Current Microbiology

, Volume 53, Issue 5, pp 401–405

Nucleotide Polymorphism Associated with Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis

  • S. Srivastava
  • A. Garg
  • A. Ayyagari
  • K. K. Nyati
  • T. N. Dhole
  • S. K. Dwivedi


Ethambutol (EMB) is a first-line drug used for antitubercular therapy in combination with other drugs as recommended by World Health Organization DOTS/DOTS-Plus regimens. EMB is also effective in the treatment of opportunistic mycobacterial infections in patients with human immunodeficiency virus. The emb locus has been considered as a drug target for EMB, and substitutions of codon 306 in Mycobacterium tuberculosis gene embB have been shown to be the most frequent and predictive mutations for EMB resistance. The aim of the present study was to detect embB and embC gene mutations in EMB-resistant clinical isolates. A total of 23 isolates of M. tuberculosis from patients with pulmonary tuberculosis were included in the study. Drug sensitivity was tested by proportion method and E-test. All 23 isolates were EMB resistant. Primers to amplify the embB and embC gene were designed, and polymerase chain reaction products were subjected for sequence analysis. H37Rv standard laboratory strain was used as control. Nucleotide sequencing showed that 16 strains had a mutation in the embB gene. The most common mutation observed in the embB gene was at codon 306, followed by mutations at codons 299 and 378 in 4 and 2 isolates, respectively. Novel mutations have been reported at codons 239, 240, 247, 282, 311, 368, 397, 446, 469, and 471. Sequence analysis of the embC gene showed mutation in 8 isolates at codon 270. Novel mutations in embC have been reported at codons 251 and 254. The most common nucleotide polymorphism in our isolates was at codons 306 and 299 in the embB gene and at codon 270 in the embC gene. A mutation at codon 306 was usually associated with high-level ethambutol resistance.

Literature Cited

  1. 1.
    Blessington B, Beiraghi A (1990) Study of the stereochemistry of ethambutol using chiral liquid chromatography and synthesis. J Chromatogr 522:195–203CrossRefGoogle Scholar
  2. 2.
    Wilkinson RG, Sheperd RG, Thomas JP, Baughn C (1961) Stereospecificity of a new type of synthetic antituberculosis agent. J Am Chem Soc 83:2212–2213CrossRefGoogle Scholar
  3. 3.
    Alcaide F, Pfyffer GE, Telenti A (1997) Role of embB in natural and acquired resistance to ethambutol in mycobacteria. Antimicrob Agents Chemother 41:2270–2273PubMedGoogle Scholar
  4. 4.
    Belanger AE, Besra GS, Ford ME, MikusováK, Belisle JT, Brennan PJ, et al. (1996) The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc Natl Acad Sci USA 93:11919–11924PubMedCrossRefGoogle Scholar
  5. 5.
    Maddry JA, Suling WJ, Reynolds RC (1996) Glycosyltransferases as targets for inhibition of cell wall synthesis in M. tuberculosis and M. avium. Res Microbiol 147:106–112PubMedCrossRefGoogle Scholar
  6. 6.
    Mikusová K, Slayden RA, Besra GS, Brennan PJ (1995) Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob Agents Chemother 39:2484–2489PubMedGoogle Scholar
  7. 7.
    Besra GS, Khoo KH, McNeil MR, Dell A, Morris HR, Brennan PJ (1995) A new interpretation of the structure of the mycolylarabinogalactan complex of Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol fragments by fast-atom bombardment, mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Biochemistry 34:4257–4266PubMedCrossRefGoogle Scholar
  8. 8.
    Brennan PJ, Nikaido H (1995) The envelope of mycobacteria. Annu Rev Biochem 64:39–63CrossRefGoogle Scholar
  9. 9.
    Khoo KH, Douglas E, Azadi P, Inamine JM, Besra GS, Mikusová K, et al. (1996) Truncated structural variants of lipoarabinomannan in ethambutol drug-resistant strains of Mycobacterium smegmatis. Inhibition of arabinan biosynthesis by ethambutol. J Biol Chem 271:28682–28690PubMedCrossRefGoogle Scholar
  10. 10.
    Takayama K, Armstrong EL, Kunugi KA, Kilburn JO (1979) Inhibition by ethambutol of mycolic acid transfer into the cell wall of Mycobacterium smegmatis. Antimicrob Agents Chemother 16:240–242PubMedGoogle Scholar
  11. 11.
    Takayama K, Kilburn JO (1989) Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 33:1493–1499PubMedGoogle Scholar
  12. 12.
    Musser JM (1995) Antimicrobial agent resistance in mycobacteria: Molecular genetic insights. Clin Microbiol Rev 8:496–514PubMedGoogle Scholar
  13. 13.
    Sander P, Bottger EC (1999) Mycobacteria: Genetics of resistance and implications for treatment. Chemotherapy 45:95–108PubMedCrossRefGoogle Scholar
  14. 14.
    Telenti A, Philipp WJ, Sreevatsan S, Bernasconi C, Stockbauer KE, Wieles B, et al. (1997) The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat Med 3:567–570PubMedCrossRefGoogle Scholar
  15. 15.
    Escalante P, Ramaswamy S, Sanabria H, Soini H, Pan X, Valiente Castillo O, et al. (1998) Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru. Tuberc Lung Dis 79:111–118CrossRefGoogle Scholar
  16. 16.
    Lee HY, Myoung HJ, Bang HE, Bai GH, Kim SJ, Kim JD, et al. (2002) Mutations in the embB locus among Korean clinical isolates of Mycobacterium tuberculosis resistant to ethambutol. Yonsei Med J 43:59–64PubMedGoogle Scholar
  17. 17.
    Ramaswamy SV, Amin AG, Goksel S, Stager CE, Dou SJ, El Sahly H, et al. (2000) Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 44:326–336PubMedCrossRefGoogle Scholar
  18. 18.
    Ramaswamy SV, Dou SJ, Rendon A, Yang Z, Cave MD, Graviss EA (2004) Genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates from Monterrey, Mexico. J Med Microbiol 53:107–113PubMedCrossRefGoogle Scholar
  19. 19.
    Sreevatsan S, Stockbauer KE, Pan X, Kreiswirth BN, Moghazeh SL, Jacobs Jr WR, et al. (1997) Ethambutol resistance in Mycobacterium tuberculosis: Critical role of embB mutations. Antimicrob Agents Chemother 41:1677–1681PubMedGoogle Scholar
  20. 20.
    Van Rie A, Warren R, Mshanga I, Jordaan AM, van der Spuy GD, Richardson M, et al. (2001) Analysis for a limited number of gene codons can predict drug resistance of Mycobacterium tuberculosis in a high-incidence community. J Clin Microbiol 39:636–641PubMedCrossRefGoogle Scholar
  21. 21.
    Chadha VK (2005) Tuberculosis epidemiology in India: A review. Int J Tuberc Lung Dis 9:1072–1082PubMedGoogle Scholar
  22. 22.
    WHO Global Tuberculosis Control–WHO Report (2002) Geneva, Switzerland. WHO/CDS/TB/2002Google Scholar
  23. 23.
    Manual of Laboratory Methods (1987) Tuberculosis Research Centre, Chennai, IndiaGoogle Scholar
  24. 24.
    van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD (1991) Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: Evaluation of an insertion sequence–dependent DNA polymorphism as a tool in epidemiology of tuberculosis. J Clin Microbiol 29:2578–2586PubMedGoogle Scholar
  25. 25.
    Masur H (1993). Recommendation on prophylaxis and therapy for disseminated M. avium complex disease inpatients infected with HIV virus. N Engl J Med 329:828–833CrossRefGoogle Scholar
  26. 26.
    Deng L, Milkusova´ K, Robuck KG, Scherman M, Brennan PJ, McNeil MR (1995) Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope. Antimicrob Agents Chemother 39:694–701PubMedGoogle Scholar
  27. 27.
    Forbes M, Kuck NA, Peets EA (1965) Effect of ethambutol on nucleic acid metabolism in Mycobacterium smegmatis and its reversal by polyamines and divalent cations. J Bacteriol 89:1299–1305PubMedGoogle Scholar
  28. 28.
    Kilburn JO, Takayama K, Armstrong EL, Greenberg J (1981) Effects of ethambutol on phospholipid metabolism in Mycobacterium smegmatis. Antimicrob Agents Chemother 19:346–348PubMedGoogle Scholar
  29. 29.
    Wolucka BA, McNeil MR, de Hoffmann E, Chojnacki T, Brennan PJ (1994) Recognition of the lipid intermediate for arabinogalactan/ arabinomannan biosynthesis and its relation to the mode of action of ethambutol on mycobacteria. J Biol Chem 269:23328–23335PubMedGoogle Scholar
  30. 30.
    Mokrousov I, Otten T, Vyshevskiy, Narvskaya O (2002) Detection of emb306 mutations in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis from Northwest Russia: Implications for genotypic resistance testing. J Clin Microbiol 40:3810–3813PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • S. Srivastava
    • 1
  • A. Garg
    • 1
  • A. Ayyagari
    • 1
  • K. K. Nyati
    • 1
  • T. N. Dhole
    • 1
  • S. K. Dwivedi
    • 2
  1. 1.Department of MicrobiologySanjay Gandhi Postgraduate Institute of Medical SciencesLucknowIndia
  2. 2.Department of Environmental ScienceBabasaheb Bhimrao Ambedkar UniversityLucknowIndia

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