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. 2010 Oct 25;55(1):355–360. doi: 10.1128/AAC.01030-10

Molecular Investigation of Resistance to the Antituberculous Drug Ethionamide in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis▿

F Brossier 1,2, N Veziris 1,2, C Truffot-Pernot 2, V Jarlier 1,2, W Sougakoff 1,2,3,*
PMCID: PMC3019671  PMID: 20974869

Abstract

Ethionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active against Mycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded by ndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETHr) isolates, 24 ETH-susceptible (ETHs) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETHSip; defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETHr isolates, we found mutations in ethA, ethR, or inhA or its promoter, which mostly corresponded to new alterations in ethA and ethR. The 9 ETHr isolates without a mutation in these three genes (9/47, 19%) had no mutation in ndh, and a single isolate had a mutation in mshA. Of the 16 ETHSip isolates, 7 had a mutation in ethA, 8 had no detectable mutation, and 1 had a mutation in mshA. Finally, of the 24 ETHs isolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in the inhA promoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETHr isolates, highlighting the complexity of the mechanisms of ETH resistance in M. tuberculosis.

Ethionamide (ETH), a second-line antituberculous drug, is a structural thioamide analogue of isoniazid (INH), the cornerstone of front-line tuberculosis (TB) treatment. ETH is considered to be the most active antituberculous drug after aminoglycosides and fluoroquinolones and is a component of most of the drug regimens used for treating multidrug-resistant (MDR) TB (MDR-TB) or suspected MDR-TB (25, 32). To date, drug susceptibility testing (DST) for ETH relies mainly on phenotypic tests because the molecular mechanisms of ETH resistance are not fully understood. In vitro phenotypic investigation of ETH resistance is experimentally difficult and can yield discordant results according to the experimental method and resistance breakpoint values used, which still remain matters of debate (26). Therefore, a genotypic approach would be of great value to improve and hasten DST and the management of MDR-TB, as well as reduce the proportion of patients inadequately treated on the basis of erroneous DST results, consequently limiting the emergence of extensively drug-resistant tuberculosis among patients treated for MDR-TB.

ETH and INH share the same molecular target, the NADH-dependent enoyl-acyl carrier protein reductase InhA of the fatty acid biosynthesis type II system, which is involved in the synthesis of mycolic acids (2, 19). Consequently, cross-resistance to these two antibiotics can be observed in clinical isolates. However, though ETH is a structural analogue of INH, cross-resistance between ETH and INH does not occur systematically. Strains with low-level resistance to INH frequently display low-level ETH resistance, whereas high-level INH-resistant (INHr) strains typically remain ETH susceptible (ETHs) (6, 7, 21). This apparent paradox is due to the fact that INH and ETH are prodrugs that need to be activated by mycobacterial enzymes in order to exert their antimicrobial activity, with each drug being activated by a specific mechanism. INH is activated by the katG-encoded catalase-peroxidase (33), whereas ETH is activated by the ethA-encoded NADPH-specific FAD-containing mono-oxygenase (3, 10, 27). In addition, the expression of EthA is negatively regulated by EthR, a regulator that interacts directly with the ethA promoter region (11).

Resistance to ETH or INH has previously been reported to result primarily from (i) mutations altering the activator proteins KatG (14, 23) and EthA (3, 10, 21), leading to resistance to INH and ETH, respectively; (ii) mutations in the InhA protein targeted by INH and ETH, which prevents the corresponding activated forms of the drugs from binding the target, leading to cross-resistance to both antibiotics (2, 29); (iii) mutations in the inhA promoter region that cause overexpression of the target InhA and cross-resistance to the two drugs (3, 10, 16); and (iv) mutations in the negative transcriptional regulator EthR, specifically leading to ETH resistance (ETHr) (3). A higher prevalence of mutations in katG (associated with a high level of INH resistance) than in the inhA/inhA promoter region (associated with a low level of INH resistance and coresistance to ETH) accounts for the fact that cross-resistance to the two drugs is not systematic in clinical strains, making the use of ETH in cases of INH resistance possible (30). Finally, some INH- and ETH-resistant clinical isolates of Mycobacterium tuberculosis have been reported to have no mutations in the genes known to be involved in INH or ETH resistance (i.e., in katG, inhA, ethA, and ethR) (4, 5, 14, 21) but display mutations in other genes possibly involved in INH/ETH resistance, such as ndh, mshA, and dfrA. ndh encodes an NADH dehydrogenase that regulates the NADH/NAD+ ratio. Mutations in ndh would result in an increased intracellular NADH concentration and, concomitantly, coresistance to INH and ETH by competitively inhibiting the binding of INH-NAD and ETH-NAD adducts to InhA (20, 30). Such mutations have been demonstrated to cause INH and ETH resistance in Mycobacterium smegmatis (20) and Mycobacterium bovis BCG (30) but not yet in M. tuberculosis (the ndh mutations reported to date in M. tuberculosis have also been observed in INH-susceptible [INHs] isolates) (4, 9, 13, 18, 23). mshA encodes a glycosyltransferase, an enzyme involved in mycothiol biosynthesis, a major low-molecular-weight thiol in M. tuberculosis. Mycothiol would promote ETH activation via the mono-oxygenase EthA. Mutations in mshA have been proposed to cause a defect in ETH activation but have never been documented in clinical isolates and have been found only in in vitro-selected INH- and ETH-resistant M. tuberculosis mutants (28). Finally, dfrA encodes dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. Recently, DHFR was proposed to be a possible target of INH because one of the isonicotinoyl adducts generated by the activation of INH by KatG has been shown to inhibit the M. tuberculosis DHFR (1). However, the importance of dfrA in INH and ETH resistance is uncertain, as a recent investigation of dfrA from INHr clinical isolates found no dfrA mutation in 127 INHr strains (15).

Few systematic studies on the distribution of nucleotide changes affecting the above-mentioned genes and focused primarily on a limited number of genes have been conducted (4, 10, 13, 17, 21, 24). These studies also included a limited number of ETHr strains, usually less than 25 (4, 10, 13, 21, 24). In the present study, we investigated a set of 87 clinical isolates showing different patterns of susceptibility to ETH, including 47 exhibiting ETH resistance and 16 displaying ETH susceptibility with an intermediate proportion of resistant cells (ETHSip), for the presence of mutations in ethA, ethR, katG, inhA and the inhA promoter region, mshA, ndh, and dfrA.

(This study was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 12 to 15 September 2009.)

MATERIALS AND METHODS

Drug susceptibility testing and strains.

DST was performed on Lowenstein-Jensen medium using the standard proportions method (8) and concentrations of 20 mg/liter for ETH and 0.1, 0.2, 1, and 10 mg/liter for INH (31). ETHr was defined, as recommended, as ≥10% resistant mutants at 20 mg/liter (31). For the purpose of this study, we distinguished a subgroup of isolates exhibiting an abnormal proportion of ETH-resistant cells (≥1%) that was still too low (<10%) to meet the WHO definition of resistance. This particular pattern was referred to as “susceptible but intermediate proportion” (Sip), i.e., ≥1% but <10% resistant mutants. Resistance to INH was defined as ≥1% resistant mutants at 0.2 mg/liter. Two levels of resistance were distinguished: a low level of resistance (LL), defined as resistance to INH at concentrations of ≥0.2 mg/liter but <1 mg/liter, and a high level (HL) of resistance, defined as resistance to INH at concentrations of ≥1 mg/liter.

A total of 87 M. tuberculosis complex isolates from TB cases diagnosed in France and received at the French National Reference Center for Mycobacteria (mainly in the framework of MDR national surveillance) were collected over a 1-year period. These isolates corresponded to all of the INHr isolates (n = 63) received and a random selection of susceptible isolates (n = 24). On the basis of the results of DST, the 87 isolates were classified as ETHr (n = 47, all of which were resistant to INH and including 39 MDR isolates), ETHs (n = 24, 13 of which were resistant to INH and including 11 MDR isolates), and ETHSip (n = 16, all of which were resistant to INH and including 13 MDR isolates).

DNA sequencing of genes associated with drug resistance.

For all isolates, the entire ethA, ethR, katG, and inhA genes and the fabG1-inhA regulatory region (up to position −189 upstream from the fabG1 gene) were sequenced. For isolates displaying wild-type ethA/ethR, inhA, and inhA promoter, the entire ndh, dfrA, and mshA genes were sequenced. Genomic DNA was isolated from bacteria grown on Lowenstein-Jensen medium. A loop of culture was resuspended in water (500 μl) and killed by heating at 95°C for 15 min.

DNA (5 μl) obtained by heat shock extraction (1 min at 95°C and 1 min in ice, repeated five times) was submitted to PCR amplification using the following protocol: an initial denaturation step of 5 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at the primer-dependent annealing temperature (Ta), and 1 min at 72°C, ending with a final extension step of 7 min at 72°C. ethA, ethR and the intergenic region, dfrA, ndh, and mshA were amplified and sequenced using the primers in Table 1. After amplification, unincorporated nucleotides and primers were removed by filtration with Microcon 100 microconcentrators (Amicon Inc., Beverly, MA), and the amplicons were sequenced using a BigDye Terminator cycle sequencing ready kit (Applied Biosystems, Courtaboeuf, France).

TABLE 1.

Oligonucleotide primers used in PCR and DNA sequencing

Resistance gene Primer Sequence (5′ → 3′) Accession no. Hybridization temp (°C) PCR product size (bp) Reference or source
ethA (1,470 bp) ETHA1 ATC ATC GTC GTC TGA CTA TGG BX842584 64 1,278 21
ETHA9 CCT CGA GTA CGT CAA GAG CAC
ETHA10 CGT TGA CGG CCT CGA CAT TAC 64 342 21
ETHA8 GGT GGA ACC GGA TAT GCC TG
ethR (651 bp) ETHR1 CGC TGA CAC CGG AGA TTC C BX842584 59 915 This study
ETHR4 CGC TCC TAT ATG ACC GCA CG
ndh (1,392 bp) Ndh1s ACT GAG TAC CTG GCA GGC TG BX842578 57 1,479 This study
Ndh4as GCT AAC TGA ACT CGC TCA TCG
mshA (1,443 bp) mshA5s GCG TGT CAC TTC GGT TCC TGC BX842573 57 749 This study
mshA6as CAC CGG GAT GGA CCA CGT CG
mshA11s GGC CGC CGT GAA GAA CGC GG 62 1,020 This study
mshA12as CAG CTC GAC GAT CAG CGC GG
dfrA (480 bp) dfr2s CGC TTG CGG GGG ACG AAG C BX842580 61 480 This study
dfr4as GCA TCC GGC GAT CAA AGC TCC
katG (2,223 bp) katA CCC GAT AAC ACC TCC TG BX842578 59 817 5
katCas GTT TCG ACG TCG TTC ATG GC
kat2 CTC GGC GAT GAG CGT TAC AG 59 1,318 5
kat4as CCA GCG GTA AGC GCT TGT AG
katC CCG AGT ACA TGC TGC TCG AC 59 609 5
katEas GGT GAT CGC ACA TCC AGC AC
fabG1-inhA promoter Pro1 TCA ATA CAC CCG CAG CCA BX842576 53 493 5
Pro2 GTC ATC CGC ATG AGG AAT
inhA (810 bp) INHA1 AGG ACG CAC ATG ACA AGC BX842576 53 412 5
INHA2 TCA TGA TCG GCA GCA GCG
INHA3 CCA CAT CTC GGC GTA TTC 53 601 5
INHAD CGA AAT GCA GGT AGT GCT C
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Statistical analysis.

The 47 ETHr, 16 ETHSip, and 24 ETHs isolates were compared for mutations using Fisher's exact test. P values were two-tailed, and P values of ≤0.05 were considered significant.

Nucleotide sequence accession numbers.

The nucleotide sequences determined for the mutant genes were deposited in the GenBank database under the following accession numbers: HM587450 to HM587471 for the ethA mutants, HM587472 and HM587473 for the ethR mutants, and HM587474 and HM587475 for the mshA mutants.

RESULTS

Of the 47 ETHr/INHr isolates, 22 were mutated in ethA. The difference in the proportion of ETHr isolates with a mutation was significant compared to differences in proportions for the ETHs and ETHSip isolates (P = 2 × 10−10 and P = 0.009, respectively). Ten of the 22 ethA mutants had at least one missense mutation (n = 8 with single mutations, n = 2 with double mutations), 7 had an AMB mutation, and 5 had an insertion of one to three nucleotides in ethA. Nearly all of these mutations (16/17) represented new mutations in EthA (Table 2), and only 1 (S266R) had been previously reported (4). Twelve of these 22 isolates also possessed a mutation in the inhA promoter region, and 1 had both a mutation in the inhA promoter region and a new mutation in EthR (F110L). Seven isolates showed a remarkable combination of mutations: Q269AMB in EthA combined with −15C → T in the inhA promoter region and A110V in KatG.

TABLE 2.

Sequencing results for ethA, ethR, inhA and its promoter region, dfrA, ndh, mshA, and katG in 87 isolates of M. tuberculosis

Resistance phenotypea
 Sequencing resultb
 Resistance level (no. of isolates)
ETH INH EthAc EthR inhA promoter InhA DfrA Ndh MshA KatG
R R M1R (ATG → AGG) wt wt wt ND ND ND S315T HL (1)
G11A (GGC → GCC)-S266R (AGC → AGG) wt wt wt ND ND ND S315T HL (1)
T61M (ACG → ATG) wt wt wt ND ND ND wt LL (1)
Q165P (CAG → CCG) wt wt wt ND ND ND S315T HL (1)
CG insertion after 754 wt wt wt ND ND ND S315T HL (1)
L272P (CTA → CCA) wt wt wt ND ND ND S315T HL (1)
S329L (TCA → TTA)-C403G (TGT → GGT) wt wt wt ND ND ND S315T LL (1)
Y386C (TAC → TGC) wt wt wt ND ND ND S315T HL (1)
Y461H (TAC → CAC) wt wt wt ND ND ND S315T HL (1)
Q269AMB (CAG → TAG) wt −15C → T wt ND ND ND A110V LL (6), HL (1)
G124D (GGC → GAC) wt −15C → T, 102G → A wt ND ND ND wt HL (1)
TC insertion after 675 wt −15C → T wt ND ND ND S315T HL (1)
A insertion after 1391 wt −15C → T wt ND ND ND S315T HL (2)
GGC insertion after 59 wt −8T → C wt ND ND ND S315T HL (1)
H22P (CAC → CCC) F110Lc −15C → T wt ND ND ND S315T HL (1)
wt A95Tc −15C → T wt ND ND ND wt HL (1)
wt wt −15C → T wt ND ND ND S315T HL (6)
wt wt −15C → T wt ND ND ND wt LL (3)
wt wt −15C → T S94A ND ND ND wt LL (3)
wt wt wt S94A ND ND ND wt LL (2)
wt wt wt S94A ND ND ND D419H LL (1)
wt wt wt wt wt wt A187V-V171Gd S315T HL (1)
wt wt wt wt wt wt wt S315T HL (6)
wt wt wt wt wt wt wt F658V LL (1)
wt wt wt wt wt wt wt wt LL (1)
Sip R V7A (GTT → GCT)-S266R (AGC → AGG) wt wt wt ND ND ND S315T HL (1)
S266R (AGC → AGG) wt wt wt ND ND ND S315T HL (2)
G43V (GGC → GTC) wt wt wt ND ND ND S315T LL (1)
F66L (TTC → CTC) wt wt wt ND ND ND S315T LL (1)
L129R (CTC → CGC)-P192T (CCG → ACG) wt wt wt ND ND ND S315T HL (1)
G124D (GGC → GAC)-A199V (GCC → GTC) wt −102G → A wt ND ND ND S315T HL (1)
wt wt wt wt wt wt N111Sd S315T HL (1)
wt wt wt wt wt wt wt S315T HL (8)
S R wt wt wt wt wt wt wt S315T HL (9)
wt wt −47 G → C wt wt wt wt S315T HL (1)
wt wt wt wt wt wt wt R595OPA HL (1)
wt wt wt wt wt wt wt G494D HL (1)
wt wt wt wt wt wt wt E553K LL (1)
S S wt wt wt wt wt wt wt wt S (11)
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a

R, resistant; Sip, susceptible but intermediate proportion; S, susceptible.

b

Amino acid (one-letter code) mutations are generally given for all genes except the inhA promoter region, and the corresponding nucleotide changes are given in parentheses. The position of the nucleotide insertions in ethA is indicated. The new mutations found in EthA and EthR are underlined. wt, wild type; ND, not done.

c

New mutations: F110L (TTC → CTC) and A95T (GCC → ACC).

d

New mutations: A187V (GCA → GTA), V171G (GTG → GGG), and N111S (AAC → AGC).

The 25 remaining ETHr isolates had a wild-type ethA (Table 2). Among these isolates, nine displayed a mutation in the inhA promoter region only, whereas four others possessed the mutation plus another mutation in either inhA (S94A; n = 3) or EthR (new mutation A95T; n = 1). Three other isolates had a mutation (S94A) in inhA only. Finally, the nine remaining ETHr isolates with a wild-type ethA had no mutation in EthR, InhA, or the inhA promoter. dfrA, ndh, and mshA were investigated in the nine isolates, and only one was found to have a mutation in one of these genes (double mutation V171G and A187V in mshA). Finally, investigating the mechanisms of INH resistance in the 47 ETHr isolates showed the presence of KatG mutations in 35 isolates (S315T in 26, A110V in 7, D419H in 1, and F658V in 1), the inhA promoter in 26 isolates, and/or InhA (S94A) in 6 isolates (see Table 2 for the details regarding mutation combinations).

The 16 ETHSip isolates showed fewer mutations than the ETHr isolates (P = 0.009) and more mutations than the ETHs isolates (P = 0.004). Among the 16 ETHSip isolates, 7 were mutated in ethA (44%), of which 3 had double mutations: V7A and S266R, L129R and P192T, or G124D and A199V. The isolate with the last mutation also had a −102G → A mutation in the inhA promoter region (Table 2). Most of these mutations (7/8) were new (Table 2). Mutations in codon 43 were also described in other studies: G43C by DeBarber et al. (10) and G43S by Morlock et al. (21). The other nine ETHSip isolates had no mutations in ethA or ethR, inhA, or the inhA promoter. One of these nine isolates had an N111S mutation in mshA, whereas the remaining eight had wild-type dfrA, ndh, and mshA sequences. Resistance to INH in the 16 ETHs isolates was accounted for by mutations in KatG (S315T).

Finally, among the 24 ETHs isolates, 23 had no mutations in ethA/ethR, inhA/inhA promoter region, dfrA, ndh, or mshA, whereas a single isolate displayed a rare mutation (−47G → C) with an unknown effect in the inhA promoter region. Resistance to INH in the 13 ETHs isolates displaying INH resistance was accounted for by mutations in KatG: S315T (n = 10), R595OPA (n = 1), G494D (n = 1), or E553K (n = 1). No KatG mutation was found in the 11 ETHs/INHs isolates.

Overall, the analysis of the distribution of mutations in our collection of clinical isolates indicated that 81% had at least one mutation in one of the major genes involved in ETH resistance, and these were distributed as follows: 39% in inhA or the inhA promoter alone, 24% in ethA alone, 31% in the inhA promoter and ethA, 3% in the inhA promoter and ethR, and 3% in the inhA promoter, inhA, and ethR.

DISCUSSION

By studying a large number of clinical isolates, the four major genes clearly involved in ETH resistance (ethA/ethR, inhA/inhA promoter), and three genes proposed to be involved in this resistance (mshA, ndh, dfrA), we have highlighted the most relevant genetic patterns involved in ETH resistance in both ETHr and ETHSip isolates. In 81% (38/47) of the ETHr clinical M. tuberculosis isolates, ETH resistance could be attributed to mutations in at least one of the four major genes or regions known to be involved in ETH resistance (ethA, ethR, inhA, or the inhA promoter region). More specifically, 47% (22/47) of the ETHr isolates had mutations in ethA, a finding that agrees with reports in three other publications that indicated the proportion of genes with mutations to range from 37% (15/41) to 100% (11/11) (4, 10, 21), with the overall proportion being 51% (39/76) when the results of the three studies are combined. In these previous publications, the mutations in ethA were nonsynonymous, affecting 22 distinct codons, and the other mutations were either deletions (n = 8) or insertions (n = 2). In our study, the alterations detected in ethA were also scattered throughout the gene and consisted of missense mutations, nonsense mutations, and insertions. Most of the mutations identified in our study were new and affected codons different from those cited in the three previous reports. Overall, these data clearly confirm a high degree of diversity in the mutations affecting ethA, as previously observed in other mycobacterial resistance genes, such as katG and pncA, in which hundreds of distinct mutations have been described thus far (5, 22). However, the S315T mutation is by far the most frequent KatG mutation observed in INHr clinical isolates, which is in contrast to the lack of a prominent mutation in EthA. The predominance of the codon 315 mutation in KatG has been explained by the need for M. tuberculosis to maintain sufficient catalase-peroxidase activity to protect against oxidizing species produced by neutrophils of the infected host. No predominant ethA mutations have emerged in the ETHr clinical isolates investigated thus far, suggesting the existence of another enzyme with redundant functions. The genome of M. tuberculosis possibly encodes more than 30 mono-oxygenases (3), and the function of EthA, as opposed to that of KatG, does not seem to be essential in M. tuberculosis.

In the present study, we found two ETHr isolates with two different mutations in EthR, F110L and A95T. These two mutations are, to the best of our knowledge, the first to be described in ethR in relation to ETH resistance. The crystal structure of EthR, which negatively regulates the expression of EthA by interacting directly with the ethA promoter region, was previously reported to be a homodimer containing a classical DNA binding domain (helix-turn-helix motif) and ligand-binding site made of two helices (α4 to α9) which is required to control the conformational change that prevents the binding of EthR to its target DNA (11, 12). On the basis of this crystal structure, F110 is localized in helix α5, with its aromatic side chain oriented in the hydrophobic tunnel constituting the ligand-binding domain, and A95 is located quite far from the pocket where the ligand interaction occurs but is in the vicinity of helices α4 and α5, which contribute to the ligand-binding domain.

Notably, 62% (29/47) of ETHr isolates had mutations in inhA (n = 6) or the inhA promoter region (n = 26), which is in agreement with the results in other publications that reported that the proportions of strains carrying mutations in inhA or the inhA promoter ranged from 25 to 100% in ETHr strains. The overall proportion is 68% (74/108) when the results from these publications are combined (4, 13, 17, 21, 24). These data confirm the significance of this mechanism in the structure or level of expression of the target InhA and in the development of resistance to ETH in clinical M. tuberculosis isolates. In our study, this mechanism is associated with mutations in EthA/EthR in a proportion of isolates equal to the proportion of isolates having mutations in inhA or the inhA promoter region without mutation in EthA/EthR.

In the two previous reports that tested inhA, the inhA promoter, and ethA, the proportions of strains with no mutation in these three genes were 21% (5/24) (4) and 7% (3/41) (21), which are close to the figures found in our study (9/47, 19%). We further investigated mshA, ndh, and dfrA in the nine ETHr isolates lacking a mutation in the three main resistance genes. One double mutation (V171G-A187V) was detected in mshA in a single isolate; the other isolates had no mutation. The significance of the presence of this double mutation remains unclear because the in vivo selection of ETHr/INHr mshA mutants has been reported in only a single study (28) prior to the present study. For ndh, two reports have shown that ndh mutations in M. smegmatis and M. bovis BCG can mediate coresistance to ETH and INH (20, 30), but the impact of ndh mutations in M. tuberculosis is still unknown. The product of dfrA, DHFR, was proposed to be a new target for INH (1), but the consequences of dfrA mutations in clinical strains of M. tuberculosis are unknown. Taken together, the data suggest that the role played by mshA, ndh, and dfrA in ETH resistance in clinical strains of M. tuberculosis is unclear, and further studies are needed to investigate this role. ETH resistance likely stems from mechanisms that remain to be discovered, highlighting the complexity of the mechanisms underlying the resistance.

The critical concentrations and proportions chosen for DST were based on clinical failure; thus, strains with more than 10% resistant mutants were undoubtedly clinically resistant (8). Currently, strains with a proportion of resistant mutants of less than 10% are considered susceptible by WHO standards. Nevertheless, we extended our study to isolates with a high enough proportion (∼1%) of ETH-resistant cells to be considered abnormal but not high enough (<10%) to be considered resistant according to WHO recommendations. Interestingly, 7 of the 16 ETHSip isolates displaying this pattern had ethA mutations known to be implicated in ETHr; these corresponded to new mutations in 6 isolates, and 1 isolate had a mutation in mshA. Even though the proportion of isolates without a mutation was higher among ETHSip (8/16) than ETHr (8/47) isolates (P = 0.009), the fact that isolates with >1% and <10% resistant mutants have mutations more often than fully susceptible isolates (P = 0.004) suggests that they should be regarded as isolates potentially resistant to ETH and might define a new clinically significant resistance category. To date, the clinical significance of these mutants remains unknown, but the existence of this class of isolates points to the potential danger of using a single cutoff for defining ETH resistance in phenotypic resistance assays.

Finally, among the 24 ETHs isolates, only 1 harbored a mutation in the major genes known to be linked to ETH resistance. The consequence of the mutation (−47G → C) in the inhA promoter is unknown (10, 13, 24).

Because the rapid determination of drug resistance is an important prerequisite for the initiation of effective chemotherapy and preventing the acquisition of additional resistance traits, the fast prediction of resistance to bactericidal second-line drugs using DNA sequencing is of great interest, particularly in the context of MDR-TB. In the present study, mutations in ethA, ethR, inhA, or the inhA promoter region accounted for ETH resistance in 81% of the isolates. Consequently, finding such mutations by DNA sequencing or other molecular approaches can be expected to be a strong indicator of ETH resistance. However, the absence of such mutations should not be interpreted as indicating that a strain, particularly an MDR strain, is susceptible to ETH. Moreover, as long as other mechanisms of ETH resistance remain to be discovered, searching the strains for mutations in dfrA, ndh, and mshA will likely add limited value to the genetic detection of ETH resistance.

Acknowledgments

This work was supported by a grant from the Ministère de la Recherche (program grant UPRES EA 1541).

Footnotes

▿

Published ahead of print on 25 October 2010.

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