Increased Virulence of an Epidemic Strain of Mycobacterium massiliense in Mice Shaobin Shang1, Sara Gibbs1, Marcela Henao-Tamayo 1 , Crystal A. Shanley1, Gerald McDonnell2, Rafael Silva Duarte3, Diane J. Ordway1*, Mary Jackson 1 * 1 Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America, 2 STERIS Limited, Basingstoke, England, 3 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Abstract Background: Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue. Methodology/Principal Finding: We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNc knockout mice exposed to a high dose aerosol with these two isolates. CRM- 0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses. Conclusions/Significance: These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host. Citation: Shang S, Gibbs S, Henao-Tamayo M, Shanley CA, McDonnell G, et al. (2011) Increased Virulence of an Epidemic Strain of Mycobacterium massiliense in Mice. PLoS ONE 6(9): e24726. doi:10.1371/journal.pone.0024726 Editor: J. Ross Fitzgerald, University of Edinburgh, United Kingdom Received February 23, 2011; Accepted August 19, 2011; Published September 12, 2011 Copyright: 2011 Shang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported in part by the National Institutes of Health/National Institute of Allergy and Infectious Diseases grant AI089718, the College of Veterinary Medicine and Biomedical Sciences Research Council CRC 0014 (Colorado State University) and the STERIS Foundation. The STERIS Foundation provided funds for equipment and materials purchased for the study. In addition, Dr. McDonnell, an employee of STERIS, assisted in the technical aspects of disinfectant- susceptibility testing and aided in the presentation of these results in the manuscript. The STERIS Foundation and the Research Council CRC 0014 had no role in the decision to publish. Competing Interests: I have read the journal’s policy and have the following conflict. Dr. Gerald McDonnell is an employee of STERIS. This affiliation does not alter our adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: Mary.Jackson@colostate.edu (MJ); D.Ordway-Rodriguez@ colostate.edu (DJO) Introduction Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and are responsible for colonization, infection and pseudo-outbreaks in health care settings throughout the world. Most NTM nosocomial outbreaks caused by rapidly growing mycobac- teria (RGM) have involved the species M. abscessus, M. chelonae, M. fortuitum and two recently recognized Mycobacterium species closely related to M. abscessus: M. massiliense and M. bolletii [1,2]. These pathogens can cause a wide range of clinical diseases including skin and soft tissue infections, pulmonary, central nervous system and disseminated infections [3,4,5,6,7,8,9,10,11,12,13,14]. While dis- seminated infections caused by RGM essentially occur in immunodeficient patients and pulmonary infections are widely thought to be opportunistic and to occur in patients with struc- tural lung disease (e.g., chronic obstructive pulmonary disease, bronchiectasis, cystic fibrosis), numerous cases of skin and soft tissue infections have been reported in otherwise immunocompetent patients, caused by contaminated materials and invasive procedures involving catheters, non-sterile surgical procedures, injections and implantations of foreign bodies. Recent published reports [15,16] and outbreaks in Brazil [13,17] suggest that the prevalence of infections caused by these microorganisms is increasing and that infections due to RGM are particularly problematic due to their ubiquitous presence in hospitals’ water sources and the difficulty of treating some of the infections they cause. Following infection, very little is known about the host immune response and what enables NTM to persist in host tissues. While some NTM species, such as M. fortuitum and M. chelonae, can be controlled by innate immunity [18,19], others like M. abscessus, require adaptive immunity for efficient control of the infection [19,20]. Our studies support the early occurrence of phagocytosis PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24726