|Year : 2015 | Volume
| Issue : 3 | Page : 387-392
Mutations at embB306 codon and their association with multidrug resistant M. tuberculosis clinical isolates
A Gupta1, SK Singh2, S Anupurba1
1 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Submission||21-Mar-2014|
|Date of Acceptance||13-Jan-2015|
|Date of Web Publication||12-Jun-2015|
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
Source of Support: Financial assistance to Dr. Anamika Gupta (SRF)
from ICMR, New Delhi., Conflict of Interest: None
Purpose: The presence of embB306 mutation in ethambutol (EMB)-susceptible (EMBs) clinical isolates questions the significance of these mutations in conferring resistance to EMB. The present study was carried out to determine the occurrence of embB306 mutation in EMB-resistant (EMBr) and EMBs strains of M. tuberculosis. One hundred and four multidrug-resistant tuberculosis (MDR-TB) strains were also included to establish the relevance of excessive use of rifampicin (RIF) and isoniazid (INH) in occurrence of embB306 mutations in EMBs M. tuberculosis isolates. Materials and Methods: Deoxyribonucleic acid (DNA) from M. tuberculosis clinical strains was isolated by cetyltrimethylammonium bromide (CTAB) method. Phenotypic and genotypic drug susceptibility testing (DST) was performed on 354 M. tuberculosis isolates by using standard proportion method and multiplex-allele-specific polymerase chain reaction assay, respectively. Results: The overall frequency of embB306 mutations in EMBr isolates was found to be five times higher than its occurrence in EMB-susceptible isolates (50% vs 10%). Further, the association between embB306 mutation and EMB-resistance was observed to be statistically significant (P = 0.000). Conclusion: The embB306 is not only the main causative mutation of EMB resistance, but is a sensitive applicant marker for EMB-resistance study.
Keywords: Adaptive mutation, embB306, multidrug resistance, tuberculosis
|How to cite this article:|
Gupta A, Singh S K, Anupurba S. Mutations at embB306 codon and their association with multidrug resistant M. tuberculosis clinical isolates. Indian J Med Microbiol 2015;33:387-92
|How to cite this URL:|
Gupta A, Singh S K, Anupurba S. Mutations at embB306 codon and their association with multidrug resistant M. tuberculosis clinical isolates. Indian J Med Microbiol [serial online] 2015 [cited 2020 May 29];33:387-92. Available from: http://www.ijmm.org/text.asp?2015/33/3/387/158560
| ~ Introduction|| |
Ethambutol (EMB) acts on cell wall of M. tuberculosis by inhibiting arabinosyl transferases enzyme encoded by the embCAB operon. embCAB operon contains three genes arranged in the order emb-C-embA-embB encoding three homologous arabinosyl transferases. ,, Point mutations at codon 306 of the M. tuberculosis embB gene (embB306) are found in 30-69% of EMB-resistant (EMBr) strains. ,,,,, Earlier studies indicated that the embB306 mutations were only observed in EMBr strains and have been proposed as both a cause of EMB resistance and a marker for EMB resistance in diagnostic assays. , On the other hand, the presence of embB306 mutations in EMB-susceptible (EMBs) clinical isolates questioned the validity of this statement. For the first time, Mokrousov et al., described the presence of embB306 mutations in EMBs M. tuberculosis isolates and noticed that embB306 mutations in EMBs isolates were limited to the isolates already resistant to other drugs. In a pilot study by Hazbon et al., upon an international collection of isolates, 46% of embB306 mutants were fully susceptible to EMB and, like other studies, reported a lack of these mutations in pan-susceptible strains. Instead, mutants in embB306 were found to be strongly associated with resistance to any antibiotic and to multidrug-resistant (MDR) strains, suggesting that embB306 mutations were responsible for broad antibiotic resistance rather than simply for EMB resistance. , Other studies have supported the association between EMB resistance and isoniazid (INH) resistance  or embB306 mutations and MDR. ,,,, The present study was carried out to determine the occurrence of embB306 mutations in all EMBr and susceptible strains of M. tuberculosis with or without having multidrug resistance and to evaluate the performance of multiplex-allele specific-polymerase chain reaction (MAS-PCR) assay The present study was carried out to determine the occurrence of embB306 mutations in all EMBr and susceptible strains of M. tuberculosis with or without having multidrug resistance and to evaluate the performance of multiplex-allele specific-polymerase chain reaction (MAS-PCR) assay and to validate the detection of drug resistance in EMBr M. tuberculosis isolates in low-resource settings.
| ~ Materials and Methods|| |
Tuberculosis-suspected specimens were collected from Sir SundarLal (SS) Hospital, a tertiary-care hospital of Banaras Hindu University (BHU) and two other tuberculosis centres of Varanasi, North India. Three hundred and fifty-four Mycobacterium tuberculosis (MTB) strains were isolated, identified and subjected to indirect drug susceptibility testing (DST) according to the gold standard proportion method (PM). , H37Rv (ATCC 27294) and a known MDR strain were used as negative and positive controls, respectively. The study was approved by the ethical committee of the Institution.
Detection of EMB resistance determinants
Deoxyribonucleic acid (DNA) was isolated according to the method as described by van Embden et al., with minor modifications. A two-step MAS-PCR assay was performed to detect simultaneously mutations in the embB306 first and third bases, which are known to confer EMB resistance, using 354 purified M. tuberculosis DNA.  A strain with the embB306 wild-type allele would produce two allele-specific bands of 160 and 210 bp [[Figure 1], lanes 1 and 6], a strain with embB306 mutated in the first base would produce a 210-bp fragment only [[Figure 1], lanes 4 and 5] and a strain with embB306 mutated in the third base would produce only a 160-bp fragment [[Figure 1], lanes 2, 3 and 7]. In addition, a 324-bp fragment is invariably amplified by the outer primers E1 and E2 [Table 1]. The primers used for a single-tube PCR targeting a portion of the embB gene (embB positions 730 to 1053 in strain H37Rv; accession number Z80343, positions 33265 to 33588) were described in [Table 1]. The compositions of reaction mixtures and PCR condition for embB codon 306 was used as described by Mokrousov et al., with few modifications.
|Figure 1: Agarose gel electrophoresis patterns of embB306 MAS-PCR assay: Lanes: 1 and 6, strains with embB306 wild-type allele; 2, 3 and 7, strains with the embB306 ATG to ATH mutation; 4 and 5, strains with embB306 ATG to BTG mutation ("B" represents G, C, or T; "H" represents A, C, or T). Lane M, 100-bp DNA ladder (Bangalore Genei)|
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|Table 1: Description of primers for the amplification of embB gene and nucleotide changes responsible for ethambutol resistance |
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Detection of rifampicin and INH resistance determinants
A two-step MAS-PCR assay was performed to detect mutations at rpoB codons (516, 526 and 531) and katG codon 315. ,
| ~ Results|| |
Among all, M. tuberculosis isolates included in this study, 117, 129, 113 and 123 were detected resistant to RIF, INH, EMB and STR (Streptomycin), respectively by PM. In addition, 104/354 (29.38%) isolates were observed to be MDR.
Of the 113 EMBr M. tuberculosis isolates, 16 (14.16%) were monoresistant to EMB, 86 and 76 isolates were additionally resistant to INH and RIF, respectively, while 75 were found to be MDR. Fifty-six (49.56%) of 113 EMBr M. tuberculosis isolates contained a mutated embB306. Moreover, 43 and 35 of these 56 strains were additionally resistant to INH and RIF, respectively. Further, 33/75 MDR-TB isolates also had mutation in embB306 while 12/16 strains which were monoresistant to EMB-harboured mutated embB306 codon [Table 2].
|Table 2: Incidence of embB306 mutations in EMB - susceptible and resistant MTB isolates resistant to INH and RIF drugs |
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In 241 EMBs M. tuberculosis isolates, INH and RIF resistance was observed in 43 and 41 M. tuberculosis isolates, respectively. Further, 29 were observed to be MDR. Twenty-three (9.54%) of 241 EMBs M. tuberculosis isolates had a mutation in embB306 codon. The distribution of embB306 allelic variants among 241 EMBs strains was as follows: ATG (wild type), 218 strains; BTG, 13 strains; ATH, 10 strains (according to the degenerated base code, B is G, C or T and H is A, C or T). Further, of these 23 strains, 11 and 8 were additionally resistant to INH and RIF, respectively, and had a mutated embB306. Further, 8/29 MDR-TB isolates also had mutation in embB306 while 12 strains which were pan-susceptible isolates had a mutated embB306. Thus, the overall frequency of embB306 mutations in North India in EMBr isolates was five times higher than its occurrence in EMBs isolates resistant to other first-line drugs (50% vs 10%). Further, of 23 EMBs strains with a mutated embB306, 13 isolates contained the mutation ATG to GTG (Met to Val) while 10 isolates had ATG to ATA (Met to Ile).
Of 241 EMBs strains, 218 (90.46%) showed a three-band profile implying no mutation in embB306 [[Figure 1], lanes 1 and 5] while 23 EMBs isolates had a mutated embB306 codon. The distribution of embB306 allelic variants among 113 EMBr strains was as follows: ATG (wild type), 57 strains; BTG, 26 strains; ATH, 30 strains (according to the degenerated base code, B is G, C or T and H is A, C or T). Of note, the assay used provides double PCR quality confirmation so as to rule out false-negative results due to a lack of amplification. The sensitivities, specificities and accuracy of MAS-PCR assay compared to that of PM for the detection of embB306 mutation was observed to be 50%, 90% and 77%, respectively [Supplementary Table].
The MAS-PCR data also showed the presence of INH- and RIF-resistant specific mutations at embB306 in mutant strains [Table 3]. Six of 23 EMBs M. tuberculosis strains with a mutated embB306 also contained a mutation at katG315 and rpoB codons while eight culture and MAS-PCR proven pan-susceptible isolates also have embB306 gene alteration. Further, 38 of 56 EMBr strains with a mutation at embB306 that were additionally resistant to INH also contained a mutation at katG315. Moreover, 25 of 56 EMBr strains with a mutation at embB306 that were additionally resistant to RIF also contained a mutation at one of three rpoB codons [Table 3].
|Table 3: Occurrence of katG codon 315 and rpoB codons 516, 526, 531 mutations in INH and RIF resistant isolates respectively with a mutated embB306 gene (n=79) |
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| ~ Discussion|| |
The presence of embB306 mutation in EMBs clinical isolates questions the significance of these mutations in conferring resistance to EMB. ,,,, We have found 10% (23/241) of EMBs isolates to contain embB306-mutated allele. While a study from Kuwait reported a very low level (2%, 3/122) and a study from Russia found a very high level (31.2%, 48/154) of mutated embB306 in EMBs isolates in their findings , while Plinke et al., showed no embB306 mutation in EMBs strains.
In our study, the occurrence (56 of 113, 50%) of embB306 mutations in EMBr M. tuberculosis isolates from North India is similar to other studies in which they have found 47-69% of embB306 mutations in genotypically distinct EMBr strains ,,,,, but higher than the occurrence of 25-30% of these mutations in other studies. ,
In some studies, EMBr and EMBs strains mostly involved either MDR-TB strains or additionally resistant to several other anti-TB drugs. ,,,,, In our study, a total of 41 (39.42%) mutated embB306 were detected in 104 MDR-TB strains. Our observation differs from the study of Plinke et al., in which they showed no embB306 mutation in EMBs MDR strains but agrees with another observation.  In the present study, 44 EMBr strains which were additionally resistant to either INH or RIF contained a mutated embB306 [Table 2]. Further, 6/23 EMBs M. tuberculosis strains with a mutated embB306 also contained a mutation at katG315 and rpoB codons (531 = 4, 526 = 2). Moreover, 38/56 and 25/56 EMBr strains with a mutation at embB306 also contained a mutation at katG315 and one of three rpoB codons (rpoB531 = 19, 526 = 4, 516 = 2) [Table 3]. Similarly, in a study from China,  38.0% (92/242 isolates) all of the strains with a mutation in embB306 also had mutations in rpoB, and 92.4% (85/92 strains) of them had a mutation in katG, inhA or ahpC.
In the present study, 6 of 241 (2.49%) EMBs isolates were found to have katG315 mutations. Similarly, Ahmad et al., found 3 of 122 (2%) EMBs isolate resistant to INH plus one or more other anti-TB drugs contained a mutated embB306. The authors suggested that M. tuberculosis isolates with katG315 mutations are more likely to acquire high-level resistance to EMB and embB306 mutations. , The resistance of M. tuberculosis to INH develops more readily and nearly 50-95% of all INH resistant isolates, irrespective of embB306 status, from some geographical locations contain katG315 mutations. ,, Since strains with katG315 mutations remain infectious and are more likely to acquire further resistance to other anti-TB drugs  so it might be a possible reason behind opposite EMB susceptibility results. However, in our study, only 6/23 EMBs isolates with embB306 alteration had additional katG315 mutation but 11/23 were phenotypically INH resistant so there may be a strong possibility of any other gene alteration in remaining five INH-resistant isolates. Although the exact role of embB306 mutations play in the development of EMB resistance and multidrug resistance in M. tuberculosis is not fully understood. It is believed that variant genetic alterations that accumulate in epidemic M. tuberculosis lead to the development of drug resistance, ,, including EMB resistance,  or the reason behind this discrepancy may be that embB306 mutations confer M. tuberculosis variable EMB MICs and clinical strains with low to moderate levels of resistance may readily show opposite EMB susceptibility results. ,,, Further, the excessive use of INH and other TB drugs such as ciprofloxacin indicates the possibility of mechanisms such as decreased permeability of the bacterial membrane or increased efflux of these antibiotics playing a role in the development of resistance in M. tuberculosis. ,
In present study, the association between embB306 mutation and EMB-resistance is statistically significant (odds ratio = 9.312; P = 0.000; 95% confidence interval, CI, =5.102-17.103), our data suggest that embB306 is not the sole causative mutation of EMB-resistance, but is a sensitive applicant marker for EMB-resistance study. Similar observations were also reported in some studies. , In contrast to our result, a study by Bakula et al.,  concluded that the embB gene is not sufficient for rapid detection of EMB resistance, and the codon 306 mutations are not good predictive markers of resistance to EMB.
MAS-PCR assay was used for the detection of embB306 mutation  as it provides double PCR quality confirmation so as to rule out false-negative results due to a lack of amplification [Figure 1].
Besides being informative, the study had some limitations like: As a significant number of phenotypically EMBr isolates do not carry mutations in the embB306 codon, investigation of mutations in other codons like embB406, embB497 and embCAB operon, can add information in the existing knowledge of mutations in the EMBr/EMBs isolates.
| ~ Conclusions|| |
We conclude that the presence of embB306 mutations in a minority of phenotypically EMBs strains may be either due to the inherent problems associated with DST for EMB or due to adaptive mutations that is influenced by excessive use of INH or RIF and their resistance. Further, our data suggests that embB306 is not only the sole causative mutation of EMB resistance, but is a sensitive applicant marker for EMB resistance study.
| ~ Acknowledgements|| |
We are thankful to all the patients and volunteers for providing clinical specimens. Financial assistance to Dr. Anamika Gupta (SRF) from Indian Council of Medical Research (ICMR), New Delhi, India is also thankfully acknowledged.
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[Table 1], [Table 2], [Table 3]