Resumo

Mycobacterium abscessus is a rapidly-growing mycobacterium (RGM) responsible for skin and soft tissue infections and pulmonary disease, especially in patients with cystic fibrosis. Since 2004, several outbreaks of surgical-site infections caused by a single strain of M. abscessus subsp. bolletii have occurred in Brazil. We are using one isolate from this strain (CRM0019), obtained at Rio de Janeiro, in studies of uptake and survival using RAW cells. There are evidences that different multiplicities of infection (MOI) can produce different intracellular developments of the infection, changing the chemokine profile, in experiments involving mycobacteria and macrophagic cells. Here, we tested if the intracellular development of this outbreak isolate in RAW cells could be affected by different MOIs. RAW 264.7 cells were infected with a transformed CRM0019 stably expressing green fluorescent protein, at four different MOIs ranging from 0.01 to 10, during 72 hours, with amikacin in the culture medium to prevent extracellular growth. Internalized bacteria were counted in paraformaldehyde fixed cells, by epifluorescence microscopy with DAPI stain; the intracellular development was quantified by colony formation unit (CFU) counts, after cells were lysed. No internalized bacteria were visualized at MOIs 0.01 and 0.1, observing at least 10 fields. At MOIs 1 and 10, the internalization rates were ∼20% and ∼80%, respectively. About 10% of the initial inoculum was recovered after six hours of infection, and no significant growth was detected during the experiment, in all groups studied. There were no differences in the growth rate of internalized mycobacteria comparing the four different MOIs tested along 72 hours. These results showed that the MOI is not a determinant factor in the intracellular development of Mycobacterium abscessus subsp. bolletii CRM0019. Funding: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)