Study of Mycobacterium abscessus isolates of subsp.massiliense environmental and outbreak model in vivo and ex alive

Micobacterias de crescimento rapido (MCR) tem sido frequentemente descritas em surtos de infeccoes em seres humanos. Mycobacterium abscessus subsp. massiliense e uma MCR que foi descrita pela primeira vez em 2004, e desde entao, 10 trabalhos identificando essa especie em surtos e casos clinicos esporadicos sao os relatos presentes na literatura. Assim, a interacao M. abscessus subsp. massiliense-hospedeiro foi o objetivo principal deste trabalho. Para isso, variantes de colonia lisa e rugosa de um isolado do surto que ocorreu em Belem, entre 2004 e 2005 (B7 L e R), e de 2 isolados nao relacionados a surtos (B67 (L) e MG3-6 (R)) foram utilizadas em infeccoes intraperitoneal e endovenosa de camundongos e ex vivo em macrofagos peritoneais e celulas epitelioides-like. No que tange a resposta imunologica apos a infeccao intraperitoneal, o nivel de citocinas foi semelhante para os 4 isolados. Entretanto, apos 14 dias da infeccao os bacos dos animais infectados pelos isolados B7 R e MG3-6 apresentavam um aumento significativo de peso em relacao aos demais grupos. Apos 90 dias, esta diferenca se manteve apenas para o grupo infectado com B7 R. A histologia dos bacos destes animais mostrou que a resposta proliferativa nos foliculos linfoides foi mais pronunciada para a infeccao por B7 R, levando a necrose central dos mesmos apos 14 dias. Nestes, bacilos alcool-acido resistentes foram encontrados penetrando a zona perifolicular, e tambem em vasos de maior calibre, sugerindo disseminacao via corrente sanguinea. A infeccao endovenosa corroborou os resultados obtidos para a infeccao intraperitoneal. Apos 2 dias de infeccao, a recuperacao de unidades formadoras de colonia (UFCs) do baco foi significativamente maior para os animais infectados por B7 R. Entretanto, a infeccao foi igualmente controlada em todos os grupos, semelhantemente a infeccao intraperitoneal. A infeccao de culturas de macrofagos e CEs-like corrobou os achados in vivo, pois B7 R foi capaz de se proliferar no interior de macrofagos e, apenas para este isolado, houve o aumento na porcentagem de CEs-like infectadas. Adicionalmente, o isolado B7 R estimulou uma producao menor de TGF-ƒÒ nas culturas quando comparado ao isolado de fenotipo liso. CEs-like nao foram capazes de conter a infeccao causada pelo isolado B7 R, talvez pela interacao micobacteria-CEs-like resultar em menor producao de TGF-£].Tomados em conjunto, os resultados apresentados sugerem que micobacterias formadoras de colonias rugosas associadas a surtos sao mais patogenicas que as de fenotipo liso e que aquelas presentes no ambiente. Os mecanismos subjacentes responsaveis por estes achados estao sendo estudados.Rapidly growing mycobacteria (RGMs) have been frequently identified in outbreaks of human infections. Mycobacterium abscessus subsp. massiliense is an RGM which was first described in 2004, and since then 10 publications identifying this species in outbreaks and sporadic clinical cases have been reported. The interaction between M. abscessus subsp. massiliense and host was the main objective of this work. Therefore, variants of smooth and rough colonies (B7 S and B7 R), isolated from an outbreak which took place in Belém, from 2004 to 2005, and two non-related isolates [B67 (S) and MG3-6 (R)] were used to produce intraperitoneal and intravenous infections in mice, and ex vivo infection in peritoneal macrophages and epithelioid-like cells. In terms of immune response following intraperitoneal infection cytokine levels were similar for the four isolates. However, after 14 days, the spleens of animals infected with B7 R and MG3-6 showed a significant increase in comparison to the spleens of animals infected with the other two isolates. After 90 days, this difference was maintained only for the group infected with B7 R. The histological analysis of the spleens showed that the proliferative response in lymphoid follicles was more evident in animals infected with B7 R, leading to central necrosis after 14 days. Acid-fast bacilli were found penetrating the perifollicular zone of these spleens, and also large blood vessels, suggesting blood dissemination. The intravenous infection corroborated the results obtained for intraperitoneal infections. After two days of infection, the recovery of colony forming units (CFUs) from spleen was significantly larger in animals infected with B7 R. Nevertheless, the infection was likewise controlled by all groups, similarly to the intraperitoneal infection. In the intravenous infection model, animals infected with B7 L, B67 and MG3-6 showed low production of TGF- in early time points. The infection of macrophages and epithelioid-like cells corroborated the in vivo findings, and B7 R was capable of proliferating inside macrophages and only for this isolate there was an increase in the percentage of infection in epithelioid-like cells. Furthermore, B7 R and B7 S stimulated a modest production of TGF- in these cultures when compared to the nonrelated isolates. Contrary to what was observed previously for M. avium, epithelioid-like cells were not able to restrain the infection caused by B7 R. Taken together, the results suggest that rough colony forming mycobacteria associated to outbreaks are more pathogenic than bacteria of smooth phenotype and those present in the environment, maybe because of the lower levels of TGF- upon mycobacteria-infected cells interaction. The underlying mechanisms are under investigation.TEDEBV UNIFESP: Teses e dissertaçõe