The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, grampositive rods. The genus comprises several species classified into three main groups. Firstly, the major group of these organisms, which poses the biggest threat, is the M. tuberculosis complex which can cause tuberculosis-like disease. These include M. bovis, M. africanum and M. microti. Members of the M. tuberculosis complex are not found in the environment. The second group is M. leprae which is the causative agent of leprosy. The last group constitutes the nontuberculous mycobacteria (NTM), which are all the environmental mycobacteria that can cause various diseases resembling tuberculosis. Due to the importance of environmental mycobacteria, 15 mycobacteria isolates were isolated from environmental samples such as soil, water and drinking water biofilms. After PCR amplification of the hsp65 gene using genus specific primers hsp65, the isolates revealed sequences similarities when compared with the well characterized mycobacteria in the GenBank. Alignment of the nucleotide sequences and homology analysis were done with Clustall. It has been suggested that mycobacteria-associated phages (mycobacteriophages) may make an important contribution to the evolution of pathogenic mycobacteria. Spontaneous induction of phage associated with mycobacteria isolates using overlay and indicator plate methods was not successful to detect the presence of any inducible phage. A phage was isolated from soil samples that was designated the name A22. After purification and characterization. A22 phage was compared morphologically to well characterized L5 phage using electron microscopy. Morphological studies revealed that A22 mycobacteriophage had a non-contractile tail approximately 150 nm long with an isometric head approximately 60 nm, the phage could be assigned to the family Siphoviridae, According to these criteria, both of the phages (A22 and L5) belong to the order Caudovirales (tailed bacteriophages). Based on PCR amplification of A22 phage DNA using L5 gp71 specific primers and the infection of M. smegmatis L5 lysogen, we believe that this novel A22 phage differs from L5 phage.

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.

Made available in DSpace on 2016-06-02T19:31:35Z (GMT). No. of bitstreams: 1 DissKYK.pdf: 979832 bytes, checksum: b4f8dd12d53ca1f1f19852dfb9661856 (MD5) Previous issue date: 2006-04-24 / Financiadora de Estudos e Projetos / Fifty-nine water samples from Feijão River, Monjolinho River and the Water Treatment Station (SAAE) were analyzed, and 51 were positive for mycobacteria. Concerning the two procedures of decontamination, we obtained 83.0% of positive samples isolated with sulfuric acid and 39.0% with cetylpyridinium chloride. We recovered 425 strains of mycobacteria, and 402 were characterized concerning growth rate and pigment production. In drinking water samples there were predominance of mycobacteria with slow growth, wherein 66.7% were scotochromogenics species and 47.5% were identified as M. lentiflavum. The identification was done through biochemical tests and phenotypics features (classical methodology), micolic acid analyses by thinlayer chromatography (TLC) and PCR-Restriction Analysis (PRA). Through the classical methodology associated to TLC 33.3% of the mycobacteria were identified and 18 species were defined. From the 402 mycobacteria 93 strains were selected and submitted to PRA, being 35 identified by the classical and molecular methodology, 51 only identified by PRA and 40% remained obscure concerned identification. The PRA pattern 440 in BstEII and 130/110 in HaeIII was defined as M. new. This pattern was found for 24 strains. For an accurate identification, the association of different methodologies is necessary. / Foram analisadas 59 amostras de água provenientes do Ribeirão do Feijão, Rio do Monjolinho e da Estação de Tratamento de Água (SAAE), das quais 51 foram positivas para o isolamento de micobactérias. Dos dois procedimentos de descontaminação, obtivemos 83,0% de amostras positivas para isolamento com ácido sulfúrico e 39,0% com cloreto de cetilpiridínio. Houve recuperação de 425 cepas de micobactérias, sendo 402 caracterizadas com relação a velocidade de crescimento e formação de pigmentação. Em amostras de água tratada, houve o predomínio de micobactérias de crescimento lento, sendo que dessas, 66,7% foram espécies escotocromógenas, e 47,5% identificadas como M. lentiflavum. A identificação foi realizada por testes bioquímicos e fenotípicos (metodologia clássica), análise de ácidos micólicos por cromatografia em camada delgada (TLC) e PCR-Restriction Analysis (PRA). Pela metodologia clássica associada ao TLC, foram identificadas 33,3% das micobactérias e definidas 18 espécies. Das 402 micobactérias, 93 foram selecionadas e submetidas ao PRA, sendo 35 identificadas pela metodologia clássica e molecular, 51 identificadas somente pelo PRA e havendo discordância de 40,0% entre as identificações. O padrão de PRA 440 em BstEII e 130/110 em HaeIII foi definido como M. new, sendo obtido em 24 cepas. Para uma identificação acurada, concluímos que é necessária a associação de mais de uma metodologia.

Abstract This thesis aimed at developing methods for tracking the environmental origins of microbial contaminants of the food chain. We worked on three targets: i) environmental mycobacteria ii) toxinogenic Bacillus species iii) post-harvest fungi in strawberry jam. Our aim was to develop methods for early detection of the above contaminants, which have the potential to endanger consumer health. We developed a novel method based on 16S rRNA hybridization for tracking the reservoirs of potentially pathogenic environmental mycobacteria in piggeries and soil. From 1010 to 1012 16S rRNA molecules of environmental mycobacteria were found per gram of peat, wood shavings and straw in piggeries with a high prevalence of infections. These beddings may thus be a source of mycobacteria for pigs. We found 1010–1011 of mycobacterial 16S rRNA molecules per gram of Finnish forest soil, indicating that the soil contained 107–109 mycobacteria per gram. These numbers exceed the previous cultivation-based estimates of mycobacterial content in Finnish soils. To elucidate the role of mastitis in the input of toxinogenic Bacillus into the dairy production chain, milks were sampled from mastitic cows. Twenty-three Bacillus isolates were screened for toxins using the sperm cell motility inhibition assay. Four of the six toxinogenic isolates found were identified as Bacillus pumilus and two as Bacillus licheniformis. The isolates produced toxic substances that were heat-stable (100 °C) and soluble in methanol, thus being of non-protein nature. The extracts prepared from the toxin-producing isolates disrupted the plasma membrane of exposed sperm cells at concentrations 1–15 μg ml-1 (B. pumilus) 20–30 μg ml-1 (B. licheniformis). The toxic action of the mastitis-associated B. licheniformis strains was similar to that of the lipopeptide lichenysin A. The genes for lichenysin synthetase were found in these strains by PCR. This study revealed that heat-stable toxin-producing strains of B. pumilus and B. licheniformis occur in milk of mastitic milking cows. They may enter the dairy production chain when milk of clinically healthy cows recovered from mastitis is sent to dairies. Many foodborne contaminant fungi are known to produce volatile organic compounds. We investigated the suitability of such metabolites as early indicators of fungal contamination of strawberry jam. We found that volatile organic compounds commonly produced by the contaminant fungi in strawberry jam were 2-pentanone, styrene, 3-methyl-1-butanol, 1,3-pentadiene and ethanol. The results indicate that these compounds could be used to detect fungal contamination of jam.