Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal againstMycobacterium ulcerans

Abstract

AbstractBackgroundBuruli ulcer (BU) is a skin neglected tropical disease (NTD) caused byMycobacterium 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 againstM. ulcerans.However,in vitrotime kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug. Colony forming units (CFU) enumeration is thein vitroreference method to measure bacterial load, although this is a cumbersome method due to the slow growth ofM. ulcerans.The aim of this study was to assess thein vitroactivity of RIF, CLA and AMX/CLV combinations against clinicalM. ulceransisolates 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/IS2404genes quantification.Methodology/Principal findingsTKA of RIF, CLA and AMX/CLV alone and in combination were performed against differentM. ulceransclinical 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 effectivein vitrothan 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 quantifyM. ulceransbacterial loads inin vitroTKA.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 anin vitromicrobiological 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 byMycobacterium ulceransmainly 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 againstM. ulcerans, being even more effectivein vitrothan 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. Thesein vitroresults 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).