Abstract
To study the evolution of drug resistance, we genetically and biochemically characterized
Mycobacterium tuberculosis
strains selected
in vitro
for ethambutol resistance. Mutations in decaprenylphosphoryl-β-
d
-arabinose (DPA) biosynthetic and utilization pathway genes
Rv3806c
,
Rv3792
,
embB
and
embC
accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number.
Rv3806c
mutations increased DPA synthesis, causing MICs to double from 2 to 4 µg/ml in a wild-type background and to increase from 16 to 32 µg/ml in an
embB
codon 306 mutant background. Synonymous mutations in
Rv3792
increased the expression of downstream
embC
, an ethambutol target, resulting in MICs of 8 µg/ml. Multistep selection was required for high-level resistance. Mutations in
embC
or very high
embC
expression were observed at the highest resistance level. In clinical isolates,
Rv3806c
mutations were associated with high-level resistance and had multiplicative effects with
embB
mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance.