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.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a mechanism of genetically encoded but rapidly reversible drug tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of 7 cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multidrug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis-infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug tolerance, treatment failure, and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a novel mechanism of genetically encoded but rapidly reversible drug-tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of seven cytosines (7C) in the glpK gene.Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multi-drug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug-tolerance, treatment failure and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.

h Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA (Rv3806c) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis. We evaluated the prevalence of ubiA and embB (Rv3795) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ؎ 6.5% of the African EMB-resistant isolates but in only 9.5% ؎ 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment. E thambutol (EMB), a first-line antituberculosis drug, is often used in combination with other drugs to treat tuberculosis and prevent the emergence of drug resistance (1). Numerous studies have shown that mutations in the embCAB operon, particularly the embB gene, are a major cause of EMB resistance in Mycobacterium tuberculosis (2-8). A second set of mutations, which occur in the ubiA gene, has been associated with EMB resistance in clinical M. tuberculosis isolates (9-11). Mutations in ubiA almost always occur in EMB-resistant strains that also contain embB mutations, and ubiA appears to have multiplicative effects with embB mutations on MICs (9). The evolutionary path leading from lowto high-level EMB resistance has been studied in the laboratory (9). In these studies, high-level EMB resistance appears to develop through the stepwise acquisition of mutations in embB, ubiA, and embC.In the study described here, we examined the prevalence of ubiA mutations in isolates from two different geographic regions. Our results confirm the association between ubiA mutations and the presence of embB mutations and EMB resistance. We also demonstrate that the prevalence of these mutations varies by geographic location, suggesting that local factors may play a role in the type of mutations which develop as M. tuberculosis strains evolve to become EMB resistant.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB)-resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces small colony variant (SCV) phenotype, but this mutation does not change the minimal inhibitory drug concentrations (MICs) of any drug in wild-type M. tuberculosis. However, the same orn mutation in a low level EMB resistant double embB-aftA mutant (MIC=8μg/ml) produces an SCV with an EMB MIC of 32μg/ml. Reversible-resistance is indistinguishable from a drug-persistent phenotype because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size and reversing the EMB MIC back to the 8μg/ml MIC of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to the parental embB-aftA mutant, whereas large colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug-resistance in human tuberculosis.