Abstract
This project is a continuation of the previous thematic project (2006-2010), entitled "Mycobacteria of medical significance in Brazil: molecular characterization, interaction with the environment and with macrophages" (Proc. N. 2006/01533-9). We will keep on studying nontuberculous mycobacteria (NTM), the main investigation line of our laboratory. In Subproject 1 we will evaluate a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus. In the previous project, we studied a significant number of isolates from outbreaks of infections in laparoscopic, arthroscopic and plastic surgeries using DNA sequencing and Pulsed Field Gel Electrophoresis (PFGE). These outbreaks had a nationwide distribution (>2,000 notified cases between 2004 and 2008) and were caused, in the majority of cases, by a single strain, now classified as a subspecies of M. abscessus named subsp. bolletii. We also studied isolates of the same subspecies not related to surgery outbreaks. Therefore we have a significant collection of isolates, which will be used here to evaluate the discriminatory power of MLST in comparison to PFGE. We aim to provide an additional scheme for M. abscessus typing in outbreak situation. In Sub-project 2 we will characterize isolates belonging to the Mycobacterium chelonae-M. abscessus group without conclusive species identification. A taxonomic reclassification of three members of this group was recently proposed, as a consequence of our study of the surgery outbreak isolates. We confirmed also that members of this group have high internal variability. With this, we will expand this study with the analysis of 40 additional isolates, whose taxonomic classification is still inconclusive. Due to an increase in the significance of the M. chelonae-M. abscessus group in Brazil, in the last years, it is important to move on with taxonomy studies, in order to contribute with epidemiologic analyses and surveillance in future outbreaks. In Sub-project 3 we will finish the bioinformatics analysis of a conjugative plasmid of Mycobacterium avium, identified during the previous project. This plasmid can be transferred by conjugation between different species of slowly-growing mycobacteria (SGM), as M. avium, Mycobacterium kansasii e Mycobacterium bovis BCG. We have demonstrated for the first time the occurrence of horizontal gene transfer between different species of mycobacteria. With the bioinformatics analysis we intend to identify genes responsible for conjugation and the replication origin of this plasmid and to develop E. coli-mycobacteria shuttle vectors, providing new tools for studies with mycobacteria. If genes related to antimicrobial resistance or other metabolic processes are identified, these activities will be tested in vitro. Sub-project 4 will give continuity to the project on identification and characterization of mycobacteriophages proposed in the last project. With the collaboration established with Fundação Parque Zoológico de São Paulo (FPZSP), we will isolate and characterize mycobacteria and mycobacteriophages present in compost processed at FPZSP. Isolated mycobacteria will be identified by phenotypic and molecular methods and phages will be characterized morphologically and sequenced. Metagenomics will be carried out with the same material, and phage sequencing and metagenomics data will be compared. Results should improve the knowledge about distribution of mycobacteria and phages in the Brazilian environment and lead to the development of diagnostic and therapeutic tools and control mechanisms for some mycobacterial diseases for which the available tools are not satisfactory. (AU)
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