Repurposing of statins for Buruli Ulcer treatment: antimicrobial activity against Mycobacterium ulcerans

Abstract

Mycobacterium ulcerans causes Buruli Ulcer, a neglected infectious skin disease that typically progresses from an early non-ulcerative lesion to an ulcer with undermined edges. If not promptly treated, these lesions can lead to severe disfigurement and disability. The standard antibiotic regimen for Buruli Ulcer treatment has been oral rifampicin combined with intramuscular streptomycin administered daily for 8 weeks. However, there has been a recent shift toward replacing streptomycin with oral clarithromycin. Despite the advantages of this antibiotic regimen, it is limited by low compliance, associated side effects, and refractory efficacy for severe ulcerative lesions. Therefore, new drug candidates with a safer pharmacological spectrum and easier mode of administration are needed. Statins are lipid-lowering drugs broadly used for dyslipidemia treatment but have also been reported to have several pleiotropic effects, including antimicrobial activity against fungi, parasites, and bacteria. In the present study, we tested the susceptibility of M. ulcerans to several statins, namely atorvastatin, simvastatin, lovastatin and fluvastatin. Using broth microdilution assays and cultures of M. ulcerans-infected macrophages, we found that atorvastatin, simvastatin and fluvastatin had antimicrobial activity against M. ulcerans. Furthermore, when using the in vitro checkerboard assay, the combinatory additive effect of atorvastatin and fluvastatin with the standard antibiotics used for Buruli Ulcer treatment highlighted the potential of statins as adjuvant drugs. In conclusion, statins hold promise as potential treatment options for Buruli Ulcer. Further studies are necessary to validate their effectiveness and understand the mechanism of action of statins against M. ulcerans.

Keywords: Buruli Ulcer; Mycobacterium ulcerans; antimicrobial activity; drug repurposing; statins.

Figure 1

Dose–response curves for the antimicrobial activity of statins against Mycobacterium ulcerans. The broth microdilution assay was used to determine the dose–response curves of M. ulcerans against statins. M. ulcerans 98–912 was incubated for 7 days with increasing concentrations of statins [0.003052–400 μg/mL] (FLU, fluvastatin; ATR, atorvastatin; SIMV, simvastatin; LOV, lovastatin) or with standard antibiotics for BU treatment [0.000763–100 μg/mL] (RIF, rifampicin; STR, streptomycin). After this period of incubation, metabolic viability of M. ulcerans was determined with resazurin. Dashed lines represent the IC50 for each statin. Four independent experiments are represented and each experiment had three technical replicates.

Figure 2

Statins have a mycobactericidal effect against Mycobacterium ulcerans infected macrophages. THP1 cells were differentiated to macrophages with PMA. M. ulcerans infected macrophages were treated with 2 μM ATR, 2 μM SIMV, 2 μM FLU, and 1 μM RIF for 72 h, after which CFU were performed. *p < 0.05; ***p < 0.001; ****p < 0.0001. One representative experiment of three independent experiments is shown. Each experiment had 3–4 technical replicates.

Figure 3

Isobologram analysis between statins and antibiotics. The plots show the fractional inhibitory concentration (FIC) values represented the drug interaction between rifampicin (RIF) or streptomycin (STR) and atorvastatin (ATR) or fluvastatin (FLU) against M. ulcerans: (A) FIC RIF vs FIC ATR; (B) FIC RIF vs FIC FLU; (C) FIC STR vs FIC ATR; (D) FIC STR vs FIC FLU. Antimicrobial combinations that result in FIC values ≤0.5 are synergistic; FIC values between 0.5 and 4 are additive; FIC values > above 4 are antagonistic. Shaded areas correspond to the above-mentioned cut-offs. One representative experiment of three independent experiments is shown. Each experiment had three technical replicates.