Rifampicin and rifabutin resistance in 1003 Mycobacterium tuberculosis clinical isolates

Abstract

Drug-resistant TB remains a public health challenge. Rifamycins are among the most potent anti-TB drugs. They are known to target the RpoB subunit of RNA polymerase; however, our understanding of how rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross-resistance between the two rifamycin drugs rifampicin and rifabutin. Methods: We performed WGS of 1003 Mycobacterium tuberculosis clinical isolates and determined MICs of both rifamycin agents on 7H10 agar using the indirect proportion method. We generated rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamarBlue reduction assay. Results: Of the 1003 isolates, 766 were rifampicin resistant and 210 (27%) of these were rifabutin susceptible; 102/210 isolates had the rpoB mutation D435V (Escherichia coli D516V). Isolates with discordant resistance were 17.2 times more likely to harbour a D435V mutation than those resistant to both agents (OR 17.2, 95% CI 10.5-27.9, P value ,10 #40). Compared with WT, the D435V in vitro mutant had an increased IC 50 of both rifamycins; however, in both cases to a lesser degree than the S450L (E. coli S531L) mutation. Conclusions: The observation that the rpoB D435V mutation produces an increase in the IC 50 of both drugs contrasts with findings from previous smaller studies that suggested that isolates with the D435V mutation remain rifabutin susceptible despite being rifampicin resistant. Our finding thus suggests that the recommended critical testing concentration for rifabutin should be revised.

Whole-genome sequence analysis and comparisons between drug-resistance mutations and minimum inhibitory concentrations of Mycobacterium tuberculosis isolates causing M/XDR-TB

“…However, in the case of RFB, a greater proportion of isolates harboring rpoB mutations had MIC values lower than the CC (0.5 μg/ml as recommended by the kit instructions). Possibly, mutations (especially rpoB Asp435Val) assumed to confer resistance to RIF might not be highly correlated with RFB resistance, as found previously by others [26,28,29]. Furthermore, we found that isolates carrying rpoB Asp435Val alone had significantly lower MIC values for RFB than did isolates carrying rpoB Ser450Leu.…”

Population structure, biogeography and transmissibility ofMycobacterium tuberculosis

“…We expect these tools to facilitate the use of the expanded Mtb classification by scholars of Mtb evolution and public health practitioners alike for applications such as the rapid assessment of potential outbreaks and isolate triage for detailed phylogenetic analyses. Further, low level susceptibility to specific Mtb drugs has been reported for some lineages 21,22 . Improving the resolution of classification at the sub-lineage level can allow for a better understanding of these findings and potentially a more refined prediction of drug resistance profiles.…”

Antibiotic resistance by high-level intrinsic suppression of a frameshift mutation in an essential gene

“…Ribosomal frameshifting errors occur rarely, but when they do the consequences for protein function are generally catastrophic, and frameshift mutations are generally only observed in nonessential genes. In this context we were intrigued by reports of clinical isolates of Mtb carrying frameshift mutations (11)(12)(13)(14), or nonsense mutations (12,(29)(30)(31), in the essential gene rpoB. Surprisingly, none of the publications reported any experimental analysis of the mutations or explained how these mutants were viable.…”

“…Considering the functional importance of reading frame maintenance during translation it is surprising that there have been published reports of frameshift mutations in the essential gene rpoB among rifampicin-resistant clinical isolates of Mycobacterium tuberculosis (Mtb) (11)(12)(13)(14). To our knowledge none of these mutants have been investigated to determine either the validity of the mutation reported or a possible mechanism that could explain bacterial viability.…”