Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates Johana Rueda,a,b Teresa Realpe,a,b Gloria Isabel Mejia,b Elsa Zapata,b Juan Carlos Rozo,c Beatriz Eugenia Ferro,c,d Jaime Robledoa,b Escuela de Ciencias de la Salud, Universidad Pontificia Bolivariana (UPB), Medellín, Colombiaa; Unidad de Bacteriología y Micobacterias, Corporación para Investigaciones Biológicas (CIB), Medellín, Colombiab; Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombiac; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, Netherlandsd
Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n ⴝ 57 ETH-resistant MDR-TB isolates; n ⴝ 3 ETH-susceptible MDR-TB isolates; and n ⴝ 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.
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thionamide (ETH), a structural analog of isoniazid (INH), is a second-line drug used in the treatment of multidrug-resistant tuberculosis (MDR-TB) (1). Both ETH and INH are classified as prodrugs that are activated by different mycobacterial enzymes. INH is activated by the katG-encoded catalase-peroxidase, and ETH is activated by the ethA-encoded monooxygenase (2, 3). The activated INH and ETH drugs share the same molecular target, i.e., the NADH-dependent enoyl-acyl carrier protein reductase InhA, which is involved in the long-chain mycolic acid biosynthesis pathway (4). Therefore, the cross-resistance between INH and ETH can be detected in Mycobacterium tuberculosis clinical isolates in the case of mutations affecting the common target, which may occur when patients have previously been treated with INH and not with ETH (5). The frequency of cross-resistance differs between countries: 100% in Korea (6), 95.12% in Argentina (7), 94% in Brazil (8), 62% in France (9), and 13.8% in Thailand (10). Resistance to INH and ETH is mainly due to the chromosomal mutations. The mutation-carrying genes, such as those encoding the enzymes KatG (11, 12) and EthA (13, 14), are associated with individual resistance to INH and ETH, respectively. Mutations at the inhA promoter region or inhA gene result in the overexpression or modification of the InhA target, causing cross-resistance to INH and ETH (8, 15). The ndh gene encodes NADH dehydrogenase, which regulates the NADH/NAD⫹ ratio. Mutations in ndh would result in an increased intracellular NADH concentration, leading to the cross-resistance to both the drugs (16, 17). Mutations in ethR, a negative transcriptional regulator of the expression of EthA, lead to ETH resistance (9, 18). Finally, mshA gene mutations that encode a glycosyl-transferase enzyme involved in mycothiol biosynthesis have been proposed to cause a defect in ETH
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activation (19, 20). ETH resistance is currently detected by the phenotypic susceptibility tests due to the partially known molecular resistance mechanisms (21). The automated system BD Bactec MGIT 960 (Becton, Dickinson, and Company, Sparks, MD, USA) is the current reference standard for drug susceptibility testing with first- and second-line drugs (22). However, this test is not currently recommended for ETH because of its poor reliability and reproducibility (23). The frequency of resistance to ETH may vary according to the geographical location: 56.3% in Kenya (24), 52.5% in India (25), 31.3% in Europe (26), 11.1% in Colombia (27), and 10.8% in Mexico (28). Therefore, it is important to identify the genes related to the resistance to ETH for defining the real usefulness of this drug in the second-line treatment regimens for patients with MDR-TB. The present study aimed to identify mutations in katG, ethA, ethR, mshA, ndh, and inhA genes and in the inhA promoter associated with independent resistance or
Received 29 April 2015 Returned for modification 27 May 2015 Accepted 7 September 2015 Accepted manuscript posted online 14 September 2015 Citation Rueda J, Realpe T, Mejia GI, Zapata E, Rozo JC, Ferro BE, Robledo J. 2015. Genotypic analysis of genes associated with independent resistance and cross-resistance to isoniazid and ethionamide in Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother 59:7805–7810. doi:10.1128/AAC.01028-15. Address correspondence to Johana Rueda, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01028-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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cross-resistance to INH and ETH in M. tuberculosis clinical isolates from two major Colombian cities. Clinical isolates. A total of 64 M. tuberculosis clinical isolates from individual patients were obtained from two Colombian cities. Of them, 57 isolates were ETH-resistant MDR-TB strains, three were ETH-susceptible MDR-TB strains, and four were fully susceptible strains. The ETH-resistant MDR-TB isolates were collected from the Unidad de Bacteriología y Micobacterias, Corporación para Investigaciones Biológicas (CIB), in the city of Medellín (n ⫽ 30), and from the Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), in the city of Cali (n ⫽ 27). All of the isolates were collected between 2000 and 2013 and stored at ⫺70°C until tested for this study. All of the available MDR-TB isolates that were resistant to ETH were previously tested using the agar proportion method (29). PCR amplification and sequencing. M. tuberculosis isolates were cultured on Middlebrook 7H11 for 3 weeks. Total genomic DNA was isolated using the cetyltrimethylammonium bromide (CTAB)/NaCl method (30) and quantified with a NanoDrop ND2000 spectrophotometer (Thermo Scientific) by measuring the absorption at 260 nm. Then, 10 l of DNA at a concentration of 10 ng/l were submitted was subjected to PCR using the following amplification protocol. An initial denaturation step of 3 min at 95°C was performed, followed by 40 cycles of 30 s at 95°C, 30 s at 57°C, and 2 min at 72°C, ending with a final extension step of 5 min at 72°C. All of the genes were amplified and sequenced using the primers shown in Table S1 in the supplemental material. The amplicons were sequenced using the Sanger method (Macrogen, Seoul, Republic of Korea). For all of the isolates, the entire ethA, ethR, mshA, katG, ndh, and inhA genes and the fabG1-inhA regulatory region (up to position ⫺200 upstream from the fabG1 gene) were sequenced. Sequence analysis. Chromatograms were analyzed and edited using the program FinchTV v1.4 (http://www.geospiza.com /Products/finchtv.shtml), and consensus sequences were edited by using Clone Manager v9.0 (Sdi-Ed Software, NC, USA). The nucleotide sequence alignments were performed using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and protein translation sequences were performed using the ExPASy program (http://expasy.org/tools/dna.html). Susceptibility testing. Each of the 64 M. tuberculosis isolates was tested for susceptibility to first- and second-line drugs using the agar proportion method (29). In addition, the 30 isolates collected from Medellín were tested using MICs to INH and ETH with the automated system BD Bactec MGIT 960 (Becton, Dickinson, and Company, Sparks, MD, USA) according to the manufacturer’s instructions (31, 32). The drugs were used at different concentrations, such as 0.1, 0.5, 1, and 5 g/ml for INH (SigmaAldrich, St. Louis, MO, USA), and 2.5, 5, 7.5, 10, 25, 50, and 100 g/ml for ETH (Sigma-Aldrich, St. Louis, MO, USA). High levels of resistance to INH and ETH were defined as a MIC of ⱖ1 g/ml and of ⱖ25 g/ml, respectively (8). The M. tuberculosis H37Rv strain was used as a susceptible control. Statistical analysis. The Epidat software, version 3.1 (Xunta de Galicia, Santiago de Compostela, Spain, and Pan American Health Organization), was used for the calculation of the Cohen kappa index between the results of susceptibility testing and sequencing. This study was approved by the CIB and CIDEIM ethic committees. DNA sequencing of ethA, mshA, ethR, katG, ndh, and inhA and its promoter. Of the 57 ETH-resistant MDR-TB isolates, 41
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showed mutations in ethA: nonsynonymous mutations were identified in 32 (78.0%) isolates, four isolates (9.7%) had insertions (one to three nucleotides), and five isolates (12.1%) had deletions in ethA. Seventeen of these isolates also showed mutations in mshA; mutations in 12 of the isolates were in the N111S codon, mutations in three isolates were in the A187V codon, mutations in one isolate were in the N111S and I460R codons, and mutations in one isolate were in the S352F codon. A synonymous mutation in ethR was detected in only one isolate. The remaining 16 ETH-resistant MDR-TB isolates were wild-type for ethA. However, nine of them had the mutation in the N111S codon of mshA. Of the seven ETH-susceptible isolates, three had mutations in mshA (N111S), and one had mutations in ethA (K448E). All MDR-TB isolates showed mutations at katG in the codons for S315T (n ⫽ 47 isolates), T275A and S315T (n ⫽ 5 isolates), S315T and R463L (n ⫽ 3 isolates), V442G (n ⫽ 1 isolate), I248M and S315T (n ⫽ 1 isolate), S315G and R463L (n ⫽ 1 isolate), W397 and S652A (n ⫽ 1 isolate), and G206D and S315T (n ⫽ 1 isolate). No mutations in katG were detected in the INH-susceptible isolates. Cross-resistance was detected in 19 out of 57 (33.3%) ETHresistant MDR-TB isolates by mutations within the inhA promoter region and/or the inhA gene at positions ⫺15C¡T (n ⫽ 11 isolates), ⫺8T¡G (n ⫽ 3 isolates), ⫺8C¡ T (n ⫽ 1 isolate), ⫺8T¡A (n ⫽ 1 isolate), ⫺17G¡T (n ⫽ 1 isolate), ⫺15C¡T and I194T codon (n ⫽ 1 isolate), or S94A codon (n ⫽ 1 isolate). Two isolates also had mutations in the ndh gene (V18A codon). The remaining 38 ETH-resistant MDR-TB isolates did not show mutations in inhA or its promoter. However, three of these isolates had mutations in ndh. One of the three ETH-susceptible MDR-TB isolates showed a mutation in inhA (S94A). No mutations in the inhA promoter or in ndh were detected in the four INH and ETH susceptible isolates. Descriptions of mutations identified in this study are provided in Table 1. Agreement between the susceptibility testing and sequencing results. Agreement between the phenotypic and genotypic tests for the detection of resistance to INH and ETH was found to be 100% (index kappa ⫽ 1) and 90% (index kappa ⫽ 0.45; 95% confidence interval, 0.08 to 0.82), respectively. However, there was a disagreement among the four isolates, which were confirmed to be susceptible to ETH by the phenotypic susceptibility tests. These isolates exhibited mutations in the mshA N111S codon (n ⫽ 3 isolates) and ethA K448E codon (n ⫽ 1 isolate); mutations were confirmed by sequencing. Of the 57 ETH-resistant isolates by the phenotypic susceptibility test, two isolates showed the absence of mutations in the genes studied. INH and ETH MICs. MIC values for INH and ETH in M. tuberculosis isolates are summarized in Table 2. High-level resistance to INH (MIC ⱖ1.0 g/ml) and to ETH (MIC ⱖ25 g/ml) was detected in 20 out of 30 ETH-resistant MDR-TB isolates. Nine isolates showed high-level resistance to INH (MIC ⱖ1.0 g/ml) and low-level resistance to ETH (MIC between 2.5 and 10 g/ml), and one isolate showed low-level resistance to INH (MIC between 0.1 and 0.5 g/ml) and high-level resistance to ETH (MIC ⱖ25 g/ml). Relationship between the INH and ETH MICs and sequencing results. Of the 29 ETH-resistant MDR-TB isolates with highlevel resistance to INH, 14 (48.3%) had mutations in two or more INH resistance-related genes, and 15 isolates had mutations in katG. One isolate with low-level resistance to INH had double
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Genes Associated with Resistance to Ethionamide
TABLE 1 Mutationsa in katG, ethA, mshA, inhA promoter region, inhA, and ndh in 64 isolates of M. tuberculosis Resistance phenotypeb INH
ETH
R
R
No. (%) of isolates
Resistance genotypec inhA Promoter
inhA
ndh
WT N111S WT N111S, I460R (ATA¡AGA1379) WT WT A187V N111S N111S WT WT WT A187V N111S WT
WT WT WT WT
WT WT WT WT
WT WT WT WT
WT WT WT WT WT WT WT WT WT WT WT
WT WT WT WT WT WT WT WT WT WT WT
WT ⫺ 15C¡T ⫺ 8T¡G ⫺ 8T¡C ⫺ 8T¡G ⫺ 8T¡A ⫺ 15C¡T ⫺ 15C¡T
WT WT WT WT WT WT WT WT
C deleted at 369 WT
WT WT N111S WT N111S WT WT S352F (TCC¡TTC1055) WT N111S
WT WT WT WT WT WT WT WT WT WT N316K (AAC¡AAA948) V18A V18A WT WT WT WT WT WT
⫺ 15C¡T ⫺ 15C¡T
WT WT
WT WT
WT WT WT WT WT
N111S WT WT WT WT
⫺ 15C¡T ⫺ 15C¡T ⫺ 15C¡T ⫺ 17G¡T ⫺ 15C¡T
WT WT WT WT I194T
WT WT WT WT WT
WT WT
N111S WT
WT WT
S94A WT
S315T G206D, S315T S315T
WT WT WT
N111S N111S WT
WT WT WT
WT WT WT
WT A226E (GCA¡GAA677) WT WT WT
katG
ethA
mshA
L301R (CTG ¡CGG902) E36Q (GAA¡CAA106)d E36Q (GAA¡CAA106) E36Q (GAA¡CAA106)
3 (5.27) 2 (3.51) 2 (3.51) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75)
T275A, S315T S315T S315T S315T, I248 M (ATT¡ATG744) S315T S315T S315T, R463L S315T S315T S315T S315T S315T S315T, R463L S315T S315T
1 (1.75) 2 (3.51) 2 (3.51) 1 (1.75) 1 (1.75) 1 (1.75) 2 (3.51) 2 (3.51)
S315T S315T S315T S315T S315T S315T S315T S315T
M409I (ATG¡ATC1227) T340N (ACC¡AAC 1019) GGG insertion at 1141 G deleted at 32 C deleted at 965 N379Y (AAC¡TAC 1135) L374R (CTT¡CGT1121) F157L (TTC¡CTC469)
1 (1.75) 1 (1.75)
1 (1.75) 1 (1.75)
S315G, R463L V442G (GTC¡GCC1325) S315T S315T S315T S315T W397Y, S652A (TCA¡GCA1954) S315T S315T
5 (8.8) 1 (1.75) 2 (3.51)
5 (8.8) 5 (8.8) 2 (3.51) 1 (1.75)
1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75)
T44A (ACC¡GCC130) Y143STOP (TAC¡TAA429) C137R (TGC ¡CGC409) C403W (TGT¡TGG1209) F302S (TTC¡TCC905) C137F (TGC ¡TTC410) H281R (CAC¡CGC 842) G deleted at 32 C deleted at 1265 Insertion GC at 755 Insertion G at 672
R
S
1 (33.33) 1 (33.33) 1 (33.33)
S315T S315T S315T
WT K448E (AAG¡GAG1342) WT
N111S WT WT
WT WT WT
S94A WT WT
WT WT WT
S
S
2 (50) 2 (50)
WT WT
WT WT
WT N111S
WT WT
WT WT
WT WT
a Amino acid (one-letter code) mutations are generally given for all genes except the inhA promoter region. The position of the nucleotide insertion or deletion in ethA is indicated. The mutations not previously reported in the literature found in katG, ethA, mshA and ndh genes are underlined, and the corresponding nucleotide changes are given in parentheses. b R, resistant. S, susceptible. c WT, wild type. d One of these five isolates had a synonymous mutation (ACT¡ACG 447) in the ethR gene.
mutations: ⫺15C¡T at the inhA promoter and V442G codon in katG (Table 3). A total of 18 (86%) of the 21 ETH-resistant MDR-TB isolates with high-level resistance to ETH had mutations in two or more
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genes related to resistance to ETH. Three isolates showed a mutation in a single gene related to resistance to ETH. Additionally, five (55.5%) of the nine isolates with low-level resistance to ETH had mutations in a single gene, and the remaining four isolates showed
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TABLE 2 INH and ETH MICs in 30 M. tuberculosis isolates for the automated system BD Bactec MGIT 960 Resistance phenotypea No. (%) of INH ETH isolates R
R
2 (6.6) 3 (10) 2 (6.6) 2 (6.6) 3 (10) 3 (10) 5 (16.7) 2 (6.6) 2 (6.6) 5 (16.7) 1 (3.4)
MICs (g/ml)b INH (0.1 g/ml c) 0.1 0.5 1 5
ETH (2.5 g/mlc)
⬎5.0 2.5 5 7.5 10 25 50 100 ⬎100 X
X X
X X X
X X X
X X
X X
X X X X X
X X X X
a
R, resistant. b X, the lowest concentration at which INH and ETH inhibit the growth of M. tuberculosis. c Critical concentration for drugs evaluated by the automated system BD Bactec MGIT 960.
mutations in two or more genes related to resistance to ETH (Table 4). ETH is a second-line drug that shares the same mechanism of action as INH and is considered one of the several useful drugs for the treatment of patients who present with MDR-TB (8). Accordingly, it is important to know the frequency of mutations associated with independent resistance and cross-resistance to INH and ETH in clinical isolates of MDR-TB, in order to define the efficacy of ETH in the recommended treatment regimens. In this study, 72% (41 out of 57) of the ETH-resistant MDR-TB isolates evaluated had mutations in ethA, which have been associated with ETH resistance. In addition, previous studies have reported about 54.2% to 100% of MDR-TB isolates with mutations on this gene, including nonsynonymous mutations, deletions, and insertions (10, 13). In our study, 90% of the mutations detected in ethA (18 of 20 mutations) have not been previously reported. These results indicate that ethA mutations are distributed across the structural gene, with no single predominating nucleotide or codon. This may suggest the existence of one or more enzymes with functional redundancy to EthA. In fact, the genome of M. tuberculosis probably encodes more than 30 monooxygenases (18). Nevertheless, more studies are required to completely explain the influence of the diverse mutations found in ethA gene and ETH resistance. In the present study, 95% of the INH-resistant isolates had mutations at codon 315 of the katG gene. The most frequent was the transitional mutation G¡C (AGC¡ACC), resulting in the substitution of serine by threonine (S¡T) in the amino acid chain. The frequency of mutations in codon 315 in the katG gene was found to differ within the geographical regions: 97% in South Africa (33), 88% in Colombia (34), 60 to 87% in Brazil (35), 46% in Spain (36), 28% in Japan (37), 7% in Finland (33), and 4% in
South Korea (38). Other studies have demonstrated that the majority of INH-resistant isolates with mutations at codon 315 retained their catalase-peroxidase activity while showing a lowered ability to activate INH. This secures a sufficient level of oxidative protection that may preserve its characteristics of virulence and transmissibility (39). The katG mutations in the codons for G206D, I248M, V442G, and S652A found in this study have not been previously reported. The frequency of inhA promoter region or inhA gene mutations was found to differ within the geographical regions but represented at least 13.8% (10) and up to 100% (6) of ETH- and INH-resistant clinical isolates. Our study confirmed mutations in these genes in 33.3% (19 of 57) of ETH-resistant MDR-TB isolates. The most commonly found mutation, ⫺15C¡T in the inhA promoter, occurred in 66.6% (12 of 18) of the isolates. This mutation is associated with a 20-fold increase in inhA mRNA levels, resulting in the overexpression of InhA, which leads to a titration of INH or ETH and a consequent increase in INH and ETH MICs in M. tuberculosis (15). Mutations in the ndh gene (R13C, V18A, T110A, R268H, and G313R codons) have been described in M. tuberculosis clinical isolates (4). In our study, we observed three mutations in this gene (V18A, A226E, and N316K codons). Mutations in V18A have been previously described in INH-susceptible and -resistant isolates (17), but the contribution of this mutation in the INH-resistant phenotype was unknown, whereas mutations in A226E and N316K have not been previously reported in M. tuberculosis or in Mycobacterium smegmatis. Therefore, the contribution of these mutations in INH and ETH resistance is uncertain. Future research should be directed toward determining the role and prevalence of mutations in ndh in INH- and ETH-resistant M. tuberculosis isolates and confirming its contribution in cross-resistance mechanisms as reported in M. smegmatis (40) and Mycobacterium bovis (16). Mutations in mshA (N111S, I460R, S352F, and A187V codons) were detected in 45.6% (26 of 57) of ETH-resistant MDR-TB isolates studied. However, the N111S mutation was also detected in three ETH-susceptible M. tuberculosis isolates. The N111S mutation has been identified in the M. tuberculosis Erdman strain belonging to the Haarlem genotype, which is susceptible to both INH and ETH. This suggested that this mutation was neutral and did not confer resistance to ETH (41). In this study, mutations in ethA, mshA, ndh, inhA, or the inhA promoter region were associated with ETH resistance in 96.5% of the phenotypically resistant isolates. This suggested that mutations in these genes might function as markers for the detection of ETH resistance. However, it is important to note that the absence of such mutations in 3.5% of the isolates may indicate the presence of other undiscovered mechanisms of ETH resistance. Numerous studies have shown that mutations in codon 315 of
TABLE 3 Relationship between low and high levels of resistance to INH and mutations in katG, ndh, and inhA genes and inhA promoter region in 30 ETH-resistant MDR-TB isolates Resistance
katG
katG and ndh
katG and inhA promoter
katG and inhA
katG, inhA, and inhA promoter
No. (%) of isolates
High level of resistance to INH, ⬎1 g/ml Low level of resistance to INH, 0.1–0.5 g/ml
15 0
2 0
10 1a
1 0
1 0
29 (97) 1 (3)
a
Mutations in katG codon encoding V442G and in the inhA promoter region encoding C-15T.
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Genes Associated with Resistance to Ethionamide
TABLE 4 Relationship between low and high levels of resistance to ETH and mutations in ethA, mshA, ndh, and inhA genes and inhA promoter region in 30 ETH-resistant MDR-TB isolates
Resistance
ethA, mshA, inhA ethA and ethA and ethA and inhA mshA and inhA inhA and inhA mshA and and inhA No. (%) ethA mshA promoter mshA ndh promoter promoter promoter inhA promoter of isolates
High level of resistance 1 to ETH ⱖ25 g/ml Low level of resistance 5 to ETH ⱖ2.5–10 g/ml a
1
1
7
1
3
2
1
0
4
21 (70)
0
0
1a
1
1
0
0
1
0
9 (30)
Synonymous mutation (ACT¡ACG 447) in the ethR gene.
katG are associated with high-level resistance to INH (35, 36); this is consistent with our results, since 100% of the tested MDR-TB isolates from Medellín with mutations in the 315 codon had INH MICs between 0.5 and 5 g/ml. According to these results, the treatment of MDR-TB patients with high doses of INH may not be effective (42). In 18 out of 21 (86%) isolates from Medellín, the high-level resistance to ETH was associated with mutations in two or more genes. A similar situation has been described in isolates with high-level resistance to ethambutol, in which resistance has been described as a multistep process (43). More studies are required in order to understand the contribution of multiple mutations to confering high-level resistance to ETH and, ultimately, to know the clinical value that this finding may have in the treatment of patients with MDR-TB. In conclusion, mutations in genes (katG, ethA, and inhA and its promoter) were associated with resistance to INH and/or ETH in the present study. Special attention must be given to the second-line therapeutic drugs, including ETH, due to the frequency of cross-resistance between INH and ETH by mutations in inhA or its promoter that were detected in 33.3% of M. tuberculosis isolates from Colombia. Future studies should aim toward determining the role of additional genes, such as ndh and mshA, and their relationships to resistance to these two drugs. ACKNOWLEDGMENTS This work was supported by Departamento Administrativo Colombiano de Ciencia, Tecnología e Innovación (Colciencias) 221356933562. We declare no conflicts of interest.
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Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n ⴝ 57 ETH-resistant MDR-TB isolates; n ⴝ 3 ETH-susceptible MDR-TB isolates; and n ⴝ 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.
E
thionamide (ETH), a structural analog of isoniazid (INH), is a second-line drug used in the treatment of multidrug-resistant tuberculosis (MDR-TB) (1). Both ETH and INH are classified as prodrugs that are activated by different mycobacterial enzymes. INH is activated by the katG-encoded catalase-peroxidase, and ETH is activated by the ethA-encoded monooxygenase (2, 3). The activated INH and ETH drugs share the same molecular target, i.e., the NADH-dependent enoyl-acyl carrier protein reductase InhA, which is involved in the long-chain mycolic acid biosynthesis pathway (4). Therefore, the cross-resistance between INH and ETH can be detected in Mycobacterium tuberculosis clinical isolates in the case of mutations affecting the common target, which may occur when patients have previously been treated with INH and not with ETH (5). The frequency of cross-resistance differs between countries: 100% in Korea (6), 95.12% in Argentina (7), 94% in Brazil (8), 62% in France (9), and 13.8% in Thailand (10). Resistance to INH and ETH is mainly due to the chromosomal mutations. The mutation-carrying genes, such as those encoding the enzymes KatG (11, 12) and EthA (13, 14), are associated with individual resistance to INH and ETH, respectively. Mutations at the inhA promoter region or inhA gene result in the overexpression or modification of the InhA target, causing cross-resistance to INH and ETH (8, 15). The ndh gene encodes NADH dehydrogenase, which regulates the NADH/NAD⫹ ratio. Mutations in ndh would result in an increased intracellular NADH concentration, leading to the cross-resistance to both the drugs (16, 17). Mutations in ethR, a negative transcriptional regulator of the expression of EthA, lead to ETH resistance (9, 18). Finally, mshA gene mutations that encode a glycosyl-transferase enzyme involved in mycothiol biosynthesis have been proposed to cause a defect in ETH
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activation (19, 20). ETH resistance is currently detected by the phenotypic susceptibility tests due to the partially known molecular resistance mechanisms (21). The automated system BD Bactec MGIT 960 (Becton, Dickinson, and Company, Sparks, MD, USA) is the current reference standard for drug susceptibility testing with first- and second-line drugs (22). However, this test is not currently recommended for ETH because of its poor reliability and reproducibility (23). The frequency of resistance to ETH may vary according to the geographical location: 56.3% in Kenya (24), 52.5% in India (25), 31.3% in Europe (26), 11.1% in Colombia (27), and 10.8% in Mexico (28). Therefore, it is important to identify the genes related to the resistance to ETH for defining the real usefulness of this drug in the second-line treatment regimens for patients with MDR-TB. The present study aimed to identify mutations in katG, ethA, ethR, mshA, ndh, and inhA genes and in the inhA promoter associated with independent resistance or
Received 29 April 2015 Returned for modification 27 May 2015 Accepted 7 September 2015 Accepted manuscript posted online 14 September 2015 Citation Rueda J, Realpe T, Mejia GI, Zapata E, Rozo JC, Ferro BE, Robledo J. 2015. Genotypic analysis of genes associated with independent resistance and cross-resistance to isoniazid and ethionamide in Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother 59:7805–7810. doi:10.1128/AAC.01028-15. Address correspondence to Johana Rueda, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01028-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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cross-resistance to INH and ETH in M. tuberculosis clinical isolates from two major Colombian cities. Clinical isolates. A total of 64 M. tuberculosis clinical isolates from individual patients were obtained from two Colombian cities. Of them, 57 isolates were ETH-resistant MDR-TB strains, three were ETH-susceptible MDR-TB strains, and four were fully susceptible strains. The ETH-resistant MDR-TB isolates were collected from the Unidad de Bacteriología y Micobacterias, Corporación para Investigaciones Biológicas (CIB), in the city of Medellín (n ⫽ 30), and from the Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), in the city of Cali (n ⫽ 27). All of the isolates were collected between 2000 and 2013 and stored at ⫺70°C until tested for this study. All of the available MDR-TB isolates that were resistant to ETH were previously tested using the agar proportion method (29). PCR amplification and sequencing. M. tuberculosis isolates were cultured on Middlebrook 7H11 for 3 weeks. Total genomic DNA was isolated using the cetyltrimethylammonium bromide (CTAB)/NaCl method (30) and quantified with a NanoDrop ND2000 spectrophotometer (Thermo Scientific) by measuring the absorption at 260 nm. Then, 10 l of DNA at a concentration of 10 ng/l were submitted was subjected to PCR using the following amplification protocol. An initial denaturation step of 3 min at 95°C was performed, followed by 40 cycles of 30 s at 95°C, 30 s at 57°C, and 2 min at 72°C, ending with a final extension step of 5 min at 72°C. All of the genes were amplified and sequenced using the primers shown in Table S1 in the supplemental material. The amplicons were sequenced using the Sanger method (Macrogen, Seoul, Republic of Korea). For all of the isolates, the entire ethA, ethR, mshA, katG, ndh, and inhA genes and the fabG1-inhA regulatory region (up to position ⫺200 upstream from the fabG1 gene) were sequenced. Sequence analysis. Chromatograms were analyzed and edited using the program FinchTV v1.4 (http://www.geospiza.com /Products/finchtv.shtml), and consensus sequences were edited by using Clone Manager v9.0 (Sdi-Ed Software, NC, USA). The nucleotide sequence alignments were performed using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and protein translation sequences were performed using the ExPASy program (http://expasy.org/tools/dna.html). Susceptibility testing. Each of the 64 M. tuberculosis isolates was tested for susceptibility to first- and second-line drugs using the agar proportion method (29). In addition, the 30 isolates collected from Medellín were tested using MICs to INH and ETH with the automated system BD Bactec MGIT 960 (Becton, Dickinson, and Company, Sparks, MD, USA) according to the manufacturer’s instructions (31, 32). The drugs were used at different concentrations, such as 0.1, 0.5, 1, and 5 g/ml for INH (SigmaAldrich, St. Louis, MO, USA), and 2.5, 5, 7.5, 10, 25, 50, and 100 g/ml for ETH (Sigma-Aldrich, St. Louis, MO, USA). High levels of resistance to INH and ETH were defined as a MIC of ⱖ1 g/ml and of ⱖ25 g/ml, respectively (8). The M. tuberculosis H37Rv strain was used as a susceptible control. Statistical analysis. The Epidat software, version 3.1 (Xunta de Galicia, Santiago de Compostela, Spain, and Pan American Health Organization), was used for the calculation of the Cohen kappa index between the results of susceptibility testing and sequencing. This study was approved by the CIB and CIDEIM ethic committees. DNA sequencing of ethA, mshA, ethR, katG, ndh, and inhA and its promoter. Of the 57 ETH-resistant MDR-TB isolates, 41
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showed mutations in ethA: nonsynonymous mutations were identified in 32 (78.0%) isolates, four isolates (9.7%) had insertions (one to three nucleotides), and five isolates (12.1%) had deletions in ethA. Seventeen of these isolates also showed mutations in mshA; mutations in 12 of the isolates were in the N111S codon, mutations in three isolates were in the A187V codon, mutations in one isolate were in the N111S and I460R codons, and mutations in one isolate were in the S352F codon. A synonymous mutation in ethR was detected in only one isolate. The remaining 16 ETH-resistant MDR-TB isolates were wild-type for ethA. However, nine of them had the mutation in the N111S codon of mshA. Of the seven ETH-susceptible isolates, three had mutations in mshA (N111S), and one had mutations in ethA (K448E). All MDR-TB isolates showed mutations at katG in the codons for S315T (n ⫽ 47 isolates), T275A and S315T (n ⫽ 5 isolates), S315T and R463L (n ⫽ 3 isolates), V442G (n ⫽ 1 isolate), I248M and S315T (n ⫽ 1 isolate), S315G and R463L (n ⫽ 1 isolate), W397 and S652A (n ⫽ 1 isolate), and G206D and S315T (n ⫽ 1 isolate). No mutations in katG were detected in the INH-susceptible isolates. Cross-resistance was detected in 19 out of 57 (33.3%) ETHresistant MDR-TB isolates by mutations within the inhA promoter region and/or the inhA gene at positions ⫺15C¡T (n ⫽ 11 isolates), ⫺8T¡G (n ⫽ 3 isolates), ⫺8C¡ T (n ⫽ 1 isolate), ⫺8T¡A (n ⫽ 1 isolate), ⫺17G¡T (n ⫽ 1 isolate), ⫺15C¡T and I194T codon (n ⫽ 1 isolate), or S94A codon (n ⫽ 1 isolate). Two isolates also had mutations in the ndh gene (V18A codon). The remaining 38 ETH-resistant MDR-TB isolates did not show mutations in inhA or its promoter. However, three of these isolates had mutations in ndh. One of the three ETH-susceptible MDR-TB isolates showed a mutation in inhA (S94A). No mutations in the inhA promoter or in ndh were detected in the four INH and ETH susceptible isolates. Descriptions of mutations identified in this study are provided in Table 1. Agreement between the susceptibility testing and sequencing results. Agreement between the phenotypic and genotypic tests for the detection of resistance to INH and ETH was found to be 100% (index kappa ⫽ 1) and 90% (index kappa ⫽ 0.45; 95% confidence interval, 0.08 to 0.82), respectively. However, there was a disagreement among the four isolates, which were confirmed to be susceptible to ETH by the phenotypic susceptibility tests. These isolates exhibited mutations in the mshA N111S codon (n ⫽ 3 isolates) and ethA K448E codon (n ⫽ 1 isolate); mutations were confirmed by sequencing. Of the 57 ETH-resistant isolates by the phenotypic susceptibility test, two isolates showed the absence of mutations in the genes studied. INH and ETH MICs. MIC values for INH and ETH in M. tuberculosis isolates are summarized in Table 2. High-level resistance to INH (MIC ⱖ1.0 g/ml) and to ETH (MIC ⱖ25 g/ml) was detected in 20 out of 30 ETH-resistant MDR-TB isolates. Nine isolates showed high-level resistance to INH (MIC ⱖ1.0 g/ml) and low-level resistance to ETH (MIC between 2.5 and 10 g/ml), and one isolate showed low-level resistance to INH (MIC between 0.1 and 0.5 g/ml) and high-level resistance to ETH (MIC ⱖ25 g/ml). Relationship between the INH and ETH MICs and sequencing results. Of the 29 ETH-resistant MDR-TB isolates with highlevel resistance to INH, 14 (48.3%) had mutations in two or more INH resistance-related genes, and 15 isolates had mutations in katG. One isolate with low-level resistance to INH had double
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Genes Associated with Resistance to Ethionamide
TABLE 1 Mutationsa in katG, ethA, mshA, inhA promoter region, inhA, and ndh in 64 isolates of M. tuberculosis Resistance phenotypeb INH
ETH
R
R
No. (%) of isolates
Resistance genotypec inhA Promoter
inhA
ndh
WT N111S WT N111S, I460R (ATA¡AGA1379) WT WT A187V N111S N111S WT WT WT A187V N111S WT
WT WT WT WT
WT WT WT WT
WT WT WT WT
WT WT WT WT WT WT WT WT WT WT WT
WT WT WT WT WT WT WT WT WT WT WT
WT ⫺ 15C¡T ⫺ 8T¡G ⫺ 8T¡C ⫺ 8T¡G ⫺ 8T¡A ⫺ 15C¡T ⫺ 15C¡T
WT WT WT WT WT WT WT WT
C deleted at 369 WT
WT WT N111S WT N111S WT WT S352F (TCC¡TTC1055) WT N111S
WT WT WT WT WT WT WT WT WT WT N316K (AAC¡AAA948) V18A V18A WT WT WT WT WT WT
⫺ 15C¡T ⫺ 15C¡T
WT WT
WT WT
WT WT WT WT WT
N111S WT WT WT WT
⫺ 15C¡T ⫺ 15C¡T ⫺ 15C¡T ⫺ 17G¡T ⫺ 15C¡T
WT WT WT WT I194T
WT WT WT WT WT
WT WT
N111S WT
WT WT
S94A WT
S315T G206D, S315T S315T
WT WT WT
N111S N111S WT
WT WT WT
WT WT WT
WT A226E (GCA¡GAA677) WT WT WT
katG
ethA
mshA
L301R (CTG ¡CGG902) E36Q (GAA¡CAA106)d E36Q (GAA¡CAA106) E36Q (GAA¡CAA106)
3 (5.27) 2 (3.51) 2 (3.51) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75)
T275A, S315T S315T S315T S315T, I248 M (ATT¡ATG744) S315T S315T S315T, R463L S315T S315T S315T S315T S315T S315T, R463L S315T S315T
1 (1.75) 2 (3.51) 2 (3.51) 1 (1.75) 1 (1.75) 1 (1.75) 2 (3.51) 2 (3.51)
S315T S315T S315T S315T S315T S315T S315T S315T
M409I (ATG¡ATC1227) T340N (ACC¡AAC 1019) GGG insertion at 1141 G deleted at 32 C deleted at 965 N379Y (AAC¡TAC 1135) L374R (CTT¡CGT1121) F157L (TTC¡CTC469)
1 (1.75) 1 (1.75)
1 (1.75) 1 (1.75)
S315G, R463L V442G (GTC¡GCC1325) S315T S315T S315T S315T W397Y, S652A (TCA¡GCA1954) S315T S315T
5 (8.8) 1 (1.75) 2 (3.51)
5 (8.8) 5 (8.8) 2 (3.51) 1 (1.75)
1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75) 1 (1.75)
T44A (ACC¡GCC130) Y143STOP (TAC¡TAA429) C137R (TGC ¡CGC409) C403W (TGT¡TGG1209) F302S (TTC¡TCC905) C137F (TGC ¡TTC410) H281R (CAC¡CGC 842) G deleted at 32 C deleted at 1265 Insertion GC at 755 Insertion G at 672
R
S
1 (33.33) 1 (33.33) 1 (33.33)
S315T S315T S315T
WT K448E (AAG¡GAG1342) WT
N111S WT WT
WT WT WT
S94A WT WT
WT WT WT
S
S
2 (50) 2 (50)
WT WT
WT WT
WT N111S
WT WT
WT WT
WT WT
a Amino acid (one-letter code) mutations are generally given for all genes except the inhA promoter region. The position of the nucleotide insertion or deletion in ethA is indicated. The mutations not previously reported in the literature found in katG, ethA, mshA and ndh genes are underlined, and the corresponding nucleotide changes are given in parentheses. b R, resistant. S, susceptible. c WT, wild type. d One of these five isolates had a synonymous mutation (ACT¡ACG 447) in the ethR gene.
mutations: ⫺15C¡T at the inhA promoter and V442G codon in katG (Table 3). A total of 18 (86%) of the 21 ETH-resistant MDR-TB isolates with high-level resistance to ETH had mutations in two or more
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genes related to resistance to ETH. Three isolates showed a mutation in a single gene related to resistance to ETH. Additionally, five (55.5%) of the nine isolates with low-level resistance to ETH had mutations in a single gene, and the remaining four isolates showed
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TABLE 2 INH and ETH MICs in 30 M. tuberculosis isolates for the automated system BD Bactec MGIT 960 Resistance phenotypea No. (%) of INH ETH isolates R
R
2 (6.6) 3 (10) 2 (6.6) 2 (6.6) 3 (10) 3 (10) 5 (16.7) 2 (6.6) 2 (6.6) 5 (16.7) 1 (3.4)
MICs (g/ml)b INH (0.1 g/ml c) 0.1 0.5 1 5
ETH (2.5 g/mlc)
⬎5.0 2.5 5 7.5 10 25 50 100 ⬎100 X
X X
X X X
X X X
X X
X X
X X X X X
X X X X
a
R, resistant. b X, the lowest concentration at which INH and ETH inhibit the growth of M. tuberculosis. c Critical concentration for drugs evaluated by the automated system BD Bactec MGIT 960.
mutations in two or more genes related to resistance to ETH (Table 4). ETH is a second-line drug that shares the same mechanism of action as INH and is considered one of the several useful drugs for the treatment of patients who present with MDR-TB (8). Accordingly, it is important to know the frequency of mutations associated with independent resistance and cross-resistance to INH and ETH in clinical isolates of MDR-TB, in order to define the efficacy of ETH in the recommended treatment regimens. In this study, 72% (41 out of 57) of the ETH-resistant MDR-TB isolates evaluated had mutations in ethA, which have been associated with ETH resistance. In addition, previous studies have reported about 54.2% to 100% of MDR-TB isolates with mutations on this gene, including nonsynonymous mutations, deletions, and insertions (10, 13). In our study, 90% of the mutations detected in ethA (18 of 20 mutations) have not been previously reported. These results indicate that ethA mutations are distributed across the structural gene, with no single predominating nucleotide or codon. This may suggest the existence of one or more enzymes with functional redundancy to EthA. In fact, the genome of M. tuberculosis probably encodes more than 30 monooxygenases (18). Nevertheless, more studies are required to completely explain the influence of the diverse mutations found in ethA gene and ETH resistance. In the present study, 95% of the INH-resistant isolates had mutations at codon 315 of the katG gene. The most frequent was the transitional mutation G¡C (AGC¡ACC), resulting in the substitution of serine by threonine (S¡T) in the amino acid chain. The frequency of mutations in codon 315 in the katG gene was found to differ within the geographical regions: 97% in South Africa (33), 88% in Colombia (34), 60 to 87% in Brazil (35), 46% in Spain (36), 28% in Japan (37), 7% in Finland (33), and 4% in
South Korea (38). Other studies have demonstrated that the majority of INH-resistant isolates with mutations at codon 315 retained their catalase-peroxidase activity while showing a lowered ability to activate INH. This secures a sufficient level of oxidative protection that may preserve its characteristics of virulence and transmissibility (39). The katG mutations in the codons for G206D, I248M, V442G, and S652A found in this study have not been previously reported. The frequency of inhA promoter region or inhA gene mutations was found to differ within the geographical regions but represented at least 13.8% (10) and up to 100% (6) of ETH- and INH-resistant clinical isolates. Our study confirmed mutations in these genes in 33.3% (19 of 57) of ETH-resistant MDR-TB isolates. The most commonly found mutation, ⫺15C¡T in the inhA promoter, occurred in 66.6% (12 of 18) of the isolates. This mutation is associated with a 20-fold increase in inhA mRNA levels, resulting in the overexpression of InhA, which leads to a titration of INH or ETH and a consequent increase in INH and ETH MICs in M. tuberculosis (15). Mutations in the ndh gene (R13C, V18A, T110A, R268H, and G313R codons) have been described in M. tuberculosis clinical isolates (4). In our study, we observed three mutations in this gene (V18A, A226E, and N316K codons). Mutations in V18A have been previously described in INH-susceptible and -resistant isolates (17), but the contribution of this mutation in the INH-resistant phenotype was unknown, whereas mutations in A226E and N316K have not been previously reported in M. tuberculosis or in Mycobacterium smegmatis. Therefore, the contribution of these mutations in INH and ETH resistance is uncertain. Future research should be directed toward determining the role and prevalence of mutations in ndh in INH- and ETH-resistant M. tuberculosis isolates and confirming its contribution in cross-resistance mechanisms as reported in M. smegmatis (40) and Mycobacterium bovis (16). Mutations in mshA (N111S, I460R, S352F, and A187V codons) were detected in 45.6% (26 of 57) of ETH-resistant MDR-TB isolates studied. However, the N111S mutation was also detected in three ETH-susceptible M. tuberculosis isolates. The N111S mutation has been identified in the M. tuberculosis Erdman strain belonging to the Haarlem genotype, which is susceptible to both INH and ETH. This suggested that this mutation was neutral and did not confer resistance to ETH (41). In this study, mutations in ethA, mshA, ndh, inhA, or the inhA promoter region were associated with ETH resistance in 96.5% of the phenotypically resistant isolates. This suggested that mutations in these genes might function as markers for the detection of ETH resistance. However, it is important to note that the absence of such mutations in 3.5% of the isolates may indicate the presence of other undiscovered mechanisms of ETH resistance. Numerous studies have shown that mutations in codon 315 of
TABLE 3 Relationship between low and high levels of resistance to INH and mutations in katG, ndh, and inhA genes and inhA promoter region in 30 ETH-resistant MDR-TB isolates Resistance
katG
katG and ndh
katG and inhA promoter
katG and inhA
katG, inhA, and inhA promoter
No. (%) of isolates
High level of resistance to INH, ⬎1 g/ml Low level of resistance to INH, 0.1–0.5 g/ml
15 0
2 0
10 1a
1 0
1 0
29 (97) 1 (3)
a
Mutations in katG codon encoding V442G and in the inhA promoter region encoding C-15T.
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Genes Associated with Resistance to Ethionamide
TABLE 4 Relationship between low and high levels of resistance to ETH and mutations in ethA, mshA, ndh, and inhA genes and inhA promoter region in 30 ETH-resistant MDR-TB isolates
Resistance
ethA, mshA, inhA ethA and ethA and ethA and inhA mshA and inhA inhA and inhA mshA and and inhA No. (%) ethA mshA promoter mshA ndh promoter promoter promoter inhA promoter of isolates
High level of resistance 1 to ETH ⱖ25 g/ml Low level of resistance 5 to ETH ⱖ2.5–10 g/ml a
1
1
7
1
3
2
1
0
4
21 (70)
0
0
1a
1
1
0
0
1
0
9 (30)
Synonymous mutation (ACT¡ACG 447) in the ethR gene.
katG are associated with high-level resistance to INH (35, 36); this is consistent with our results, since 100% of the tested MDR-TB isolates from Medellín with mutations in the 315 codon had INH MICs between 0.5 and 5 g/ml. According to these results, the treatment of MDR-TB patients with high doses of INH may not be effective (42). In 18 out of 21 (86%) isolates from Medellín, the high-level resistance to ETH was associated with mutations in two or more genes. A similar situation has been described in isolates with high-level resistance to ethambutol, in which resistance has been described as a multistep process (43). More studies are required in order to understand the contribution of multiple mutations to confering high-level resistance to ETH and, ultimately, to know the clinical value that this finding may have in the treatment of patients with MDR-TB. In conclusion, mutations in genes (katG, ethA, and inhA and its promoter) were associated with resistance to INH and/or ETH in the present study. Special attention must be given to the second-line therapeutic drugs, including ETH, due to the frequency of cross-resistance between INH and ETH by mutations in inhA or its promoter that were detected in 33.3% of M. tuberculosis isolates from Colombia. Future studies should aim toward determining the role of additional genes, such as ndh and mshA, and their relationships to resistance to these two drugs. ACKNOWLEDGMENTS This work was supported by Departamento Administrativo Colombiano de Ciencia, Tecnología e Innovación (Colciencias) 221356933562. We declare no conflicts of interest.
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