Whole-genome sequencing of two clinical strains of <i>Mycobacterium tuberculosis</i> with phenotypic susceptibility to rifampicin but predicted resistance by Xpert MTB/RIF

Introduction. More than 40% of Mycobacterium tuberculosis strains are resistant to rifampicin (RIF) and isoniazid, the first-line drugs. The tuberculosis pathogen becomes resistant to RIF mainly due to mutations in the rpoB gene. The aim of the study was to search for the most probable compensatory mutations in the rpoA, rpoB and rpoC genes encoding α-, β- and β′-subunits of M. tuberculosis RNA polymerase. Materials and methods. A cross-sectional analysis of phenotypic and genetic resistance to RIF among 2298 clinical strains of M. tuberculosis revealed 8 cases in which resistance as determined by the Xpert Ultra MTB/RIF test was not confirmed bacteriologically. In all cases, these were chronic multidrug-resistant or extensively drug-resistant M. tuberculosis patients in whom RIF was discontinued due to the detection of resistance to this drug in the isolated strains. Two strains were obtained for genotype testing, Sanger sequencing and whole-genome sequencing. Results. Repeat Xpert Ultra MTB/RIF test, Sanger sequencing and whole genome sequencing revealed the presence of a single S450L mutation in the rpoB gene with phenotypic sensitivity in both strains. Phylogenetic analysis revealed that both genomes belonged to the Beijing B0/W148 genotype. The strains were characterized by a higher growth rate than the other isolates. Two potential compensatory mutations V483G and H748P in the groC gene were identified in the absence of other significant changes in the rpoA and rpoB genes. Conclusion. It is suggested that the phenomenon of discrepancy between results of bacteriological and molecular genetic tests is associated with the acquisition of compensatory mutations in the groC gene during RIF treatment of Beijing B0/W148 strains, and the identified mutations affect the conformation of the β'-subunit, restoring the transcription efficiency of affected by the major S450L mutation.