Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 24-26).

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-30T21:17:29Z No. of bitstreams: 1 Juliana Perrone Bezerra De Menezes Avaliacao do papel... - 2005.pdf: 32348298 bytes, checksum: 109d71b2fb835421caaa135becd309de (MD5) / Made available in DSpace on 2012-11-30T21:17:29Z (GMT). No. of bitstreams: 1 Juliana Perrone Bezerra De Menezes Avaliacao do papel... - 2005.pdf: 32348298 bytes, checksum: 109d71b2fb835421caaa135becd309de (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Micobactérias ambientais podem ser encontradas em água, solo, poeira, alimentos e animais. A importância do estudo dessas micobactérias tem aumentado nos últimos anos, principalmente, devido a predisposição de pacientes com imunodeficiência à infecção por essas espécies de micobactéria. Além disso, a exposição a micobactérias ambientais pode constituir um dos fatores associados à baixa eficácia da imunização com a vacina BCG. As manifestações da doença, assim como a manutenção da infecção micobateriana, dependem da interação entre a micobactéria e o sistema imune do hospedeiro. O presente trabalho teve como objetivo avaliar a resposta de macrófagos peritoneais de camundongos susceptíveis BALB/c infectados com M intracellulare ou M fortuitum. Macrófagos peritoneais de camundongos BALB/c foram infectados por M intracellulare ou M fortuitum e as diferenças entre essas duas espécies quanto à capacidade de infectar e sobreviver no interior de macrófagos primários, tratados ou não com IFN-y, e produzir óxido nítrico foram avaliadas. Foi observado que os macrófagos infectados com M fortuitum apresentam um maior percentual de células infectadas que aqueles infectados com M. intracellulare, após 4, 24 e 48 horas de infecção. Entretanto, tanto M. fortuitum quanto M intracellulare são capazes de sobreviver no interior de macrófagos peritoneais, pois não há alteração da carga bacilar dessa duas espécies de micobactéria ao longo da infecção. Observamos ainda que M. intracellulare induziu uma maior produção de óxido nítrico por macrófagos primários infectados e tratados por IFN-y que M fortuitum. No entanto, o pré-tratamento com IFN-y não alterou o percentual de células infectadas nem a viabilidade de M intracellulare ou M. fortuitum. Os dados obtidos neste trabalho mostram que, in vitro, M. fortuitum e M. intracellulare interagem de formas distintas, levando á diferentes respostas do macrófago e a destinos intracelulares distintos. Além disso, mostramos que M intracellulare e M. fortuitum são resistentes ao óxido nítrico produzido por macrófagos após ativação por IFN-y. / Environmental mycobacteria are found in water, soil, dust, food and animals. Environmental Mycobacterium importance has increased in the last few years, mostly because of immunodeficient patient predisposition to infection. Moreover, exposure to environmental mycobacteria could be associated to low levels of protection induced by immunization with BCG. Disease manifestations as well as infection outcome depend on interaction between mycobacteria and host immune system. The goal of this work was to evaluate peritoneal macrophage response, from the susceptible BALB/c mice, to M. intracellulare or M. fortuitum infection. Peritoneal inflammatory macrophages, pre-activated or not with IFN-y, were infected by M. intracellulare or M fortuitum and diferences between these two species related to the capacity to infect macrophages, to survive intracellularly and to induce NO production were evaluated. It was observed that the percentage of M. fortuitum-xnÍQoXQá cells was higher related to M. intracellulare-míecieá ones, after 4, 24 and 48 hours of infection. In addition, both M. fortuitum and M. intracellulare presented the ability to survive in peritoneal macrophages. It was also observed that in response to IFN-y activation, M. intracellulare induced higher NO production thanM fortuitum. However, pre-activation with IFN-y did not modify, neither the percentage of M. intracellulare and M. fortuitum infected cells, nor intracellular bacillum survival. These data demonstrate that, in vitro., M. fortuitum and M. intracellulare differently interact with macrophages, inducing diferent macrophage reponses and that both M. intracellulare and M fortuitum are resistant to NO production upon IFN-y activation.

Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrobial resistance because (1) cells are protected by layers of other cells and extracellular material (2) and differences in physiologic state of cells as a consequence of growing on surfaces. The objectives of the work were to (1) establish methods for reproducible growth of mycobacterial biofilms (2) measure the formation of biofilms on surfaces by cells of M. avium and M. intracellulare (3) measure the antibiotic- and chlorine- susceptibility of M. avium and M. intracellulare strain TMC1406T in cell grown in suspension, cells grown in biofilms and suspended and of cells grown in biofilms (4) measure the hydrophobicity of M. avium and M. intracellulare grown in suspension and in biofilms. Methods were developed for growing mycobacteria in biofilms in polystyrene flasks and on glass beads. Although both strains formed biofilms, M. intracellulare strain TMC 1406T more readily formed biofilms than M. avium strain A5 in polystyrene flasks. The majority of M. intracellulare strain TMC 1406T cells grew on the walls of the flasks rather than in suspension like M. avium strain A5. The susceptibility of M7H9 medium-grown cells of M. avium strain A5 and M. intracellulare strain TMC 1406T cells grown in suspension, cells grown in biofilms and suspended and cells grown in biofilms was measured against clarithromycin, ethambutol, kanamycin, rifampicin and streptomycin. Cells grown in biofilms and exposed to antibiotics in biofilms were five-fold resistant to antibiotics than were cells grown in biofilms and exposed in suspension. Cells grown and exposed in suspension were ten-fold more sensitive to antibiotics than were cells grown in biofilms and exposed in suspension. The chlorine susceptibility of cells grown in medium and water was also measured. Cells grown in biofilms were more resistant to chlorine than cells grown in biofilms and suspended. Cells grown in suspension were more sensitive to chlorine than cells grown in biofilms and suspended. The hydrophobicity data (i.e., hexadecane adherence and contact angle measurements) showed that cells grown in biofilms are more hydrophobic than cells grown in biofilms and suspended and cells grown in suspension. It is clear that there are physiological changes between cells grown in suspension, cells grown in biofilms and suspended and cells in biofilms. / Master of Science

Bacteria of the <i>Mycobacterium avium, M. intracellulare, and M. scrofulaceum</i> group (MAIS) are opportunistic human pathogens and widespread in the environment. The first objective of this study was to demonstrate that plasmids from clinical isolates are closely related to plasmids from environmental MAIS isolates. A 12.9 kb plasmid, pVT2, from a clinical <i>M. avium isolate</i>, MDl, was cloned and used a a DNA probe to examine the relationship of MAIS plasmids. The pVT2 probe hybridized with plasmids isolated from MAIS strains from the environment, from patients without AIDS with pulmonary infections, and from AIDS patients with disseminated MAIS infections. Similar results were seen with a second probe derived from pLR 7, a 15.3 kb plasmid from clinical <i>M. intracellulare</i> strain LR 113. The similarity of plasmids from environmental and clinical isolates supports the hypothesis that the environment is a source of MAIS infection in humans. / Ph. D.

Mycobacterium avium and Mycobacterium intracellulare, (M. avium complex, MAC) are human pathogens causing disease in individuals with acquired immunodeficiency syndrome (AIDS) or with thoracic abnormalities. MAC bacteria are difficult to kill because of the resistance of the pathogens to chemotherapeutic agents. One factor affecting treatment of MAC disease is the presence of interconvertible colonial variants. Transparent (T) variants have greater resistance to antibiotics and higher pathogenicity; opaque (O) variants are more susceptible to antibiotics and less pathogenic. The overall goal of this study was to investigate the mechanism for colonial variation. Based on an observation that T variants of M. intracellulare strain Va14 contained a plasmid which was 6 kb smaller than the 68 kb plasmid in O variants, it had been suggested that a transposable element might be responsible for colonial variation. The first objective was to clone the unique DNA fragment present in the 68 kb plasmid but absent from the 62 kb plasmid. The second and third objectives were to determine if the unique fragment contained a transposable element and to analyze the role of that element in the mechanism of colonial variation in M. intracellulare strain Va14. The fourth objective was to determine the distribution of IS1141 in MAC isolates. Fragments containing copies of the putative element were sequenced and a region 1596 basepairs in length with 23 basepair imperfect inverted repeats was designated as insertion sequence IS1141. IS1141 is the first insertion sequence identified in M. intracellulare. Data base searches using open reading frames (ORF) of IS1141, identified ORFb as significantly similar to the transposases of the IS3 family. The presence or absence of IS1141 in strain Va14 plasmids appeared unrelated to colonial variation, but IS1141 was present in another plasmid and the chromosome of the Va14 variants. Hybridization studies with IS1141 identified three chromosomal copies in O variants and two chromosomal copies in T variants. Va14 T variants each had a common IS1141 restriction fragment length polymorphism (RFLP) pattern which was different than the single RFLP pattern found in opaque variants. Based on these differences, it appears that IS1141 may integrate into the gene(s) responsible for the T phenotype preventing their expression. A survey of 64 James River basin non-AIDS, clinical and James River environmental MAC isolates identified 4 of 24 (17%) M. intracellulare isolates as containing IS1141. IS1141 has not been detected in any clinical or environmental M. avium or Mycobacterium species X isolates and may be limited to M. intracellulare. / Ph. D.

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