Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans

BackgroundBuruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a cumbersome method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against clinical M. ulcerans isolates by TKA, while comparing four methodologies: CFU/mL enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. Methodology/Principal findingsTKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified after 1, 3, 7, 10, 14, 21 and 28 days of treatment with different methodologies. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combination were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies provided similar results with a general good correlation between CFU counting and the other methodologies. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. Conclusions/SignificanceOur study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to the design of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment. Author summarySince 2004, when only surgery was available, Buruli ulcer (BU) treatment has improved reaching to the efficient 8-weeks all oral antibiotic course of rifampicin and clarithromycin together with wound care and, sometimes, tissue grafting and surgery. This skin neglected tropical disease caused by Mycobacterium ulcerans mainly affects people living in rural areas in under-resourced countries with limited access to health services and medicines, thus compromising patients treatment adherence. The inclusion of amoxicillin/clavulanate in BU therapy was previously described with the potential to shorten BU treatment. In this study, we confirmed the high bactericidal activity over time of rifampicin and amoxicillin/clavulanate-containing combinations against M. ulcerans, being even more effective in vitro than the antibiotics currently used to treat BU. A comparison of different methodologies that are applied in the laboratory and in the clinical environment showed good correlation between them, leading to a wide variety of biomarkers for BU research and giving the opportunity for clinical translation; the choice of the most suitable one being driven by the purpose and the context of the study. These in vitro results provide further support to the ongoing clinical trial in West Africa to evaluate if BU treatment can be shortened from 8 to 4 weeks (BLMs4BU trial: NCT05169554, PACTR202209521256638).