The ribosomal RNA synthesis ratio biomarker in Mycobacterium ulcerans for drug activity evaluation

Buruli ulcer (BU), a neglected tropical disease caused by Mycobacterium ulcerans (Mul), is treated with an 8-week regimen of rifampicin (RIF) and clarithromycin (CLA). Clinical trials are currently evaluating amoxicillin/clavulanate (AMX/CLV) co-administration to reduce treatment duration. However, conventional methods for assessing in vitro drug efficacy against Mul, like colony-forming units (CFUs), are slow and cumbersome. The ribosomal RNA synthesis ratio (RS-ratio) measures ribosome biogenesis and serves as a proxy of metabolic activity. While it is a promising predictive biomarker for treatment shortening in tuberculosis, its application in Mul remains unexplored. Here, we evaluated the RS-ratio for Mul drug activity assessment through RNA extractions from time-kill assays using RIF, CLA, and AMX/CLV, alone or in combinations. RIF + AMX/CLV-containing combinations produced a potent, rapid RS-ratio reduction, decreasing from a baseline of ≈2000 to ≈200 as early as day 3, and reaching their maximal inhibition (≈50-60) between days 7 and 10. Notably, this metabolic decline preceded the CFUs and luminescence drops observed in prior studies. Interestingly, the RS-ratio detected a metabolic recovery between days 14 and 28 (≈400), suggesting remaining bacterial viability, a phenomenon not observed by CFUs or luminescence. In summary, this is the first report using the RS-ratio to evaluate antibiotic activity against Mul. Our findings validate the RS-ratio as a molecular tool for assessing the sterilizing potential of new regimens to inform future research and clinical trial designs for the treatment of BU. Our results support the RIF + CLA + AMX/CLV regimen selection for BU treatment shortening in the BLMs4BU clinical trials (NCT05169554, PACTR202209521256638).