Sensitivity of phages to <i>M. ulcerans</i> isolates.

<p>P-plaque formation.</p

Mycobacteriophage D29 dissemination in footpads and DLN of mycobacteriophage D29-treated mice.

<p>Mice were infected subcutaneously in the left footpad with 5.5 log₁₀ AFB of <i>M. ulcerans</i> strain 1615. After the emergence of macroscopic lesion (33 days post infection; footpad swelling of 3.0 mm) mice were subjected to treatment with a single dose of subcutaneous injection of mycobacteriophage D29. Phage titres were assessed by plaque forming units. n.d., not detected. Results are from one representative experiment of two independent experiments. The bars represent the mean ± SD (n = 5). Significant differences were performed using Student's t test (**, p≤0.01, ***, p≤0.001).</p

Histology of mice footpads of non-treated mice or mycobacteriophage D29-treated mice.

<p>Histological sections of footpads collected at different time points were stained with HE (A, B, D, E, H, I and J) or with ZN (C, F, G and K). For panels A, D, H and J, the scale bars represent 100 µm. For panels B, E and I, the scale bars represent 10 µm. For panels C, F, G and K the scale bars represent 5 µm. dpi, days post-infection. At 68 days post-infection (A–C), footpads of non-treated mice show necrotic areas (asterisks). Magnifications of panel A (rectangles) show mononuclear cells adjacent/in necrotic areas (B). Panel C show bacteria in necrotic areas (C; arrowheads). At day 35 after treatment (day 68 post-infection) (D–G), footpads of mycobacteriophage D29-treated mice show abundant cellular infiltration (D), composed mainly by mononuclear cells (E). Staining for bacteria in the same tissue areas and magnifications of the bacilli (arrowheads) are shown in panels F and G. At 150 days after treatment (H–K), footpads of mycobacteriophage D29-treated mice show a persistent inflammatory infiltrate (H–I). Staining for bacteria in remaining necrotic areas (J) are shown in panel K.</p

Lesion progression and <i>M. ulcerans</i> proliferation in the footpads and DLN of infected mice.

<p>Mice were infected subcutaneously in the left footpad with 5.5 log₁₀ AFB of <i>M. ulcerans</i> strain 1615. After the emergence of macroscopic lesion (33 days post infection; footpad swelling of 3.0 mm) mice were subjected to treatment with a single dose of subcutaneous injection of mycobacteriophage D29 (dashed line). Lesion progression was assessed by measurement of footpad swelling (panel A) (n = 15). Bacterial proliferation was assessed by colony forming units in footpads (panel B) and in DLN (panel C) (n = 5). †, mice were sacrificed for ethical reasons after the emergence of ulceration of non-treated mice (68 days post infection). Results are from one representative experiment of two independent experiments. Data points and bars represent the mean ± SD (n = 5). Significant differences between treated and non-treated mice were performed using Student's t test (*, p≤0.05, **, p≤0.01, ***, p≤0.001).</p

Histological analysis of mice footpads infected with <i>M. ulcerans</i> and treated or non-treated with RS.

<p>Histological sections of footpads collected at different time points post-infection were stained with HE or ZN and the percentage of fields with AFB localized predominantly inside (A) or outside (B) cells and the percentage of fields with predominance of mononuclear (C) or neutrophilic cells (D) was evaluated. (E) Relative abundance of inflammatory infiltrates. Analyses were performed in 6 different sections of each sample of mice footpad, in a total of 3 footpads per group. Asterisks represent significant differences between treated and non-treated mice at 21 days post-infection (*, <i>P</i><0.05; ***, <i>P</i><0.001). Significant differences over time for the non-treated or RS group were determined by comparing each time point with the following (*, <i>P</i><0.05; **, <i>P</i><0.01, ***, <i>P</i><0.001). NT; non-treated mice. RS; mice treated with rifampicin and streptomycin. Time points represented are days post-infection. Treatment duration was as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032740#pone-0032740-g002" target="_blank">Figure 2</a> legend. Data bars represent the mean ± SEM.</p

Immunohistochemistry for NOS2 of mice footpads infected with <i>M. ulcerans</i> and treated or non-treated with RS.

<p>Histological sections of footpads collected at different time points were stained with DAPI (blue; nuclei staining), anti-NOS2 antibody (red) and auramine-O (green; mycobacteria staining). Magnifications ×10 (A, D, G, H, K and N) and ×40 (B, C, E, F, I, J, L, M and O). Treatment duration was as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032740#pone-0032740-g002" target="_blank">Figure 2</a> legend. (A) Footpads of non-treated mice at 12 days post-infection with no evidence for NOS2 presence in the tissue in areas with numerous bacilli (B, green) and only few cells are stained at peripheries (C). (D) At 21 days post-infection huge clumps of bacilli are observed with no evidence for NOS2 staining (F), which is neither present at the periphery (E). (G and H) Footpads of mice treated with RS at 21 days post-infection show little NOS2 staining in areas co-localized with few (J) or absent bacilli staining (I). (K) Footpads of mice treated with RS at 82 days post-infection show more numerous and larger areas of NOS2 with little co-localization with bacilli (L), whereas areas with numerous extracellular bacilli show little staining for NOS2 (M). (N and O) Footpads of mice treated with RS at 5 months after finalizing the treatment (245 days post-infection) show NOS2 staining co-localized with bacilli. Results are from one representative experiment of two independent experiments.</p

Lesion progression and bacterial proliferation of <i>M. ulcerans</i> in the footpads of mice treated or non-treated with RS.

<p>Mice were infected with <i>M. ulcerans</i> strain 98–912 and were left untreated (closed circles) or subjected to treatment with RS for 10 weeks (open circles). (A) Lesion progression was assessed by measurement of footpad swelling (n = 15). Bacterial proliferation was assessed by CFU (B) and AFB counts (C) (n = 5). (D) Percentage of well stained (black bars) versus poorly stained (including beaded) (grey bars) AFB in the footpad for the antibiotic treated group of mice. At day 12 post-infection, treatment was initiated until day 82. (E) AFB at day 12 post-infection, with well stained (arrowheads) and beaded (arrow) bacilli. (F) AFB at day 156 post-infection, with beaded (arrow) and poorly stained (triangle) bacilli. Asterisks represent significant differences between treated and non-treated mice on panels B and C (**, <i>P</i><0.01, ***, <i>P</i><0.001). Significant differences over time for the well stained or poorly stained group in panel D were determined by comparing each time point with the following (***, <i>P</i><0.001). Grey bar on panels A, B and C represents the time period of antibiotic administration. d.l., detection limit; n.d., not detected. †, mice were sacrificed for ethical reasons after the emergence of ulceration. Results are from one representative experiment of two independent experiments. Data points and bars represent the mean ± SEM.</p

Immunization increases iNOS positivity in the footpad of <i>M. ulcerans</i> challenged mice.

<p>Mice were either non-immunized (A and D) or immunized with BCG (B, E, and G) or with mycolactone-negative <i>M. ulcerans</i> 5114 (C, F, and H) two months before challenge. All mice were infected in the footpad with 2 log₁₀ CFU of <i>M. ulcerans</i> 98–912. The footpads were harvested at on day 60 (A–C), day 70 (D–F), or day 100–200 (G–H and J–K) and processed for histology. Cells were stained for the presence of iNOS (red) and nuclei were stained with DAPI (blue). In footpads of non-immunized mice, scarce or no iNOS positive cells were found in the tissue (A, D). The footpads of vaccinated mice presented foci of iNOS positive cells during the initial weeks of infection (B–C and E–F). With progression of infection, iNOS positivity became sparse. Images are representative of 4 footpads per group. Original magnification: 20×.</p

BCG vaccination induces an early Th1 cytokine profile in <i>M. ulcerans</i>-infected footpads.

<p>Mice were either non-immunized (•) or immunized with BCG (○) two months before challenge. All mice were infected in the footpad with 4 log₁₀ CFU of <i>M. ulcerans</i> 98–912. At different times post-infection, total RNA from the footpad was extracted and the presence of mRNA for IFN-ã (A), TNF (B), MIP2 (C) was assessed by real-time PCR. Data points represent the mean ± SEM (<i>n</i> = 5–8) for each time point. Statistical significance was calculated with Student's <i>t</i> test (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Leukocyte kinetics, bacterial proliferation and DLN histology of mice infected with <i>M. ulcerans</i> and treated or non-treated with RS.

<p>(A) Total number of leukocytes in the DLN were determined at different time points post-infection in non-treated (closed circles) and antibiotic-treated mice (open circles) (n = 5). (B) Total number of B cells (CD19⁺) and (C) T cells (CD3⁺) in the DLN were determined by flow cytometry (n = 5). (D) Bacterial proliferation was assessed by CFU counts; d.l., detection limit; n.d., not detected. Significant differences between values over time in non-treated or treated mice on panel A, B and C were determined by comparing each time point with the following (*, <i>P</i><0.05; **, <i>P</i><0.01). Significant differences between treated and non-treated mice on panel D (***, <i>P</i><0.001). Grey bar represents the time period of antibiotic administration. Histological sections of DLN collected at different time points were stained with HE (E, G, I and K) or with FITC fluorochrome-labeled PNA (green) counterstained with DAPI (blue; nuclei staining) for the labeling of germinal centers (F, H, J and L). Magnifications ×4. (E and I) DLN of non-treated mice at day 12 and treated mice at day 21 post-infection, respectively, showing enlargement of the DLN with germinal centers (F and J). (G) DLN of non-treated mice at day 21 post-infection showing extensive cell depletion and the absence of germinal centers (H). (K) DLN of treated mice at day 42 post-infection showing a normal structure and absence of germinal centers (L). Results are from one representative experiment of two independent experiments. Data points represent the mean ± SEM.</p