Bedaquiline and clofazimine resistance in Mycobacterium tuberculosis: an in-vitro and in-silico data analysis
Lindsay Sonnenkalb, Joshua James Carter, Andrea Spitaleri, Zamin Iqbal, Martin Hunt, Kerri M. Malone, Christian Utpatel, Daniela María Cirillo, Camilla Rodrigues, Kayzad Nilgiriwala, Philip W. Fowler, Matthias Merker, Stefan Niemann, Ivan Barilar, Simone Battaglia, Emanuele Borroni, Angela Pires Brandão, Alice Brankin, Andrea Maurizio Cabibbe, Joshua A. Carter, Daniela María Cirillo, Pauline Claxton, David A. Clifton, Ted Cohen, Jorge Coronel, Derrick W. Crook, Viola Dreyer, Sarah G. Earle, Vincent Escuyer, Lucilaine Ferrazoli, Philip W. Fowler, George F. Gao, Jennifer L. Gardy, Saheer E. Gharbia, Kelen Teixeira Ghisi, Arash Ghodousi, Ana Lúıza Gibertoni Cruz, Louis Grandjean, Clara Grazian, Ramona Groenheit, Jennifer L. Guthrie, Wencong He, Harald Hoffmann, Sarah Hoosdally, Martin Hunt, Zamin Iqbal, Nazir Ismail, Lisa Jarrett, Lavania Joseph, Ruwen Jou, Priti Kambli, Rukhsar Khot, Jeff Knaggs, Anastasia Koch, Donna Kohlerschmidt, Samaneh Kouchaki, Alexander S. Lachapelle, Ajit Lalvani, Simon Grandjean Lapierre, Ian F. Laurenson, Brice Letcher, Wan-Hsuan Lin, Chunfa Liu, Dongxin Liu, Kerri M. Malone, Ayan Mandal, Mikael Mansjö, Daniela Matias, Graeme Meintjes, Flávia de Freitas Mendes, Matthias Merker, Marina Mihalic, James Millard, Paolo Miotto, Nerges Mistry, David Moore, Kimberlee A. Musser, Dumisani Ngcamu, Ngoc Nhung Hoang, Stefan Niemann, Kayzad Soli Nilgiriwala, Camus Nimmo, Nana Okozi, Rosângela Siqueira de Oliveira, Shaheed Vally Omar, Nicholas I. Paton, Tim Peto, Juliana Maíra Watanabe Pinhata, Sara Plesnik, Zully M. Puyén, Marie Sylvianne Rabodoarivelo, Niaina Rakotosamimanana, Paola M. V. Rancoita, Priti Rathod, Gillian Rodger, Camilla Rodrigues, Timothy C. Rodwell, Eaysha Roohi, David Santos-Lázaro, Sanchi Shah
Abstract
BACKGROUND: Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data. METHODS: For this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations. FINDINGS: We discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5-MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand. INTERPRETATION: Our findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments. FUNDING: Leibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions.