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Efflux-mediated pretomanid high-level resistance in Mycobacterium tuberculosis

D Machado(1) J Perdigão(2) I Portugal(2) T G Clark(3) M Viveiros(1)

1:Laboratório de Micobactérias, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisboa, Portugal; 2:iMed.ULisboa, Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal; 3:London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, United Kingdom

In this study, we investigate the contribution of efflux to pretomanid high-level resistance in a naive M. tuberculosis pretomanid-resistant MDR clinical strain.

First- and second-line drug susceptibility testing (DST), quantitative DST, and MICs, w/without efflux inhibitors, were performed using MGIT960/TB eXIST. Whole-genome sequencing was performed. MIRU-VNTR and spoligotyping were used for typing. The contribution of efflux mechanisms was studied by MIC determination, real-time ethidium bromide efflux activity evaluation, and analysis of mRNA transcriptional levels of selected efflux pump genes in response to pretomanid. Cytotoxicity was evaluated in human-monocyte-derived macrophages.

Mtb30 was isolated from a tuberculosis patient in 2008 and belonged to the Beijing genotype. Is resistant to all first-line drugs plus the second-line drugs rifabutin and ethionamide. Susceptibility to bedaquiline, linezolid, and fluoroquinolones was confirmed, however, it shows high-level resistance to pretomanid with a MIC of 64 µg/ml. Sequencing revealed the presence of Trp88Stop in the ddn gene. Synergy assays showed synergistic interactions between efflux inhibitors (thioridazine and verapamil) with pretomanid resistance and ethidium bromide efflux. Real-time efflux assays demonstrated increased efflux activity which could be inhibited in the presence of inhibitors. Efflux pumps were found overexpressed in response to pretomanid. Pretomanid is non-cytotoxic to concentrations of 80 µg/ml.

This study shows efflux contributes to high-level resistance to pretomanid in a naive Mtb pretomanid-resistant clinical strain. Clinical isolates from untreated patients with high-level pretomanid resistance highlight the need for accurate DST prior to pretomanid treatment.

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