Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid

Abstract

Objectives: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug.

Methods: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid.

Results: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes.

Conclusions: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

Figure 1.

Pretomanid (Pa) MIC distribution for the M. tuberculosis H37Rv laboratory reference strain (n = 117).

Figure 2.

Pretomanid (Pa) MIC distributions for (a) Groups A and D, and (b) Groups B and C (excluding H37Rv). The 99th percentiles for Groups B and C based on ECOFFinder and visual estimation for Groups A and D are indicated with arrows. (c) The relationship between these MICs, represented by the heat map on the outer circle, and the underlying MTBC phylogeny (BCG is shown as part of M. bovis). The phylogenetic tree was rooted at the midpoint. The M. canettii branch lengths (dotted lines) were scaled to be 5× shorter for the purpose of visualization. An unscaled tree is shown in Figure S1.