doi:10.1186/s40249-026-01449-2engCalvet-Seral, JuanSáez-López, EmmaLópez-Expósito, Patricio R.Ferrer-Bazaga, SantiagoVaquero, Juan JoséRamón-García, SantiagoMendoza-Losana, AlfonsoThe ribosomal RNA synthesis ratio biomarker in Mycobacterium ulcerans for drug activity evaluationART-2026-149415Buruli 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).2026http://zaguan.unizar.es/record/17166410.1186/s40249-026-01449-2http://zaguan.unizar.es/record/171664oai:zaguan.unizar.es:171664info:eu-repo/grantAgreement/EC/H2020/853989/EU/EUROPEAN REGIMEN ACCELERATOR FOR TUBERCULOSIS/ERA4TBThis project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 853989-ERA4TBInfectious diseases of poverty 15 (2026), 51 [11 pp.]byhttps://creativecommons.org/licenses/by/4.0/deed.esinfo:eu-repo/semantics/openAccess