017 / Electron transport and respiration in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) occurs through the use of the aa3 type cytochrome c oxidase (CcO) under normoxic conditions or cytochrome bd oxidase (CbdO) under microaerophillia. Using these oxidases, Mtb couples substrate level oxidation to generation of metabolic energy in the form of adenosine triphosphate (ATP) under aerobic conditions. The presence of the CbdO is expected to allow for growth and survival of Mtb in the oxygen restricted environment of the human TB granuloma, thus rendering it an important enzyme for further study. CbdO is comprised of two structural subunits, CydA (subunit I) and CydB (subunit II), which are encoded by the cydAB operon. Both subunits span the cytoplasmic membrane to form part of the mycobacterial electron transport chain. Notably, two other genes that are transcribed separately from the cydAB operon, the cydDC operon, have been proposed to be required for the synthesis of a functional CbdO. Based on amino acid sequence and structural predictions, the cydDC encode heterodimeric members of the ATP Binding Cassette ABC-type transporters. In other organisms, the cydDC functions to transport reducing agents to the periplasm, thus contributing to periplasmic redox homeostasis. In this study we aimed to analyze the function of the cydDC genes in Mycobacterium smegmatis. Through bioinformatics analyses, it was demonstrated that the CydDC subunits retain conserved residues associated with the ABC domain and adopt a three-dimensional fold that is similar to their counterparts in Escherichia coli. However, the published sequence of M. smegmatis suggests that cydC is a pseudogene, which was inconsistent with the demonstrated evidence of a functional CbdO in this organism. In this study, using standard DNA sequencing, it was demonstrated that the CBTBR laboratory strain of M. smegmatis does not harbor a cydC pseudogene but rather has a functional cydC gene. Next, we interrogated the function of the M. smegmatis and Mtb cydDC genes by heterologous expression in an E. coli cydD mutant. Heterologous expression of the Mtb cydDC genes restored CbdO biogenesis in the E. coli mutant. Using various microbiological approaches, we demonstrated that the mycobacterial cydDC was able to revert the stationary phase exit defect, high temperature sensitivity and increased oxidative stress susceptibility defects of the E. coli cydD mutant. Collectively, these data provided strong evidence that the mycobacterial cydDC genes encode a functional transporter that contributes to periplasmic redox homeostasis. Following this, we generated a double deletion mutant of the cydDC operon in M smegmatis. We confirmed the genetic integrity of the ΔcydDC strain by Southern Blot analysis and proved by absorption difference spectroscopy that this strain is defective in the ability to synthesize a functional CbdO, as measured by the lack of a heme d peak in membrane preparations from the mutant. In addition, the ΔcydDC mutant displayed increased sensitivity to oxidative stress and a reduced ability to exit stationary phase, phenotypic defects that were consistent with the lack of CbdO. In summary, this study provides the first evidence that loss of the M. smegmatis cydDC genes affects CbdO biogenesis. These data also confirm that the CydDC ABC-type transporter most likely transports reducing equivalents that allow for maturation of CbdO in the periplasm. Collectively, our observations advance the understanding of the mycobacterial electron transport chain and provide new evidence to assist in the development of CbdO as a TB drug target. / MT2017

Mycobacterium tuberculosis (MTB) encodes three enzymes that are dependent on vitamin B12–derived cofactors for activity, including a B12-dependent methionine synthase (MetH). Previously, work in the Molecular Mycobacteriology Research Unit (MMRU) demonstrated vitamin B12 auxotrophy in a mutant strain disrupted in the alternative, B12-independent methionine synthase, MetE. This observation established the ability of MTB to transport corrinoids despite the absence of an identifiable B12-specific transporter. In addition, it suggested that MTB does not synthesize vitamin B12 in vitro. Notably, bioinformatic analyses identified PPE2 as the only B12-related transport candidate in MTB, though as a putative B12-regulated cobalt transporter. PPE2 is unusual in possessing directly upstream of its predicted start codon one of only two B12-dependent riboswitches in the MTB genome, and it lies in a putative operon with B12 biosynthetic genes, cobU and cobQ1. In this study, the possibility that PPE2 functions in the transport of vitamin B12 or cobalt was investigated. Transcriptional and phenotypic data suggested that PPE2 was not involved in B12 transport. Instead, it was shown that cobalt can supplement the growth of an MTB metE mutant in liquid medium, strongly supporting the ability of MTB to synthesize B12 de novo. Moreover, the ability to utilise exogenous cobalt was dependent on functional PPE2, thereby establishing a role for a PPE-family member in cobalt assimilation in MTB. Vitamin B12 comprises a central corrin ring co-ordinated to 5,6-dimethylbenzimidazole (DMB) as α-axial ligand. Substituting DMB with adenine yields the alternate form, pseudo-B12. The ability of mycobacteria to utilize pseudo-B12 precursors (cobinamide and adenine) to support full function of B12-dependent metabolic pathways was evaluated. Although the pseudo-B12 precursors appeared to complement chemically the mycobacterial B12 auxotrophs, growth of the mutants on cobinamide alone complicated this interpretation. To address this limitation, DMB synthesis was targeted by disrupting the MTB bluB homologue, Rv0306. Neither site-directed mutagenesis of key Rv0306 residues, nor full-gene deletion was sufficient to eliminate growth on cobinamide. Instead, this observation highlights the need to establish biochemically the nature of the active B12 form synthesized and utilized by MTB under different conditions. In combination, the results presented here support the inferred flexibility of vitamin B12 biosynthesis in MTB, and reinforce the potential role of B12-dependent metabolism in mycobacterial pathogenesis.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Medicine (Internal Medicine) Johannesburg 2001 / Introduction and Aim: The waxy outer cell wall of mycobacteria consists mainly of mycolic acids (MA). The unique immuno-stimulatory properties of MA via the CD 1-restricted antigen presentation pathway have been demonstrated in humans. Purification and isolation of M.tuberculosis (MTB) MA has allowed them to be applied as an antigen in an ELISA-based sero-diagnostic assay to detect specific antibodies in the sera of humans. The aim of the study was to measure the levels of antibody to MA in the sera of patients with culture proven pulmonary tuberculosis (PTB), and in control subjects without evidence of tuberculosis. / IT2018

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-07-26T14:44:00Z No. of bitstreams: 1 JOSE_EDUARDO_SACCONI_NUNES_DIS.pdf: 1721834 bytes, checksum: 00ef2ffd5994d7fa6e05348a79aaced6 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-07-31T14:27:33Z (GMT) No. of bitstreams: 1 JOSE_EDUARDO_SACCONI_NUNES_DIS.pdf: 1721834 bytes, checksum: 00ef2ffd5994d7fa6e05348a79aaced6 (MD5) / Made available in DSpace on 2018-07-31T14:47:08Z (GMT). No. of bitstreams: 1 JOSE_EDUARDO_SACCONI_NUNES_DIS.pdf: 1721834 bytes, checksum: 00ef2ffd5994d7fa6e05348a79aaced6 (MD5) Previous issue date: 2011-04-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / In 2009, tuberculosis (TB) was responsible for 1.3 million deaths worldwide. The incidence rates reached 9.4 millions and the World Health Organization (WHO) estimative indicates that one third of the world population is infected by Mycobacterium tuberculosis, the main agent responsible for the disease. The lack of new drugs released on market, the long period treatment presenting side effects (causing the abandon by the patients) and the cases with HIV co-infection contributed to the appearance of multi drug resistant strains (MDR-TB) and extensively drug resistant strains (XDR-TB). It's clear, thus, that the development of new drugs to fight TB is necessary and fundamental to the success in eradicating this disease. The histidine biosynthesis pathway emerge in this context offering attractive targets, given that its present in prokaryotes, lower eukaryotes and plants, but absent in animals. The last enzyme in the route is called Histidinol Dehydrogenase and is responsible for the conversion of L-Histidinol into LHistidine. Its essentiality to the bacilli was confirmed by gene knockout, confirming its potential for the development of inhibitory compounds. In this work, a purification protocol was developed, producing the enzyme in the homogeneous form in quantities sufficient to carry its biochemical characterization. The enzyme needs a divalent metal ion in the active site to catalyze the reaction. The kinetic constants were determined, as well as the mechanism, the pH rate profiles and the interaction of its substrates and products by isothermal titration calorimetry. A tridimensional model for its structure was constructed by sequence homology, allowing the analysis of the interaction of the substrates and metal in the active site. The results obtained will allow the rational design of molecules that act as inhibitors. / Em 2009 a tuberculose (TB) foi respons?vel por 1,3 milh?es de mortes no mundo inteiro. A incid?ncia de casos chegou ao patamar de 9,4 milh?es e as estimativas da Organiza??o Mundial da Sa?de (OMS) indicam que aproximadamente 1/3 da popula??o mundial est? infectada pelo Mycobacterium tuberculosis, principal agente causador da doen?a. A falta de novas drogas no mercado, o tratamento longo e com efeitos colaterais (levando ao abandono por parte dos pacientes) e os quadros de co-infec??o com HIV tem colaborado para o surgimento de novas cepas resistentes as drogas atualmente em uso (MDR-TB e XDR-TB). Fica claro, portanto, que o desenvolvimento de novas drogas para o combate da TB ? necess?rio e fundamental para que se tenha sucesso na erradica??o desta doen?a. A via de bioss?ntese de histidina aparece nesse contexto oferecendo alvos atrativos, visto que est? presente em organismos procari?ticos, em organismos eucari?ticos inferiores e em plantas, mas ausente em animais. A ?ltima enzima pertencente ? via ? chamada de Histidinol Desidrogenase e ? respons?vel pela convers?o de L-Histidinol em L-Histidina. Sua essencialidade para a viabilidade do bacilo foi comprovada atrav?s de nocaute g?nico, confirmando sua potencialidade para o desenvolvimento de compostos inibidores de sua atividade. Neste trabalho, um protocolo depurifica??o foi desenvolvido, produzindo a enzima na forma homog?nea em quantidades suficientes para realizar a caracteriza??o bioqu?mica da mesma. A enzima necessita de um ?on met?lico divalente no s?tio para catalisar a rea??o. Suas constantes cin?ticas foram determinadas, assim como o mecanismo, os perfis de pH, e a intera??o com os substratos e produtos atrav?s de calorimetria de titula??o isot?rmica. Um modelo tridimensional da sua estrutura foi constru?do por homologia de sequ?ncia, permitindo uma an?lise da intera??o dos substratos e do metal no s?tio ativo da enzima. Os resultados obtidos permitir?o o desenho racional de mol?culas que atuem como inibidores.

by Ho Iok Ieng Yolanda. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 116-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Yip Chi Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 70-87). / ABSTRACT --- p.i / ACKNOWLEDGEMENT --- p.iv / TABLE OF CONTENTS --- p.v / LIST OF TABLES --- p.ix / LIST OF FIGURES --- p.xiii / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.3 / Chapter I. --- Mycobacterial Infections --- p.3 / Chapter A. --- Mycobacterium tuberculosis --- p.3 / Chapter B. --- Atypical mycobacteria --- p.3 / Chapter II. --- Identification of Mycobacteria --- p.5 / Chapter A. --- Conventional methods --- p.6 / Chapter 1. --- Mycobacterium tuberculosis --- p.6 / Chapter 2. --- Atypical mycobacteria --- p.7 / Chapter B. --- Rapid identification methods --- p.8 / Chapter 1. --- Identification by fatty acid analysis --- p.8 / Chapter 2. --- Identification by mycolic acid analysis --- p.9 / Chapter III. --- In vitro Susceptibility Testing of Mycobacteria --- p.11 / Chapter A. --- Mycobacterium tuberculosis --- p.11 / Chapter 1. --- Principle --- p.12 / Chapter 2. --- Methods of susceptibility testing --- p.13 / Chapter a. --- The absolute concentration method --- p.13 / Chapter b. --- The resistance ratio method --- p.15 / Chapter c. --- The 1% proportion method --- p.16 / Chapter d. --- Radiometric method --- p.18 / Chapter e. --- Other methods --- p.19 / Chapter B. --- Atypical mycobacteria --- p.20 / Chapter IV. --- Plasmid Analysis in Mycobacteria --- p.22 / Chapter A. --- Discovery of plasmids in mycobacteria --- p.22 / Chapter B. --- Methodologies in the studies of mycobacterial plasmids --- p.23 / Chapter C. --- Possible roles of plasmid in epidemiology of mycobacteria --- p.24 / MATERIALS AND METHODS --- p.26 / Chapter I. --- Bacterial Strains and Strain Maintenance --- p.26 / Chapter A. --- Strains collection --- p.26 / Chapter B. --- Strains maintenance --- p.26 / Chapter II. --- Culture Media and Culture Conditions --- p.26 / Chapter III. --- Identification of Mycobacteria --- p.26 / Chapter A. --- Conventional methods --- p.26 / Chapter B. --- Fatty acid profile analysis --- p.27 / Chapter 1. --- Bacterial isolates --- p.27 / Chapter 2. --- Standards and reagents --- p.27 / Chapter 3. --- Preparation of methyl ester for GC/GC-MS --- p.28 / Chapter 4. --- Instrumentation --- p.28 / Chapter a. --- Gas chromatography-mass spectrometry (GC-MS) --- p.28 / Chapter b. --- Gas liquid chromatography (GLC) --- p.28 / Chapter 5. --- Fatty acid profile analysis --- p.29 / Chapter a. --- Calibration --- p.30 / Chapter b. --- Identification of mycobacterial fatty acids --- p.30 / Chapter c. --- Construction of mycobacterial fatty acid profiles --- p.30 / Chapter 6. --- Discriminant analysis --- p.31 / Chapter IV. --- In Vitro Drug Susceptibility Test --- p.31 / Chapter A. --- Test strains --- p.31 / Chapter B. --- Preparation of drug-containing media --- p.32 / Chapter C. --- Minmum inhibition concentration (MIC) determination --- p.32 / Chapter V. --- Heavy Metal Tolerance Test --- p.33 / Chapter A. --- Bacterial strains --- p.33 / Chapter B. --- Reagent and media preparation --- p.34 / Chapter 1. --- Heavy metal stock solution preparation --- p.34 / Chapter 2. --- Media preparation --- p.34 / Chapter C. --- Minimum inhibition concentration (MIC) determination --- p.34 / Chapter VI. --- Plasmid Analysis of Mycobacteria --- p.35 / Chapter A. --- Bacterial strains --- p.35 / Chapter B. --- Extraction procedures --- p.35 / Chapter 1. --- Modified Kado & Liu method --- p.35 / Chapter 2. --- French press procedure --- p.36 / Chapter 3. --- Spheroplasts preparation procedure --- p.37 / Chapter C. --- Electrophoresis procedure --- p.37 / Chapter D. --- Statistical analysis for correlation between plasmid and drug resistance or heavy metal tolerance --- p.38 / RESULTS --- p.39 / Chapter I. --- Identification of Mycobacteria --- p.39 / Chapter A. --- General characteristics of the chromatographic profile --- p.39 / Chapter B. --- Discriminant analysis --- p.40 / Chapter 1. --- Gas chromatography-mass spectrometry (GC-MS) --- p.40 / Chapter a. --- Slowly growing non-pigmented mycobacteria --- p.40 / Chapter b. --- Rapidly growing mycobacter-ia --- p.41 / Chapter c. --- Pigmented mycobacteria --- p.41 / Chapter 2. --- Gas liquid chromatography (GLC) --- p.41 / Chapter a. --- Slowly growing non-pigmented mycobacteria --- p.42 / Chapter b. --- Rapidly growing mycobacteria --- p.42 / Chapter c. --- Pigmented mycobacteria --- p.43 / Chapter II. --- Vitro Drug Susceptibility Test --- p.43 / Chapter A. --- Mycobacterium tuberculosis --- p.43 / Chapter B. --- Atypical mycobacteria --- p.45 / Chapter 1. --- General characteristics --- p.45 / Chapter 2. --- Sensitivity pattern of different species --- p.46 / Chapter a. --- Mycobacterium kansasii --- p.46 / Chapter b. --- Mycobacterium avium- intracellulare complex --- p.46 / Chapter c. --- Mycobacterium scrofulaceum --- p.47 / Chapter d. --- Mycobacterium terrae complex --- p.47 / Chapter e. --- Mycobacterium fortuitum --- p.47 / Chapter f. --- Mycobacterium chelonae --- p.48 / Chapter III. --- Heavy Metal Tolerance Test --- p.48 / Chapter IV. --- Plasmid in Mycobacteria --- p.48 / Chapter A. --- Mycobacterium tuberculosis --- p.48 / Chapter B. --- Atypical mycobacteria --- p.49 / Chapter 1. --- General characteristics --- p.49 / Chapter 2. --- Correlation between drug resistance and plasmid --- p.50 / Chapter 3. --- Correlation between heavy metal tolerance and plasmid --- p.50 / DISCUSSION --- p.52 / Chapter I. --- Identification of Mycobacteria --- p.52 / Chapter II. --- In Vitro Drug Susceptibility Test --- p.56 / Chapter A. --- Mycobacterium tuberculosis --- p.56 / Chapter B. --- Atypical mycobacteria --- p.60 / Chapter 1. --- Mycobacterium kansasii --- p.61 / Chapter 2. --- Mycobacterium avium- intracellulare complex --- p.61 / Chapter 3. --- Mycobacterium scrofulaceum --- p.62 / Chapter 4. --- Mycobacterium terrae complex --- p.62 / Chapter 5. --- Mycobacterium fortuitum --- p.63 / Chapter 6. --- Mycobacterium chelonae --- p.64 / Chapter III. --- Plasmid Analysis in Mycobacteria --- p.64 / Chapter A. --- Mycobacterium tuberculosis --- p.64 / Chapter B. --- Atypical mycobacteria --- p.66 / SUMMARYS AND CONCLUSIONS --- p.68 / LITERATURE CITED --- p.70 / Chapter APPENDIX - --- Tables --- p.88 / Figures --- p.144

O gênero Mycobacterium spp. compreende microrganismos saprófitas e patogênicos de interesse em saúde animal e humana. A espécie M. bovis, que causa tuberculose nos animais, é excretada através do leite de bovinos infectados e tem no consumo de leite cru e seus derivados uma importante via de transmissão para o homem, causando uma doença tão grave quanto à causada pelo M. tuberculosis. Como a doença nos animais é endêmica no Brasil e o queijo minas meia cura é normalmente fabricado com leite cru e muito apreciado pelo consumidor paulistano, amostras desse produto, obtidas em feiras-livres, foram analisadas quanto à ocorrência de micobactérias. As amostras foram descontaminadas pelo método HPC 1,5%, semeadas em meio Stonebrink-Leslie (incubadas a 37ºC/90 dias) e as colônias suspeitas, submetidas à reação de PCR TB multiplex e sequenciamento nucleotídico. Em 12% das amostras (16/133) foram isoladas 26 colônias de Mycobacterium spp., tendo sido identificadas 6 espécies, todas ambientais: Mycobacterium fortuitum, M. confluentis, M. elephantis, M. novocastrense, M. sphagni e M. arupense; 7 isolados, no entanto, permaneceram sem caracterização quanto à espécie. O M. fortuitum é um patógeno oportunista importante em saúde pública, sem que haja, entretanto, evidências de transmissão alimentar; o M. novocastrense, M. arupense e M. elephantis também têm sido consideradas espécies com potencial patogênico ao ser humano. Os resultados sugerem, tal como era esperado, que a frequência e a carga inicial de M. bovis em queijo Minas meia cura sejam baixas, mas se deve considerar que a metodologia empregada, por falta de outra específica, não privilegia a detecção em cenário de baixa carga inicial do agente acompanhada por alta carga contaminante. Sugerem também a necessidade de se avaliar a importância da transmissão alimentar de micobactérias não tuberculosas, especialmente para indivíduos imunossuprimidos. / Mycobacterium genus consists of saprophytic and pathogenic microorganisms of interest in animal and human health. M. bovis specie, which causes tuberculosis in animals, is excreted through the milk of infected cattle and the consumption of raw milk and its derivatives is an important route of transmission to humans, causing a disease as serious as the one caused by M. tuberculosis. Considering that the disease in animals is endemic in Brazil and that minas meia cura cheese cure is usually made from raw milk and much appreciated by paulistano consumer, samples of the product acquired in open markets, were analyzed for the occurrence of mycobacteria. The samples were decontaminated by the method HPC 1.5%, sown in Stonebrink-Leslie medium (incubated at 37 ° C/90 da ys) and the suspected colonies, submitted to PCR TB multiplex and nucleotide sequencing reaction. In 12% of samples (16/133) were isolated 26 colonies of Mycobacterium spp. and 6 species have been identified, all of them are environmental Mycobacterium fortuitum, M. confluentis, M. elephantis, M. novocastrense, M. sphagni and M. arupense; 7 isolates, however, remained without characterization for the specie. M. fortuitum is an important opportunistic pathogen in public health, without, however, evidence of being transmitted by food; M. novocastrense, M. arupense and M. elephantis species have also been considered potentially pathogenic to humans. The results suggest that the frequency and/or contamination load of M. bovis in meia cura Minas cheese is (are) low (s). We have to consider, however, that this result may have been influenced by the absence of an analytical method capable of identifying the agent in the food matrix in which a low load of microorganism is expected, accompanied by high load of contaminants. Also suggest the need to evaluate the possible importance of foodborne non-tuberculous mycobacteria, especially for immunocompromised individuals.