Abstract
BackgroundA comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST).MethodsWe investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing.ResultsOut of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5).ConclusionsOur findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.
Highlights
A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret wholegenome sequencing data for genotypic drug susceptibility testing (DST)
Owing to the inherently slow growth rate of MTBC, the only realistic way to diagnose the majority of drugresistant cases is to use rapid genotypic drug-susceptibility testing, which ranges from targeted assays to whole-genome sequencing (WGS) [2]
It is becoming increasingly clear that genotypic drug-susceptibility testing (gDST) assays are better suited than phenotypic DST to rule-in resistance caused by known mechanisms that only confer modest minimum inhibitory concentration (MIC) increases, such as for ethambutol (EMB) [3,4,5]
Summary
A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret wholegenome sequencing data for genotypic drug susceptibility testing (DST). The accuracy of gDST depends on the ability to distinguish valid markers for resistance (i.e. mutations that directly confer resistance or, alternatively, play a compensatory role in resistance) from neutral mutations that do not alter the susceptibility to an antibiotic [6, 7] In this context, one of the major confounders is the pre-existing variation in genes associated with resistance, which comprises neutral mutations and, more rarely, changes that confer intrinsic/natural resistance (i.e. resistance that arose by chance/genetic drift prior to the clinical use of a drug or a related agent with a shared resistance mechanism) [8]. The World Health Organization (WHO) has just revised the reporting language for line probe assays to reflect this possibility (e.g. gyrA A90G causes false-resistance reports for fluoroquinolones with the Hain GenoType MTBDRsl assay) [12, 13]
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