Figure 1. Increased intracellular replication of the Brazilian outbreak strain M. massiliense CRM-0019 in mouse bone marrow macrophages. Intracellular growth of M. massiliense reference strain CIP 108297 and M. massiliense outbreak strain CRM- 0019 in BMDM. BMDM were infected with M. massiliense strains at a MOI of 5, and the numbers of intracellular bacteria were determined using the bacterial colony count method (CFU) immediately after 2 hours of infection or at 1, 2, 3, 4, 5, 6 and 7 days after infection. Values shown are the mean 6 SD from two independent experiments. Growth of the M. massiliense outbreak strain CRM-0019 was significantly higher than the other isolate (*, P,0.05). doi:10.1371/journal.pone.0024726.g001 TNFa blockers are at increased risk for soft tissue infections and pulmonary NTM infections, including the RGM [21]. In addition, it has been shown that individuals infected with the human immunodeficiency virus show reduced T cell IFNc production and Increased Virulence in M. massiliense Infection TH1 immunosuppression by CD4+Foxp3+ regulatory T cell [31,32]. Lastly, the choice of using GKO mice instead of C57BL/ 6 ones owes to the reported difficulty of infecting through the aerosol route wild-type C57BL/6 mice that eliminate the bacteria very rapidly, whereas GKO mice aerosolized with about 1,000 CFU develop a successful infection [33]. Following a high dose aerosol infection with M. massiliense reference strain CIP 108297 and Brazilian outbreak strain CRM-0019 in GKO mice (,1,000 bacilli per animal), the bacterial loads in the lungs and spleens were quantified on days 1, 30 and 60 after the challenge. Aerosol infection with the outbreak strain M. massiliense CRM-0019 resulted in a sustained infection in the lungs and spleens with more than 103 organisms recovered from both organs at 60 days (Figures 2A and 2B). In comparison, GKO mice infected with the reference strain of M. massiliense showed complete clearance of this strain in the lungs and spleens by day 60 (Figures 2A and 2B). Lung histopathology in GKO mice after aerosol challenge with Mycobacterium massiliense Lung histopathology following high dose aerosol challenge with the reference and outbreak M. massiliense strains was characterized in GKO mice (Figure 3). At day 30 following the challenge, when there was bacterial persistence in the lungs for both strains, lung tissues demonstrated a local inflammatory response denoted by peribronchiolar inflammatory infiltrates (Figure 3, panel A, A-D, arrow). However, by day 60, while the reference M. massiliense strain showed complete bacterial clearance and healthy lung tissue (Figure 3, panel A, E–F), the lungs of the CRM-0019-infected GKO mice showed large areas of consolidation (Figure 3, panel A, G–H). In addition, CRM-0019-infected mice showed peribron- chiolar inflammatory infiltrates with large aggregates of foamy cells and the presence of macrophages with multiple intracellular acid fast staining bacilli (Figure 3B). Macrophage and dendritic cell cytokine expression during Mycobacterium massiliense infection Given the clear differences seen in cell influx indicated by the histological analysis, we conducted a comparative flow cytometric Figure 2. Increased bacteria in the lungs and spleens of the Brazilian epidemic strain M. massiliense CRM-0019 infected IFNc-KO mice. Bacterial counts in the lungs (A) and spleens (B), on days 1, 30 and 60 from IFNc-KO mice infected with a high dose aerosol of the epidemic strain M. massiliense CRM-0019 (open circle) and M. massiliense CIP 108297 (solid square) were compared. Results are expressed as the average (n = 5) of the bacterial load in each group expressed as Log10CFU, 6 standard error mean (SEM). Student t-test, *p,0.050. doi:10.1371/journal.pone.0024726.g002 PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24726