Phage therapy is effective against infection by Mycobacterium ulcerans in a murine footpad model

Abstract

Background: Buruli Ulcer (BU) is a neglected, necrotizing skin disease caused by Mycobacterium ulcerans. Currently, there is no vaccine against M. ulcerans infection. Although the World Health Organization recommends a combination of rifampicin and streptomycin for the treatment of BU, clinical management of advanced stages is still based on the surgical resection of infected skin. The use of bacteriophages for the control of bacterial infections has been considered as an alternative or to be used in association with antibiotherapy. Additionally, the mycobacteriophage D29 has previously been shown to display lytic activity against M. ulcerans isolates.

Methodology/principal findings: We used the mouse footpad model of M. ulcerans infection to evaluate the therapeutic efficacy of treatment with mycobacteriophage D29. Analyses of macroscopic lesions, bacterial burdens, histology and cytokine production were performed in both M. ulcerans-infected footpads and draining lymph nodes (DLN). We have demonstrated that a single subcutaneous injection of the mycobacteriophage D29, administered 33 days after bacterial challenge, was sufficient to decrease pathology and to prevent ulceration. This protection resulted in a significant reduction of M. ulcerans numbers accompanied by an increase of cytokine levels (including IFN-γ), both in footpads and DLN. Additionally, mycobacteriophage D29 treatment induced a cellular infiltrate of a lymphocytic/macrophagic profile.

Conclusions/significance: Our observations demonstrate the potential of phage therapy against M. ulcerans infection, paving the way for future studies aiming at the development of novel phage-related therapeutic approaches against BU.

Figure 1. Lesion progression and M. ulcerans proliferation in the footpads and DLN of infected mice.

Mice were infected subcutaneously in the left footpad with 5.5 log₁₀ AFB of M. ulcerans 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).

Figure 2. Mycobacteriophage D29 dissemination in footpads and DLN of mycobacteriophage D29-treated mice.

Mice were infected subcutaneously in the left footpad with 5.5 log₁₀ AFB of M. ulcerans 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).

Figure 3. Cytokine profile in DLN and footpads of non-treated mice or mycobacteriophage D29-treated mice.

Mice were infected subcutaneously in the left footpad with 5.5 log₁₀ AFB of M. ulcerans 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. Levels of the TNF (panel A and B), IL- 6 (panel C and D), IFN-γ (panel E and F) and IL-10 (panel G and H) in DLN (panel A, C, E and G) and footpads (panel B, D, F and H) of mice were quantified by ELISA assay. n.d., not detected. Results are from one representative experiment of two independent experiments. 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).

Figure 4. Histology of mice footpads of non-treated mice or mycobacteriophage D29-treated mice.

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.

Figure 5. Histology and leukocyte kinetics in DLN of non-treated mice or mycobacteriophage D29-treated mice.

Histological sections of DLN collected at different time points were stained with HE. For panels A and B the scale bars represent 500 µm. At 68 days post-infection DLN of non-treated mice show severe damage of the lymphoid tissue (panel A). At day 35 after treatment (day 68 post-infection), DLN structure of mycobacteriophage D29 treated animals was maintained (panel B). Total number of cells in the DLN was determined in DLN suspensions (panel C). Results are from one representative experiment of two independent experiments. n.d., not determined. In panel C data points represent the mean ± SD (n = 5). Significant differences between treated and non-treated mice were performed using Student's t test (*, p≤0.05).