Abstract
Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P < 0.001). In contrast, the mutations detected in embC and embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions −8 and −21.
INTRODUCTION
The emergence of multidrug-resistant tuberculosis (MDR-TB), which is resistant to at least rifampin (RIF) and isoniazid (INH), and, more recently, extensively drug-resistant tuberculosis (XDR-TB), which is resistant to any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin), is widely considered to be a serious threat to global TB control (1). Rapid detection of drug resistance is essential to designing appropriate treatment regimens, preventing treatment failure, and reducing the spread of drug-resistant isolates. Molecular assays for the detection of mutations that confer resistance (e.g., based on DNA sequencing, real-time PCR, and strip technologies such as the GenoType MTBDR line probe assay) have been increasingly used and have the potential to shorten the time to detection of resistance to one working day (2–6). However, these molecular assays require precise knowledge of the genetic variations involved in the development of resistance to particular anti-TB drugs.
Ethambutol (ETB) [dextro-2,29-(ethylenediimino)-di-1-butanol] was introduced in 1961 and is a first-line anti-TB agent used in drug combinations to prevent the emergence of drug resistance. ETB is also included in second-line regimens for MDR-TB when susceptibility is demonstrated (7). ETB interferes with mycobacterial cell wall synthesis and integrity (8–13) by inhibiting arabinosyl transferases encoded by the embCAB locus (≈10 kb), which encompasses three contiguous genes, embC, embA, and embB (14). These enzymes are essential for the synthesis of arabinogalactan (EmbA and EmbB) and lipoarabinomannan (EmbC) in the cell wall of Mycobacterium tuberculosis complex (MTBC) isolates (10, 12). Although the embCAB locus is also called the embCAB operon, it is not a real operon because the promoter of embA and embB is thought to be located in the embC-embA intergenic region (85 bp) (15, 16), and the promoter of embC is in the region upstream of embC, possibly in the Rv3792 gene (11, 17), while the embR gene of MTBC isolates is located 2 Mb from the embCAB locus (18, 19).
Resistance to ETB in MTBC isolates has been associated with chromosomal mutations in the embCAB locus, mainly embB (5, 14, 16, 20). The majority of the detected mutations are concentrated in a 576-bp region of embB, called the ETB resistance-determining region (ERDR). This region includes codons 306, 406, and 497 and is predicted to be the recognition site of the enzyme EmbB (21). Mutations in this region cause structural changes in the enzyme and alterations in the ETB-binding site and drug-protein interactions (22, 23), which result in the development of ETB resistance. Nucleotide changes in the ERDR of embB are found in ∼50% to 70% of ETB-resistant (ETB-R) isolates of the MTBC, mainly in codon 306, with previous reports estimating that 18% to 78% of isolates presenting with embB mutations have an EmbB codon 306 substitution (2–5, 13, 16, 17, 23–37). A meta-analysis of the Genotype MTBDRsl line probe assay, which allows rapid diagnosis of ETB resistance by analyzing EmbB codon 306, showed that the sensitivity and specificity (with 95% confidence interval) for ETB are 0.679 (0.652 to 0.706) and 0.799 (0.773 to 0.823), respectively (3). However, ∼30% to 50% of ETB-resistant clinical isolates do not carry a mutation in EmbB codon 306 and are not detectable by molecular methods based only on the polymorphisms in EmbB codon 306 (2–5). Although other mutations in the embCAB locus have been suggested to confer resistance, limited data have been available until now, with most studies analyzing only a short fragment of the embB gene encompassing codon 306 (2–4, 24, 25, 28, 30, 34, 35, 38).
Our main objective was to study the mutations in the entire embCAB locus and embR that are involved in ETB resistance in clinical isolates of the MTBC in France because the majority of previous studies have been based on partial sequencing of the 10-kb region containing the embCAB locus. The results were challenged with phenotypic drug susceptibility testing, phylogenetic analysis, data from the literature, and the PolyTB Web-based tool. We also assessed the association between the presence of embB mutations and resistance to first-line drugs.
MATERIALS AND METHODS
Mycobacterium tuberculosis complex clinical isolates.
A total of 131 MTBC clinical isolates collected throughout France and received by the French National Reference Center for Mycobacteria (NRC) between 2009 and 2014 were included in this study. Seventy-one isolates were ETB-R, of which 68 were MDR (including 7 XDR), 1 was resistant to RIF but susceptible to INH, and 2 were resistant to INH but susceptible to RIF. Among the 71 patients with ETB-R isolates, TB treatment history was positive for 31 (43%), negative for 36 (51%), and unknown for 4 (6%) patients. Sixty-nine patients with ETB-R isolates had 26 different countries of birth, and two had an unknown country of birth (see Table S1 in the supplemental material). Sixty of the patients had isolates that were susceptible to ETB (ETB-S), of which 16 were MDR, 5 were resistant to RIF, 9 were resistant to INH, 1 was resistant to streptomycin (STR), and 29 were susceptible to all anti-TB drugs. Among the 60 patients with ETB-S isolates, TB treatment history was positive for 13 (22%), negative for 26 (43%), and unknown for 21 (35%) patients. Forty-nine of the patients with ETB-S isolates had 25 different countries of birth, and 11 had an unknown country of birth (see Table S1 in the supplemental material).
Phenotypic drug susceptibility testing.
In vitro drug susceptibility testing for RIF, INH, STR, ofloxacin, amikacin, kanamycin, capreomycin, and ETB was performed on Lowenstein-Jensen medium according to the proportion method, with the following concentrations: 40 mg/liter RIF, 0.2 and 1 mg/liter INH, 4 mg/liter STR, 2 mg/liter ofloxacin, 20 mg/liter amikacin, 30 mg/liter kanamycin, 40 mg/liter capreomycin, and 2 mg/liter ETB (39).
DNA sequencing of ethambutol resistance-associated genes.
Genomic DNA was isolated from bacteria grown on Lowenstein-Jensen medium. A loopful of culture was suspended in water (500 μl) and heated at 95°C for 15 min. The DNA used for PCR amplification was obtained by heat shock extraction (1 min at 95°C and 1 min on ice, repeated five times). PCR amplification of the embCAB locus (9,949 bp) and the embR gene (1,167 bp) was performed with a volume of 5 μl, using the 15 oligonucleotide primer pairs listed in Table S2 in the supplemental material. After amplification, unincorporated nucleotides and primers were removed by filtration with Microcon 100 microconcentrators (Amicon Inc., Beverly, MA), and the amplicons were sequenced by using the BigDye Terminator cycle sequencing-ready kit according to the manufacturer's instructions.
Determination of phylogenetic lineage.
The phylogenetic lineages of our 131 MTBC isolates were determined by using the mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) molecular typing method for all isolates. Spoligotyping was performed for some isolates for which the MIRU-VNTR results were ambiguous. Standard 24-locus-based MIRU-VNTR typing was performed as described previously (40), with the MIRU-VNTR typing kit from GenoScreen. The amplified fragments were analyzed on a 16-capillary Applied Biosystems 3130 genetic analyzer. To determine the lineages of the isolates, the 24 numerical values generated by MIRU-VNTR analysis were compared to those in the MIRU-VNTRplus database (http://www.miru-vntrplus.org/). Spoligotyping was performed as described previously by Abadia et al., using the Luminex microbead-based approach (41). Spoligotypes in binary format were converted to an octal code based on the signatures given by the 43-spacer spoligotyping patterns for comparisons with the SITVIT/SpolDB4 international spoligotype database (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo), which contains all of the spoligotype international types (SITs) described previously for MTBC isolates. The different phylogenetic lineages were described previously by Gagneux and Small (42).
PolyTB Web-based tool.
PolyTB is a Web-based resource designed to explore MTBC genomic variation on a global scale (http://pathogenseq.lshtm.ac.uk/polytb) (43). Genomic polymorphisms and important metadata, such as in silico-inferred strain types and locations, are presented in browser, geographic map, and phylogenetic views.
Statistical analysis.
embCAB mutations and ETB resistance on one hand, and embB mutations and resistance to first-line drugs on the other hand, were compared by using the chi-square test. P values were two tailed, and a P value of ≤0.05 was considered significant. For statistical analysis, 67/71 ETB-R and 60/60 ETB-S isolates were compared (4 related ETB-R isolates were excluded).
Nucleotide sequence accession numbers.
The nucleotide sequences determined for the mutant genes included in the present report were deposited into the GenBank database under the following accession numbers: GU323395 to GU323398, KJ571490 to KJ571499, KJ571510 to KJ571513, KJ571515 to KJ571519, KM189805, and KR092803 for the nonsynonymous M306V, M306I (ATC/ATA/ATT), G406C, G406S, G406A, G406D, Q497R, L402V, F330I, E378A, N296H, N399I, Y334H, D354A, M423T, A19D plus N296H, S426N, and N13S EmbB mutants and the synonymous D534D (c1602t; nucleotide c replaced with nucleotide t at position 1602), T1027T (g3081a), L986L (c2956t), P965P (g2895a), T44T (g132t), V117V (c351t), and N760N (t2280c) EmbB mutants, respectively; KJ571500, KJ571509, KR092801, KJ571523 to KJ571525, and KR092802 for the nonsynonymous A426T, V981L, and V987A EmbC mutants and the synonymous G559G (t1677c), L121L (g363t), R345R (c1035g), and T1036T (c3108t) EmbC mutants, respectively; KJ571501 to KJ571506 for the embC-embA intergenic region nucleotides -c8a (nucleotide c replaced with nucleotide a at position −8), -c8t, -c11a (plus EmbC V981L), -c12t, -c16t, and nucleotides cg deleted at positions −21 and −20, respectively; and KJ571507, KJ571508, KJ571514, and KJ571521 for the nonsynonymous EmbA G5V mutant and the synonymous C76C (c228t), Q38Q (a114g), and H764H (c1995t) EmbA mutants, respectively.
RESULTS
Ethambutol-susceptible isolates.
The 60 ETB-S strains had different spoligotypes and MIRU-VNTR codes, representing 60 unrelated clinical isolates. Twenty isolates had no mutation in the embCAB locus (Table 1). Among the 40 other isolates, 1 of the 2 type S isolates and 1 of the 8 LAM isolates had undescribed synonymous mutations: embC g363t (L121L) and embB t2280c (N760N), respectively (Table 1). Eleven Beijing isolates harbored the synonymous embA c228t (C76C) mutation, which is known to be associated with the Beijing lineage (PolyTB database [http://pathogenseq.lshtm.ac.uk/polytb]) (20), and one of the Beijing isolates carried an undescribed mutation, EmbC V987A (Table 1). Twenty-two ETB-S isolates with the nonsynonymous V981L mutation in EmbC belonged to the Haarlem (n = 15), Ghana (n = 2), T3 variant (n = 3), T2 (n = 1), or X (n = 1) phylogenetic lineage (Table 1). This mutation was previously reported in the Haarlem lineage (16, 20) in all H1, X, and ambiguous T2T3 and T2X1 isolates as well as in one Manu2, one T5, some T1, and some T2 isolates (PolyTB database). Among the ETB-S isolates with the V981L mutation, one T3 variant isolate carried a not-yet-described synonymous mutation, embB g132t (T44T), whereas an X isolate carried two mutations, g2895a (P965P) in embB and c1035g (R345R) in embC, which were previously reported in the X and X2 lineages, respectively (PolyTB database). The last isolate, carrying the EmbC V981L polymorphism, belonged to the Haarlem family and harbored an uncharacterized mutation, EmbC A426T (Table 1). Among the five remaining ETB-S isolates, two Delhi/CAS isolates carried the EmbC R738Q mutation reported for the CAS lineage (PolyTB database) (20); a Cameroon strain had a mutation in EmbB associated with ETB resistance, M306I; and a West africanum 2 strain and a Mycobacterium bovis strain had both the EmbB E378A and EmbC T270I mutations, which were previously reported to be phylogenetic markers of ancestral MTBC isolates (in M. bovis and lineages 1, 5, and 6 of the MTBC) (PolyTB database) (16, 44, 45). The M. bovis strain also carried the EmbB N13S and embC c3108t (T1036T) mutations previously reported for M. bovis (PolyTB database) and the embB c351t (V117V) mutation previously reported for M. bovis and AFRI-1 strains (PolyTB database). Finally, no embR mutation was detected in ETB-S isolates.
TABLE 1.
Distribution of the embCAB mutations in 60 ETB-S and 71 ETB-R isolates of the MTBC
| Resistance phenotype (total no. of isolates) | Phylogenetic lineage | No. of isolates | Mutation(s) in the embCAB locusa |
||
|---|---|---|---|---|---|
| Marker(s) of phylogenetic lineages | Mutation(s) potentially associated with ETB resistanceb | Synonymous mutation (excluding marker of phylogenetic lineage) | |||
| Susceptible (60) | T | 3 | |||
| T1 | 2 | ||||
| Cameroon | 3 | ||||
| LAM | 7 | ||||
| Ural | 1 | ||||
| S | 1 | ||||
| Uganda | 3 | ||||
| S | 1 | embC g363t (L121L) | |||
| LAM | 1 | embB t2280c (N760N) | |||
| Beijing | 10 | embA c228t (C76C) | |||
| Beijing | 1 | embA c228t (C76C) | EmbC V987A | ||
| Haarlem | 14 | EmbC V981L | |||
| Ghana | 2 | EmbC V981L | |||
| T3 variant | 2 | EmbC V981L | |||
| T3 variant | 1 | EmbC V981L | embB g132t (T44T) | ||
| T2 | 1 | EmbC V981L | |||
| X | 1 | EmbC V981L, embB g2895a (P965P), embC c1035g (R345R) | |||
| Haarlem | 1 | EmbC V981L | EmbC A426T | ||
| Delhi/CAS | 2 | EmbC R738Q | |||
| Cameroon | 1 | EmbB M306Ic | |||
| West africanum 2 | 1 | EmbB E378A, EmbC T270I | |||
| M. bovis | 1 | EmbB N13S, EmbB E378A, embB c351t (V117V), EmbC | |||
| T270I, embC c3108t (T1036T) | |||||
| Resistant (71) | Beijing | 3 | embA c228t (C76C) | EmbB M306V,c embCA -c12t | |
| LAM | 1 | EmbB M423T | EmbB M306V,c embCA -c16t | ||
| Beijing | 14 | embA c228t (C76C) | EmbB M306Vc | ||
| Beijing | 1 | embA c228t (C76C) | EmbB M306Vc | embB c1602t (D534D) | |
| LAM | 3 | EmbB M306Vc | |||
| T1 | 1 | EmbC V981L | EmbB M306Vc | ||
| S | 1 | EmbB M306Vc | |||
| Beijing | 5 | embA c228t (C76C) | EmbB M306Ic | ||
| Haarlem | 3 | EmbC V981L | EmbB M306Ic | ||
| T1 | 1 | EmbB M306Ic | |||
| T2 | 1 | EmbB M306Ic | embB g3081a (T1027T) | ||
| LAM | 5 | EmbB M306Ic | |||
| Ghana | 1 | EmbC V981L | EmbB M306I,c embCA -c11a | ||
| Beijing | 1 | embA c228t (C76C) | EmbB G406Cc | ||
| Beijing | 1 | embA c228t (C76C) | EmbB G406Sc | ||
| LAM | 1 | EmbB G406Ac | embB c2956t (L986L) | ||
| Haarlem | 1 | EmbC V981L | EmbB G406Ac | ||
| Beijing | 3 | embA c228t (C76C) | EmbB G406Dc | ||
| LAM | 1 | EmbB G406Dc | |||
| Beijing | 1 | embA c228t (C76C) | EmbB Q497Rc | ||
| Cameroon | 1 | EmbB Q497Rc | |||
| Ural | 1 | EmbB F330I | |||
| Beijing | 1 | embA c228t (C76C) | EmbB D354Ad | ||
| Ural | 1 | EmbB N399I | |||
| Beijing | 1 | embA c228t (C76C) | EmbB L402V | ||
| Ural | 1 | EmbB S426N | |||
| Beijing | 3e | embA c228t (C76C), embA a114g (Q38Q) | EmbB N296H | embA c1995t (H764H) | |
| X | 1 | EmbC V981L, embB g2895a (P965P) | embCA-c8a | ||
| Beijing | 1 | embA c228t (C76C) | embCA -c8t | ||
| Beijing | 1 | embA c228t (C76C) | embCA -c12t | ||
| LAM | 1 | embCA -c12t | |||
| Beijing | 1 | embA c228t (C76C) | embCA -c16t | ||
| Delhi/CAS | 1 | EmbC R738Q | embCA -c16t | ||
| Beijing | 1 | embA c228t (C76C) | embCA with deletion of cg at positions −21 and −20 | ||
| Beijing | 3e | embA c228t (C76C) | embCA -c12t, EmbB Y334H | ||
| Haarlem | 1 | EmbC V981L | embCA -c12t, EmbB D354Ad | embC t1677c (G559G) | |
| Uganda | 1 | EmbB A19D plus N296H, EmbA G5V | |||
| NEW-1 | 1 | ||||
Excluding the synonymous embC c2781t mutation (R927R) present in all ETB-S and ETB-R isolates.
embCA is the embC-embA intergenic region.
Implication in ETB resistance proven by site-directed mutagenesis or allelic exchange.
Involvement in ETB resistance suggested by the in vitro-selected mutant.
Isolates sharing identical MIRU codes and spoligotypes.
Ethambutol-resistant isolates.
Based on spoligotyping and MIRU-VTNR typing, the 71 ETB-R isolates represented 67 unrelated strains; in the Beijing family, two distinct subsets each included three isolates sharing the same MIRU code or spoligotype, respectively.
Regarding the mutations corresponding to phylogenetic markers, the 41 ETB-R isolates carrying the synonymous embA c228t (C76C) mutation belonged to the Beijing phylogenetic lineage, as expected. Among these isolates, three also harbored the embA a114g (Q38Q) mutation reported to be phylogenetic marker of a subgroup of Beijing strains (20) (PolyTB database [http://pathogenseq.lshtm.ac.uk/polytb]). The ETB-R isolates with the EmbC V981L mutation reported previously in several phylogenetic lineages (PolyTB database) (16, 20) belonged to the Haarlem (n = 5), T1 (n = 1), Ghana (n = 1), or X (n = 1) strain family, with the latter also harboring the embB g2895a (P965P) phylogenetic marker that is specifically present in the X lineage (PolyTB database). Furthermore, one LAM ETB-R isolate obtained from a patient born in Portugal had the EmbB M423T mutation commonly reported for LAM strains from Portugal (PolyTB database), and one Delhi/CAS ETB-R isolate carried the EmbC R738Q mutation reported for the CAS lineage (PolyTB database) (20) (Table 1).
Regarding mutations potentially associated with ETB resistance, 40/71 (56%) ETB-R isolates had mutations in codon M306 of EmbB, including 24 isolates with the M306V mutation (34%) and 16 with the M306I mutation (22%). For the remaining ETB-R isolates, 30 (42%) had mutations in the embCAB locus outside EmbB codon 306, and 1 isolate belonging to the NEW-1 lineage had no mutation (Table 1). Among the 40 isolates with a mutation in codon M306 of EmbB, 5 had additional mutations in the embC-embA intergenic region (n = 3 for -c12t, n = 1 for -c16t, and n = 1 for -c11a), whereas 2 had undescribed synonymous mutations in embB, c1602t (D534D) and g3081a (T1027T) (Table 1).
Among the 30 isolates with mutations in the embCAB locus outside codon 306 of embB, 18 harbored nonsynonymous mutations in EmbB exclusively, 2 of which harbored the undescribed synonymous mutations c2956t (L986L) in embB and c1995t (H764H) in embA (Table 1). The distribution of EmbB mutations into 18 isolates was as follows: 8 G406A/C/D/S, 2 Q497R, 1 F330I, 1 D354A, 1 N399I, 1 L402V, 1 S426N, and 3 N296H. For the remaining 12 isolates with mutations in the embCAB locus, 7 had mutations in the embC-embA intergenic region exclusively (n = 1 for -c8a, n = 1 for -c8t, n = 2 for -c12t, n = 2 for -c16t, and n = 1 for the cg deletion at positions −21 and −20), with 2 of them harboring the X and CAS phylogenetic markers embB g2895a (P965P) and EmbC R738Q, respectively. Four isolates had a -c12t mutation in the embC-embA intergenic region in association with nonsynonymous mutations in EmbB outside codon 306, Y334H (n = 3) and D354A (n = 1), with the latter also carrying the undescribed silent t1677c (G559G) mutation in embC. Finally, one Uganda isolate harbored three nonsynonymous mutations in EmbA (G5V) and EmbB (A19D and N296H) (Table 1). No embR mutation was detected in the 71 ETB-R isolates.
Notably, the synonymous embC c2781t (R927R) mutation was present in all ETB-S and ETB-R isolates included in the present study. This polymorphism has also been reported for all isolates in the PolyTB database (http://pathogenseq.lshtm.ac.uk/polytb). This single nucleotide polymorphism (SNP) could be the result of a sequencing discrepancy in M. tuberculosis reference strain H37Rv deposited in GenBank (accession number AL123456.3).
DISCUSSION
Few publications have reported sequencing of the entire embCAB locus (16, 20, 46); most previous studies investigated only part of the embCAB locus (5, 26), part of embC-embB and embR almost entirely (23), or the embC-embA intergenic region entirely and part of embB (15). In these studies, the percentage of ETB-R isolates with mutations in the embCAB locus ranged from 65% (16) to 96% (46). In our study, 70/71 (98%) ETB-R isolates harbored mutations in the embCAB locus that likely contribute to ETB resistance.
Regarding mutations that do not generate ETB resistance, we identified 12 mutations that are associated with phylogenetic lineages of the MTBC (Table 2), 4 of which are well described in the literature: the EmbC T270I and EmbB E378A mutations in ancestral MTBC lineages 1, 5, and 6 and M. bovis (PolyTB database [http://pathogenseq.lshtm.ac.uk/polytb]) (16, 44, 45); the embA c228t (C76C) mutation in the Beijing lineage (PolyTB database) (20); and the EmbC V981L mutation in the Haarlem lineage (16, 20). We found the EmbC V981L mutations not only in Haarlem strains but also in type X, T1, T3 variant, and Ghana isolates. In the PolyTB database, this mutation has been reported for all H1; all X; all ambiguous T2T3 and T2X1; some T1, T2, and T5; and one Manu2 (but not Manu1) strain. Taken together, these data suggest that the EmbC V981L mutation is not a phylogenetic marker restricted to Haarlem strains, but it could correspond to a SNP that occurred earlier in the evolutionary pathway of MTBC lineages at the level of a branch leading to a group encompassing Haarlem, X, Ghana, and some T strains. Five other mutations found in our study are also associated with phylogenetic lineages of the MTBC according to the PolyTB database. Several of the mutations were found in ETB-S strains, including the embC c1035g (R345R) and embB g2895a (P965P) mutations in the X lineage (PolyTB database), the embB c351t (V117V) mutation in all AFRI-1 isolates and M. bovis, and the embC c3108t (T1036T) and EmbB N13S mutations in M. bovis (PolyTB database), whereas others were detected in ETB-R isolates, including the embA a114g (Q38Q) mutation specific to a subgroup of the Beijing lineage and associated with the EmbB N296H mutation in three Beijing isolates in our study (PolyTB database) (20), the CAS-specific EmbC R738Q polymorphism associated with the embCA -c16t mutation (PolyTB database) (20), and the EmbB M423T polymorphism, which characterizes the LAM4 lineage from Portugal and was associated with the EmbB M306V and embCA -c16t mutations in our study (Table 2). Finally, eight SNPs that are probably not involved in ETB resistance were also identified in our strains, but they are unlikely to represent phylogenetic markers because they correspond mostly to synonymous SNPs found in single isolates within a given phylogenetic group. Four of the SNPs were found in ETB-S isolates, including the embB g132t (T44T) and t2280c (N760N) mutations, the embC g363t (L121L) mutation, and the nonsynonymous EmbC A426T mutation (Table 1). Five sporadic synonymous mutations, embB c1602t (D534D), g3081a (T1027T), and c2956t (L986L); embC t1677c (G559G); and embA c1995t (H764H), were also detected in ETB-R isolates in association with mutations known to confer ETB resistance (Table 1). These synonymous mutations are unlikely to participate in drug resistance.
TABLE 2.
Mutations in the embCAB locus that are markers of phylogenetic lineages or potentially associated with ETB resistance in 60 ETB-S and 71 ETB-R isolates of the MTBC
| Mutation in the embCAB locus | Mutation |
Phylogenetic lineage(s) | |
|---|---|---|---|
| Amino acid | Nucleotidea | ||
| Markers of phylogenetic lineages | EmbC R738Q | CAS | |
| EmbC V981L | Haarlem; all H1; all X; all ambiguous T2T3 and T2X1; some T1, T2, and T5; and one each of Manu2, Ghana, and T3 variant | ||
| EmbA C76C | embA c228t | Beijing | |
| EmbA Q38Q | embA a114g | Subgroup of Beijing | |
| EmbC T1036T | embC c3108t | M. bovis | |
| EmbB N13S | M. bovis | ||
| EmbB V117V | embB c351t | AFRI-1 and M. bovis | |
| EmbB E378A | Ancestral MTBC lineages 1, 5, and 6 and M. bovis | ||
| EmbC T270I | Ancestral MTBC lineages 1, 5, and 6 and M. bovis | ||
| EmbB M423T | LAM4 from Portugal | ||
| EmbB P965P | embB g2895a | X | |
| EmbC R345R | embC c1035g | X2 | |
| Mutations potentially associated with ETB resistancea | EmbB M306V/Ib | ||
| EmbB G406C/S/A/Db | |||
| EmbB Q497Rb | |||
| EmbB F330I | |||
| EmbB Y334H | |||
| EmbB D354Ac | |||
| EmbB N399I | |||
| EmbB L402V | |||
| EmbB S426N | |||
| EmbB N296H | |||
| EmbB A19Dd | |||
| EmbA G5Vd | |||
| embCA -c8a/t | |||
| embCA -c12t | |||
| embCA -c16t | |||
| embCA with deletion of cg at positions −21 and −20 | |||
embCA is the embC-embA intergenic region.
Implication in ETB resistance proven by site-directed mutagenesis or allelic exchange.
Involvement in ETB resistance suggested by the in vitro-selected mutant.
Implication in ETB resistance remains to be experimentally verified.
In the literature, between 38 and 94% of ETB-R isolates are reported to have mutations in EmbB (5, 13, 15–17, 20, 23, 26, 28, 29, 31, 32, 34, 36, 46). In our study, 90% of ETB-R isolates had mutations in 18 EmbB codons, corresponding to 22 mutations including the above-mentioned phylogenetic markers (n = 3) and synonymous mutations (n = 4). Among the 15 remaining nonsynonymous EmbB mutations likely involved in ETB resistance, 7 (M306V/I, G406D/A/S/C, and Q497R) have been unequivocally proven by site-directed mutagenesis or allelic exchange to increase MICs and can be considered canonical EmbB mutations in ETB resistance (37, 47–50). Accordingly, in our study, only one isolate that was phenotypically susceptible to ETB harbored the M306I mutation, and a strong statistical association was found between ETB resistance and a mutation at codon 306 alone (P < 0.001) or EmbB mutations at codons 306, 406, and 497 (P < 0.001). Some authors (38, 50–52) have argued that mutations at codon 306 in EmbB do not cause ETB resistance but predispose M. tuberculosis isolates to the development of drug resistance and increase the capacity of resistant isolates to be transmitted (38, 50–52). Accordingly, several reports have described isolates with mutations at codon 306 of EmbB that remain susceptible to ETB (2, 4, 8, 13, 17, 25, 29, 30, 34, 36, 46, 51, 52). There are two explanations for the presence of a mutation at codon 306 in EmbB in an ETB-S isolate: the ETB concentration recommended for drug susceptibility testing varies from 2 to 7.5 mg/liter depending on the phenotypic method used for susceptibility testing (39), and the ETB MICs induced by mutations in codon 306 could be close to the breakpoint defining ETB resistance (37, 50).
Other mutations in EmbB that are likely responsible for ETB resistance were identified in our study: the D354A mutation was previously reported for ETB-R strains selected in vitro (37). For the F330I and N399I mutations, other mutations (F330V/S/L and N399T/H/D) were reported in the same codons in ETB-R strains (n = 9) in five studies (16, 17, 23, 36, 53) and in a single ETB-S strain in one study (53). Similarly, the L402V and Y334H mutations were previously described for ETB-R (n = 3) strains in three studies (16, 20, 35) and an ETB-S (n = 1) strain in one study (53), respectively. A total of three new EmbB mutations were identified in our study: S426N, in a Ural isolate harboring no other mutation in embCAB; N296H, in a Beijing isolate with three additional synonymous mutations in EmbA (C76C, Q38Q, and H764H); and A19D, in a Uganda strain harboring N296H in EmbB and G5V in EmbA. Causal relationships between these three mutations and ETB resistance are yet to be experimentally verified. Overall, a strong statistical association was found between ETB resistance and all the embB mutations reported in our study (P < 0.001).
It is worth highlighting here that ETB-R-conferring mutations in embB could be regarded as sensitive markers for the prediction of MDR-TB. By performing trend analysis correlating any ETB-R-conferring mutations in embB to first-line drug resistance, we found a statistically significant association between these mutations and resistance to INH plus RIF (P < 0.001). However, among the 23 strains showing resistance to 2 to 3 first-line anti-TB drugs, excluding ETB, 16 of which were MDR, only 1 was found to have an embB mutation, supporting the idea that resistance to ETB is not a prerequisite for the development of multidrug resistance.
According to the literature, the proportion of ETB-R isolates carrying a mutation in the embC-embA intergenic region varies from 0 to 27% (5, 15, 16, 20, 46, 53). In our study, 23% of ETB-R isolates had mutations at five positions in the embC-embA intergenic region, with a total of six distinct mutations: -c8a/t, -c11a, -c12t, -c16t, and a deletion of 2 nucleotides at positions −21 and −20. None of these mutations was observed in ETB-S isolates, and we found a strong statistical association between ETB resistance and mutations in the embC-embA intergenic region (P < 0.001). Excluding the -c11a mutation, the mutations at positions −8, −12, and −16 and the deletion of cg at positions −21 and −20, which are located within/adjacent to a predicted TATA box (15, 16, 53), were found in ETB-R strains carrying no other mutations involved in ETB resistance, confirming their involvement in ETB resistance. The role of the -c11a mutation is less clear because this mutation was identified in a strain that also has the EmbB M306I mutation, but Cui et al. demonstrated that mutations in the embC-embA intergenic region (including -c11a) increase ETB resistance by enhancing the transcription of embA and embB, with the MICs of ETB for strains with both embC-embA intergenic region mutations and embB mutations being much higher than those for strains with only an embB mutation (53).
Notably, we detected no mutation that is clearly associated with ETB resistance in embC and embA, despite the percentages of ETB-R isolates harboring a mutation in these two genes varying in the literature from 0.6 to 77% and 0 to 12%, respectively (5, 16, 20, 23, 46). In our study, 13% (9/71) of ETB-R isolates and 47% (28/60) of ETB-S isolates had mutations in embC, affecting eight codons, but six of them corresponded to synonymous mutations (n = 3) or markers of phylogenetic lineages (V981L, T270I, and R738Q). Among these mutations, the absence of a role of the EmbC T270I mutation in ETB resistance was experimentally confirmed by site-directed mutagenesis (54). Regarding the last two mutations in EmbC (A426T and V987A), a similar mutation in codon 426 but not with the same substitution (A426P) was described (PolyTB database [http://pathogenseq.lshtm.ac.uk/polytb]). In our study, the A426T and V987A mutations were found in clinical isolates that were susceptible to ETB, suggesting that these mutations play no significant role in ETB resistance. With respect to EmbA, 58% of ETB-R and 18% of ETB-S isolates included in our study had mutations in embA in four codons, but all were synonymous mutations, except G5V. Codon 5 in embA was previously reported by two different groups to have a G5S mutation in ETB-R isolates (16, 17). In our study, this mutation was detected in association with two EmbB mutations, A19D and N296H, the latter being detected alone in another of our ETB-R isolates. Therefore, we cannot definitively state that ETB resistance is associated with the occurrence of the G5V mutation in EmbA.
Finally, we found no isolate with a mutation in embR, despite its presence in 1% to 36% of ETB-R isolates in the literature (5, 16, 23). Based on the data generated from the panel of clinical isolates included in the present study, embR plays no significant role in the development of ETB resistance in M. tuberculosis.
In conclusion, our results strongly suggest that molecular analysis of the embCAB locus should prioritize the search for mutations at the level of two “hot spots” for mutations in order to efficiently detect ETB resistance in clinical isolates of M. tuberculosis. These two regions correspond to (i) the ERDR in EmbB that could be tentatively extended to a 606-bp region spanning residues 296 to 497 and encompassing the canonical mutations at the levels of M306, G406, and Q497 and (ii) the DNA segment extending from positions −8 to −21 in the embC-embA intergenic region. According to our data and excluding synonymous mutations and those corresponding to phylogenetic markers, 70% of ETB-R isolates had amino acid substitutions affecting the M306 (56%), G406 (11%), or Q497 (3%) codon of EmbB; 13% had EmbB mutations outside positions 306, 406, and 497 that are very likely involved in ETB resistance (i.e., N296H, F330I, D354A, N399I, L402V, and S426N); and 15% had nucleotide substitutions in the embC-embA intergenic region between positions −8 and −21 (without taking into account the isolates harboring a mutation in codon 306, 406, or 497). Importantly, the commercial Genotype MTBDRsl DNA strip assay, which is widely used in routine laboratories to detect ETB resistance in clinical M. tuberculosis isolates, evaluates only the EmbB M306 codon, resulting in its limited sensitivity (68%) in the detection of ETB resistance (3). Taken together, the molecular results from testing of the embB ERDR and the embC-embA intergenic region and the results obtained from the MTBDRsl line probe assay should markedly increase the percentage of ETB-R clinical isolates of M. tuberculosis detected by molecular testing, by 85% by testing codons 306, 406, and 497 and nucleotide positions −8, −12, and −16 and by up to 98% by testing the entire embB gene and the embC-embA intergenic region from positions −8 to −21.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by grants from the Ministère de la Recherche (grant UPRES EA 1541) and by an annual grant from the Institute National de la Santé et de la Recherche Médicale (INSERM) and the Université Pierre et Marie Curie (UPMC).
We gratefully acknowledge Gérald Millot for his technical assistance.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00150-15.
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