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, 6 (6), e21673

Difference in Virulence of Mycobacterium Avium Isolates Sharing Indistinguishable DNA Fingerprint Determined in Murine Model of Lung Infection

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Difference in Virulence of Mycobacterium Avium Isolates Sharing Indistinguishable DNA Fingerprint Determined in Murine Model of Lung Infection

Eduardo Pinheiro Amaral et al. PLoS One.

Abstract

Background: Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. The high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence.

Methods and findings: In this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. The mice, C57BL/6 wild- type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. The obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild-type mice, whereas the P104 strain established a chronic infection. In the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner.

Conclusions/significance: The results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Capacity of H27 and P104 M. avium strains to grow in macrophages and 7H9 broth.
Bacterial growth in the broth within 24 days was evaluated by spectrophotometry (A). Bone marrow cells were obtained from C57BL/6 mice and differentiated to macrophages in vitro. The resulted cells were infected at a MOI 1∶1 bacteria/macrophage, incubated for 3 h at 37°C, washed, tested for the mycobacterial uptake, and cultivated for additional 6 days. The resulted cells were lysed and CFU numbers (B) were quantified as indicated in Material and methods. The percentage of macrophages with internalized mycobacteria in the culture was quantified 3 h after infection by Ziehl-Neelsen method (C). The data obtained in three independent experiments are presented as mean ± SD of samples in triplicate. Asterisk represent statistical significance (*p<0.05).
Figure 2
Figure 2. Lung bacillary loads in C57BL/6 WT and GKO mice infected with M. avium strains.
Mice were infected by an i.t. injection of two M. avium strains, H27 and P104, isolated from different hosts (2.5×106 bacilli). At the indicated time-points, six mice were sacrificed, and bacterial burdens (CFU) in the lungs were measured. The data were obtained in three independent experiments. Asterisks represent statistical significance (**p<0.01, ***p<0.001).
Figure 3
Figure 3. Comparison of lung, liver and spleen bacillary loads in C57BL/6 WT and GKO mice infected with M. avium strains.
Mice were infected as indicated in the legend to Fig. 2. At the indicated time-points, six mice were sacrificed, and bacterial burdens (CFU) in the lungs, livers and spleens were measured. The data were obtained in two independent experiments. Asterisks represent statistical significance (**p<0.01, ***p<0.001).
Figure 4
Figure 4. Lung, liver and spleen histopathology of C57BL/6 WT and GKO mice infected with the P104 or H27 M. avium strains.
Representative lung histopathology at 60 days after P104 strain challenge in the WT (B) and GKO (A, C–E) mice and spleen (F) of the GKO mice. Lung presented parenchyma consolidation by epithelioid granulomatous infiltration (A), multifocal lesions with typical granuloma (B - arrows), cellular infiltration containing multinucleated giant cells (C - arrows) with intracellular fuchsinophilic acid-fast bacilli (D); granuloma containing multinucleated giant cells (E) and presence of BAAR inside the intrafollicular granuloma in spleen (F). Representative liver histopathology at 90 days after the challenge of the WT mice with H27 (G) and P104 (H) strains. The tissue samples were stained with H&E (A, B, C, E, G and H) or by Ziehl-Neelsen method (D and F). Total original magnification: A (4x); B, G, H (10x); C, E (20x); F (40x); D (100x).
Figure 5
Figure 5. Expression of cytokine genes in lung cells of mice infected with P104 strain.
C57BL/6 and GKO mice were infected as indicated in the legend to Fig.2. mRNA was extracted from lungs of these mice on days 5, 15, 30 and 60 post-infection. Levels of mRNA were quantified by real-time PCR using specific primers for inflammatory cytokine genes and normalized to the level of β-actin expression. Cytokine gene expression determination was performed with four animals per time-point. The data were obtained in two independent experiments. Results are expressed as fold increase ± SEM compared to non-infected animals. Asterisks represent statistical significance (**p<0.01, ***p<0.001) when compared WT and GKO mice.
Figure 6
Figure 6. Expression of cytokine genes in lung cells of mice infected with H27 strain.
Experiments were performed and results of cytokine gene expression determination were presented exactly as described in the legend to Fig. 5.
Figure 7
Figure 7. Production of pro-inflammatory mediators in culture of cells obtained from lungs of C57BL/6 WT mice infected with M. avium H27 or P104 strains.
Lung cells were isolated on day 30 after i/t infection of mice and cultured in 96 well-plates, 5×104 cells/well. Culture supernatants were collected after 48 h incubation and concentrations of IFN-γ and TNF-α were measured by sandwich ELISA. Nitric oxide production was measured by Griess reaction. The data were obtained in two independent experiments. Asterisks represent statistical significance (***p<0.001).

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