Formation of Mycobacterium abscessus colonies in cellular culture in an in vitro infection model

Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections. • Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs. • The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents. • Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.