Our finding of 15 different mutations among 15 ethA mutants combined with the 9 previously described mutations suggests that a high degree of genetic diversity occurs within the ethA genes of ETH-resistant M. tuberculosis isolates. The 15 ethA mutants we describe represent 10 spoligotype patterns, including clusters of two and five isolates. All members of the five-isolate cluster had single-nucleotide frameshift mutations. These results indicate that ethA mutations appear to be widely disbursed across the structural gene, with no single nucleotide or codon predominating. This distribution contrasts with the situation seen in katG, where the majority of the mutations occur at codon 315, with Ser 315→Thr being the most prevalent. The predominance of the codon 315 mutations has been explained by the need for the cell to maintain a minimum basal level of catalase-peroxidase activity to protect against organic peroxides. Alterations that reduce KatG activity below this critical level would be lethal, and changes that lead to little or no reduction in enzyme activity would result in little or no decrease in INH susceptibility.
The fact that no such well-adapted ethA
mutation has emerged in the ETH-resistant bacilli investigated suggests the existence of one or more enzymes with functional redundancy to EthA. In fact, the genome of M. tuberculosis
possibly encodes more than 30 monooxygenases (4
). The proliferation of such enzymes in M. tuberculosis
may have evolved as a protective mechanism against various xenobiotic substances (10
). The exact role of EthA is not known, but the gene is highly conserved throughout the genus, suggesting it serves an important function (4
). Diminution, or loss, of EthA activity would thus be expected to have a deleterious effect on the cell. Given the proliferation of EthA homologs, it seems likely that one or more of these enzymes may be capable of compensating for a loss of EthA activity.
Clearly further study is needed to substantiate the association between ethA
mutations and ETH resistance and to establish the extent of genetic diversity in this gene. Should the initial finding that a wide array of mutations occur in ETH-resistant strains be verified by future investigation, such a phenomenon would resemble that seen in the pncA
gene of M. tuberculosis
strains resistant to PZA (20
). This gene encodes pyrazinamidase, the enzyme responsible for the conversion of the PZA into its metabolically active derivative pyrazinoic acid (26
). ETH, INH, and PZA are all nicotinamide analogs, and all three drugs rely on fortuitous enzymatic conversion to their respective active metabolites.
The predominance of a single, well-adapted mutation in the katG
gene of high-level INH-resistant strains reflects this enzyme's critical function of detoxifying reactive oxygen species. Under those rare circumstances in which KatG expression is completely lost, this loss occurs in conjunction with a mutation in the ahpC
promoter that up-regulates expression of AhpC, an enzyme also involved in antioxidant defense (27
). In contrast, PZA-resistant strains display a wide diversity, both in number and spatial distribution, of pncA
mutations, and no particular mutation predominates. The ethA
genes of high-level ETH-resistant strains appear to possess a similar degree of genetic diversity, and no evidence of selective pressure favoring a particular mutation has emerged.
While all of the ethA
mutants identified were resistant to ≥50 μg of ETH per ml, together they accounted for only 15 (52%) of the 29 isolates displaying that phenotype. An inhA
missense mutation was found in half of the remaining 14 isolates. Two isolates (isolates 1 and 7) with a shared spoligotype pattern had mutations in adjacent nucleotides of inhA
codon 21 that resulted in different amino acid substitutions. These two isolates have very different levels of INH resistance. The higher INH MIC for isolate 1 may result from the replacement of an aliphatic isoleucine residue with a weakly polar hydroxyl-containing threonine residue. Such a replacement can produce a greater disruption of InhA structure than is produced when an aliphatic valine residue is substituted, as in isolate 7. X-ray crystallography of InhA has shown that Ile 21 is located in the NADH binding site (11
). The fact that no similar disparity was seen in ETH MICs may result from subtle differences in drug-target interactions between ETH, INH, and the InhA-NADH complex. Alternatively, differences in ETH resistance between the two mutants may have gone undetected because they occur at concentrations >200 μg/ml. An Ile 21→Thr substitution occurred in an unrelated strain from Brazil (isolate 14) that was also resistant to >200 μg of ETH per ml and >32 μg of INH per ml, suggesting that the phenotype associated with that particular mutation is consistent across strains. The phenotype associated with the Ile 21→Val substitution also recurred in a second strain (isolate 6); however, this strain differed from the matched pair by one spacer.
structural gene mutations were far more prevalent in this study than in previous investigations. This inconsistency presumably reflects the different criteria used for selecting the study specimens. We selected our isolates on the basis of ETH resistance, whereas in previous investigations, isolates were selected on the basis of INH resistance (21
We identified a Ser 94→Ala mutation in the inhA
structural gene of three strains (isolates 2, 20, and 35). This mutation was first described in the seminal paper identifying InhA as the target of ETH and INH (1
). Curiously, we are not aware of any prior report describing the originally identified Ser 94→Ala alteration in clinical isolates. In one strain (isolate 2), this was the only mutation identified, while in the others, it occurred in combination with either a katG
(isolate 35) or an inhA
promoter mutation (isolate 20). The resistance phenotypes of these strains differed dramatically. The inconsistency of these results is difficult to reconcile but suggests the involvement of other, strain-specific factors.
An inhA promoter mutation was identified in 15 isolates with wild-type ethA and inhA structural genes. The ETH MICs for eight of these isolates were in the range of 10 to 25 μg/ml, a moderate increase in ETH resistance that is consistent with a drug titration mechanism. Four of the 15 promoter mutants were resistant to >200 μg of ETH per ml, and the MIC for 2 mutants each was100 μg/ml. It is highly improbable that the promoter mutation alone can account for the high-level ETH resistance seen in those isolates. This assertion is supported by the fact that the INH MICs for five of these strains were ≤4 μg/ml. Were the promoter mutation alone responsible for the high-level ETH resistance seen in these strains, we would expect a concomitant and proportional increase in INH resistance.
A more plausible explanation for the high-level ETH resistance of those strains is that other, ETH-specific mechanisms of resistance are involved. Expression of EthA is under the negative regulatory control of the protein repressor EthR. An increase in EthR expression would then down-regulate ethA
, ultimately leading to less drug activation and increased resistance to ETH. Hyperexpression of EthR has been experimentally proven to cause ETH resistance (4
) and could therefore explain the highly ETH-resistant phenotype of the six strains with only an inhA
promoter mutation. How EthR production is controlled and to what stimuli it responds are unknown. The potentially important involvement of ethR
in clinical ETH resistance shows the need for additional studies to determine which factors mediate EthR production.
The MABA method proved itself a very useful research tool for correlating specific mutations in ETH and INH drug resistance markers with relative resistance phenotypes. The INH MICs obtained were highly reproducible between tests. Establishing a precise end point for ETH was somewhat technically challenging because of “trailing” effect, but there was good reproducibility between replicates. The different end point characteristics of ETH and INH presumably reflect the in vitro bactericidal potency of the two drugs: INH is considered to be bactericidal at or near its MIC, while ETH is bactericidal at concentrations 2 to 4 times its MIC (13
). The MICs we report here are specific to the MABA method, and we caution the reader against extrapolating these results to other drug susceptibility testing methods.
In summary, our finding of ethA mutations in 52% of clinical isolates for which ETH MICs were ≥50 μg/ml provides substantial new evidence confirming the role of this gene in ETH resistance. As expected, mutations in ethA had no detectable association with INH resistance. The level of INH resistance in the study isolates was explainable by and consistent with mutations in katG and inhA. Twenty-four percent of the high-level ETH-resistant strains had mutations in the inhA structural gene. With the exception of Ser 94→Ala, these mutations always occurred in combination with inhA promoter mutations. Only an inhA promoter mutation was identified in approximately a third of the isolates. The majority of those isolates displayed intermediate levels of ETH and INH resistance. Six of the promoter mutants were resistant to ≥100 μg of ETH per ml, a high level of resistance that we believe is not exclusively attributable to the promoter mutations but rather results from another mechanism. Because the regulatory protein EthR mediates ethA expression, it seems reasonable that activator and target mutations alone cannot account for all observed high-level ETH resistance. While mechanisms of ETH resistance exclusive of the ethAR loci cannot be discounted, it seems probable that mutation in ethA is not the only ETH resistance-associated mechanism involving these loci. While the identification of the ethAR loci has contributed greatly to the understanding of ETH resistance, additional investigation is clearly needed.