of M. abscessus by antigen-presenting cells such as alveolar macrophages and dendritic cells [20,21]. Autocrine and paracrine activation of dendritic cells by IL-12 p40 and migration of the infected dendritic cells to the regional mediastinal lymph nodes and subsequent mycobacterial antigen presentation to naive T cells results in differentiation and further activation of effector T cells. The accumulation in the lung of protective T cells capable of expressing IFNc and TNFa are crucial for phagocyte activation and intracellular killing [19,22]. The development of a pulmonary granuloma is orchestrated by chemokines and cytokines produced by local cells causing a continuous recruitment of lymphocytes, macrophages, dendritic cells and granulocytes to the lung. Control of the infection relies on this granulomatous response, which is an organized cellular network acting to restrain NTM growth and limit dissemination. In the context of M. tuberculosis persistence and chronic disease, it has been shown that CD4+Foxp3+ regulatory T cells are important for regulation of immunopathology [23,24,25]. The induction of CD4+Foxp3+ regulatory T cells has never been reported in the case of Mycobacterium chelonae-abscessus-massiliense group persistence and we propose it is one of the strategies adopted by these microorganisms to enhance their persistence in the human host. The immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of clinical disease caused by NTMs by preventing the induction of specific protective immune responses required to kill the bacterium. The most important outbreaks – reaching epidemic proportions - of nosocomial M. massiliense infections ever reported occurred between 2004 and 2007 in Brazil. Interestingly, these outbreaks seem to have been caused by one single strain of M. massiliense, named BRA100 [12,13,17], which displays high level resistance to glutaraldehyde (GTA), the chemical used for disinfection of surgical instruments in all of the hospital sites that had confirmed cases. While the resistance of this particular strain to high-level disinfection has been advanced as the most likely reason for its selection and dissemination across the country [12,13,17], the unusual size of the outbreaks also suggests that BRA100 may represent a particularly invasive and pathogenic strain of M. massiliense. Such variability in pathogenicity among M. tuberculosis clinical isolates has been well documented [23,26,27,28]. The potential increase in pathogenicity of the epidemic BRA100 isolates is further supported by our recent findings that one of the mechanisms through which RGM species develop high-level resistance to GTA involves a remodeling of the composition and/ or organization of their cell surface susceptible of affecting their intracellular survival and interactions with the host [29]. We undertook this study with the goal of comparing the M. massiliense BRA100 epidemic isolate from Brazil to the glutaralde- hyde-susceptible reference strain, M. massiliense CIP 108297, in their abilities to replicate inside macrophages and to infect, replicate, persist, induce organ pathology and immune responses in mice. Results Morphological traits, disinfectant resistance and in vitro growth characteristics of the M. massiliense isolates used in this study M. massiliense CIP 108297, the type strain used in this study, was isolated from the sputum and the bronchoalveolar fluid of a patient with hemoptoic pneumonia in France in 2004 [2]. CRM- 0019 is a BRA100 M. massiliense isolate from Brazil. It was responsible for a nosocomial infection following hepatic tumor resection by videolaparoscopy, the supposed index case of the epidemic in the state of Rio de Janeiro [13]. The female patient Increased Virulence in M. massiliense Infection was 45 years old and presented the initial symptoms 90 days after the videosurgery. She was submitted to clarithromycin-based antimicrobial therapy and two additional laparotomies for granuloma resections, resulting in complete recovery. M. massiliense CIP 108297 and CRM-0019 display different pulse field gel electrophoresis (PFGE) patterns confirming their lack of genetic relationship [13]. The species assignment of both isolates was confirmed by partial sequencing of their hsp65 and rpoB genes (view Information S1). The plating of CIP 108297 yielded uniform colonies interme- diate between rough and smooth, as described earlier [2]. Plating of CRM-0019 onto 7H11-OADC agar, in contrast, yielded a mixed population of nonphotochromogenic rough and smooth colonies. Suggestive of genetic instability, the culturing and re- plating of individual rough and smooth CFUs led to colonies with mixed phenotypes. Comparison of the growth rate of the two strains in 7H9-OADC broth at 30uC revealed only a marginal delay in growth associated to the BRA100 isolate during exponential phase (view Information S1). Their growth rate on 7H11-OADC agar plates was comparable. Suspension suscepti- bility tests confirmed the high level resistance of the BRA100 isolate, CRM-0019, to 2.2% GTA (view Information S1). Smooth colonies of CRM-0019 displayed the same level of resistance as the rough ones. Growth of M. massiliense strains in mouse bone marrow derived macrophages (BMDM) We investigated the capacity of M. massiliense reference strain CIP 108297 and the M. massiliense outbreak strain CRM-0019 to grow in BMDM to evaluate their virulence. The growth of each isolate was determined by the method of plating to measure colony-forming units (CFU; Figure 1). The M. massiliense outbreak strain CRM-0019 compared to the reference strain CIP 108297 demonstrated a significantly increased ability to replicate in BMDM after 24 hours of infection and steadily increased peaking at 7 days of infection. The reference strain CIP 108297 demonstrated nominal bacterial replication for 48 hours of infection and thereafter began to steadily increase in bacterial numbers in BMDM. Lastly, we evaluated the BMDM cellular viability the percentage of propidium iodide positive dead cells induced by the M. massiliense reference strain CIP 108297 and the M. massiliense outbreak strain CRM-0019 was below 6.0% for the BMDM (data not shown). Bacterial loads in GKO mice We chose a pulmonary infection model using interferon-gamma (IFN-c) gene disrupted mice (GKO) to compare the two M. massiliense strains in vivo for several reasons. Firstly, the reference strain CIP 108297 was isolated from a patient with pulmonary infection. Since our goal was to compare the pathogenicity of a BRA100 isolate from Brazil to that of CIP 108297, we wanted to ensure that the reference isolate was placed under conditions where it would perform best in terms of establishing an infection. Importantly, BRA100 isolates have been recovered both from surgery patients with skin and soft tissue infections and from patients with pulmonary infections not associated with the post-surgical epidemic [30] indicating that they are pathogenic in pulmonary infections as well. The second reason has to do with the fact that the GKO model more closely reflects an RGM infection which would occur in immunocompromised patients, a population particularly at risk for NTM infections [15]. Clinical observations and/or genetic studies in humans actually corroborate many of the findings in animals in that those with cell-mediated immunodeficiency, genetic defects in IFNc-interleukin-12 (IL-12) axis, and those individuals on PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24726