Increased virulence of an epidemic strain of Mycobacterium massiliense in mice

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 IFNγ 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.

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 ± SD from two independent experiments. Growth of the M. massiliense outbreak strain CRM-0019 was significantly higher than the other isolate (*, P<0.05).

Figure 2. Increased bacteria in the lungs and spleens of the Brazilian epidemic strain M. massiliense CRM-0019 infected IFNγ-KO mice.

Bacterial counts in the lungs (A) and spleens (B), on days 1, 30 and 60 from IFNγ-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, ± standard error mean (SEM). Student t-test, *p<0.050.

Figure 3. Lung histology from M. massiliense infected IFNγ-KO mice.

Panel A, A-H shows representative lung histopathology from the M. massiliense CIP 108297- and epidemic CRM-0019-infected animals on day 30 and 60 of the infection. Tissue sections are stained with hematoxylin and eosin. The lower photograph depicts the magnified region denoted by the square (upper photograph). CRM-0019-infected mice (panel A: D and H) showed an increased rate of granuloma (arrows) progression and involvement of larger areas of the lung compared to the control mice (panel A: B and E). Panel B shows representative lung acid fast staining bacilli at day 60 after M. massiliense CIP 108297 and CRM-0019 infection. Total magnification, panel A [A, C, E, G]  =  10x; panel A [B, D, F, H]  =  20x; panel B  =  100x.

Figure 4. Decreased macrophage and dendritic cell expression of IL-12 and TNFα in the lungs and spleens of IFNγ-KO mice infected with the epidemic strain M. massiliense CRM-0019.

Mice infected with M. massiliense CIP 108297 (closed square) and the epidemic strain M. massiliense CRM-0019 (open circle) were assayed by flow cytometry on days 30 and 60. Panel A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD11b+ macrophages cells expression of IL-12. Panels B and C show the total number of macrophage (CD11b+) and dendritic (CD11c+) cells expressing IL-12 or TNFα in the lungs (A) and spleens (B) of both types of infected mice. Results are expressed as the mean number of CD11b⁺IL-12⁺, CD11b⁺ TNFα ⁺,CD11c⁺IL-12⁺, CD11c⁺ TNFα ⁺ (± SEM, n = 5) T cells in the lungs and spleens. Student t-test, *p<0.050.

Figure 5. Decreased early CD4+ and CD8+ T cell expression of TNFα in the lungs and spleens of IFNγ-KO mice infected with the epidemic strain M. massiliense CRM-0019.

Mice infected with an aerosol dose of M. massiliense CIP 108297 (closed square) and CRM-0019 (open circle) were assayed by flow cytometry on days 30 and 60. Panel A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD4+ T cells expression of TNFα. Panels B and C show decreased CD4+ and CD8+ T cell expression of TNFα in the lungs (A) and spleens (B) of mice infected with the epidemic strain CRM-0019 compared to CIP 108297 (± SEM, n = 5). Results are expressed as the mean number of CD4⁺TNFα⁺, CD8⁺TNFα⁺, (± SEM, n = 5) T cells in the lungs and spleens. Student t-test, *p<0.050.

Figure 6. Increased CD4+ Foxp3+IL-10+ regulatory T cell expression in the lungs and spleens of IFNγ-KO mice infected with M. massiliense CRM-0019.

Lung cells obtained from mice infected with M. massiliense CIP 108297 (closed square) and CRM-0019 (open circle) were assayed by multi-parametric flow cytometry on days 30 and 60. Panel A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD4+ T cells expression of Foxp3+. Panel B shows that mice infected with M. massiliense CRM-0019 had a significant increase in CD4+Foxp3+ and CD4+Foxp3+ IL-10+ regulatory T cells in the lungs and spleens at 30 and 60 days of infection compared to M. massiliense CIP 108297-infected mice. Results are expressed as the mean number of CD4+Foxp3+ and CD4⁺Foxp3+ IL-10+ (± SEM, n = 5) T cells in the lungs and spleens. Student t-test, *p<0.050.