Molecular Detection of Drug-Resistant Mycobacterium tuberculosis with a Scanning-Frame Oligonucleotide Microarray

  • Dmitriy V. Volokhov
  • Vladimir E. Chizhikov
  • Steven Denkin
  • Ying Zhang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 465)

Abstract

The increasing emergence of drug-resistant Mycobacterium tuberculosis poses significant threat to the treatment of tuberculosis (TB). Conventional drug susceptibility testing is time-consuming and takes several weeks because of the slow growth rate of M. tuberculosis and the requirement for the drugs to show antimycobacterial activity. Resistance to TB drugs in M. tuberculosis is caused by mutations in the corresponding drug resistance genes (e.g., katG, inhA, rpoB, pncA, embB, rrs, gyrA, gyrB), and detection of these mutations can be a molecular indicator of drug resistance. In this chapter, we describe the utility of a microarray-based approach exploiting short overlapping oligonucleotides (sliding-frame array) to rapidly detect drug resistance–associated mutations (substitutions, deletions, and insertions) in the pncA gene responsible for resistance ofM. tuberculosis to pyrazinamide (PZA) as an example for this approach. Hybridization of pncA-derived RNA or DNA with the microarray enables easy and simple screening of nucleotide changes in the pncA gene. Sliding-frame microarrays can be used to identify other drug-resistant TB strains that have mutations in relevant drug resistance genes.

Keywords

drug resistance microarray mutations tuberculosis 

Notes

Acknowledgments

This work was supported by NIH grants AI44063, AI/HL49485, and AI61908.

References

  1. 1.
    Brudey, K., Driscoll, J. R., Rigouts, L., Prodinger, W. M., Gori, A., Al-Hajoj, S. A., Allix, C., Aristimuno, L., Arora, J., Baumanis, V., Binder, L., Cafrune, P., Cataldi, A., Cheong, S., Diel, R., Ellermeier, C., Evans, J. T., Fauville-Dufaux, M., Ferdinand, S., Garcia de Viedma, D., Garzelli, C., Gazzola, L., Gomes, H. M., Guttierez, M. C., Hawkey, P. M., van Helden, P. D., Kadival, G. V., Kreiswirth, B. N., Kremer, K., Kubin, M., Kulkarni, S. P., Liens, B., Lillebaek, T., Ho, M. L., Martin, C., Martin, C., Mokrousov, I., Narvskaia, O., Ngeow, Y. F., Naumann, L., Niemann, S., Parwati, I., Rahim, Z., Rasolofo-Razanamparany, V., Rasolonavalona, T., Rossetti, M. L., Rusch-Gerdes, S., Sajduda, A., Samper, S., Shemyakin, I. G., Singh, U. B., Somoskovi, A., Skuce, R. A., van Soolingen, D., Streicher, E. M., Suffys, P. N., Tortoli, E., Tracevska, T., Vincent, V., Victor, T. C., Warren, R. M., Yap, S. F., Zaman, K., Portaels, F., Rastogi, N., Sola, C. (2006) Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6, 23.PubMedCrossRefGoogle Scholar
  2. 2.
    Rusen, I. D., Enarson, D. A. (2006) FIDELIS––innovative approaches to increasing global case detection of tuberculosis. Am J Public Health 96, 14–16.PubMedCrossRefGoogle Scholar
  3. 3.
    Zignol, M., Hosseini, M. S., Wright, A., Weezenbeek, C. L., Nunn, P., Watt, C. J., Williams, B. G., Dye, C. (2006) Global incidence of multidrug-resistant tuberculosis. J Infect Dis 194, 479–485.PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang, Y., C. Vilcheze, W. R. Jacobs Jr. (2005) Mechanisms of drug resistance in Mycobacterium tuberculosis. In Tuberculosis and the Tubercle Bacillus, ed. Stewart T. Cole, K. D. E., David N. McMurray, William R. Jacobs, Jr., pp. 115–140. Washington, D.C: ASM Press.Google Scholar
  5. 5.
    Telenti, A., Iseman, M. (2000) Drug-resistant tuberculosis: what do we do now? Drugs 59, 171–179.PubMedCrossRefGoogle Scholar
  6. 6.
    Wade, M. M., Volokhov, D., Peredelchuk, M., Chizhikov, V., Zhang, Y. (2004) Accurate mapping of mutations of pyrazinamide-resistant Mycobacterium tuberculosis strains with a scanning-frame oligonucleotide microarray. Diagn Microbiol Infect Dis 49, 89–97.PubMedCrossRefGoogle Scholar
  7. 7.
    Wade, M. M., Zhang, Y. (2004) Mechanisms of drug resistance in Mycobacterium tuberculosis. Front Biosci 9, 975–994.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang, Y. (2005) The magic bullets and tuberculosis drug targets. Annu Rev Pharmacol Toxicol 45, 529–564.PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang, Y., Amzel, L. M. (2002) Tuberculosis drug targets. Curr Drug Targets 3, 131–154.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang, Y., Post-Martens, K., Denkin, S. (2006) New drug candidates and therapeutic targets for tuberculosis therapy. Drug Discov Today 11, 21–27.PubMedCrossRefGoogle Scholar
  11. 11.
    Garcia de Viedma, D. (2003) Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches. Clin Microbiol Infect 9, 349–359.PubMedCrossRefGoogle Scholar
  12. 12.
    Musser, J. M. (1995) Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 8, 496–514.PubMedGoogle Scholar
  13. 13.
    Telenti, A., Persing, D. H. (1996) Novel strategies for the detection of drug resistance in Mycobacterium tuberculosis. Res Microbiol 147, 73–79.PubMedCrossRefGoogle Scholar
  14. 14.
    Cardoso, R. F., Cooksey, R. C., Morlock, G. P., Barco, P., Cecon, L., Forestiero, F., Leite, C. Q., Sato, D. N., Shikama Mde, L., Mamizuka, E. M., Hirata, R. D., Hirata, M. H. (2004) Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil. Antimicrob Agents Chemother 48, 3373–3381.PubMedCrossRefGoogle Scholar
  15. 15.
    Kiepiela, P., Bishop, K. S., Smith, A. N., Roux, L., York, D. F. (2000) Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa. Tuber Lung Dis 80, 47–56.PubMedCrossRefGoogle Scholar
  16. 16.
    Felmlee, T. A., Liu, Q., Whelen, A. C., Williams, D., Sommer, S. S., Persing, D. H. (1995) Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting. J Clin Microbiol 33, 1617–1623.PubMedGoogle Scholar
  17. 17.
    Shi, R., Otomo, K., Yamada, H., Tatsumi, T., Sugawara, I. (2006) Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease. Microbes Infect 8, 128–135.PubMedCrossRefGoogle Scholar
  18. 18.
    Johnson, R., Jordaan, A. M., Pretorius, L., Engelke, E., van der Spuy, G., Kewley, C., Bosman, M., van Helden, P. D., Warren, R., Victor, T. C. (2006) Ethambutol resistance testing by mutation detection. Int J Tuberc Lung Dis 10, 68–73.PubMedGoogle Scholar
  19. 19.
    Bockstahler, L. E., Li, Z., Nguyen, N. Y., Van Houten, K. A., Brennan, M. J., Langone, J. J., Morris, S. L. (2002) Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance. Biotechniques 32, 508–510, 512, 514.PubMedGoogle Scholar
  20. 20.
    Marme, N., Friedrich, A., Muller, M., Nolte, O., Wolfrum, J., Hoheisel, J. D., Sauer, M., Knemeyer, J. P. (2006) Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Nucleic Acids Res 34, e90.PubMedCrossRefGoogle Scholar
  21. 21.
    Van Rie, A., Warren, R., Mshanga, I., Jordaan, A. M., van der Spuy, G. D., Richardson, M., Simpson, J., Gie, R. P., Enarson, D. A., Beyers, N., van Helden, P. D., Victor, T. C. (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, 636–641.PubMedCrossRefGoogle Scholar
  22. 22.
    Denkin, S., Volokhov, D., Chizhikov, V., Zhang, Y. (2005) Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis. J Med Microbiol 54, 1127–1131.PubMedCrossRefGoogle Scholar
  23. 23.
    Ivshina, A. V., Vodeiko, G. M., Kuznetsov, V. A., Volokhov, D., Taffs, R., Chizhikov, V. I., Levandowski, R. A., Chumakov, K. M. (2004) Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method. J Clin Microbiol 42, 5793–5801.PubMedCrossRefGoogle Scholar
  24. 24.
    Sergeev, N., Volokhov, D., Chizhikov, V., Rasooly, A. (2004) Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay. J Clin Microbiol 42, 2134–2143.PubMedCrossRefGoogle Scholar
  25. 25.
    Volokhov, D. V., George, J., Liu, S. X., Ikonomi, P., Anderson, C., Chizhikov, V. (2006) Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing. Appl Microbiol Biotechnol 71, 680–698.PubMedCrossRefGoogle Scholar
  26. 26.
    Neverov, A. A., Riddell, M. A., Moss, W. J., Volokhov, D. V., Rota, P. A., Lowe, L. E., Chibo, D., Smit, S. B., Griffin, D. E., Chumakov, K. M., Chizhikov, V. E. (2006) Genotyping of measles virus in clinical specimens on the basis of oligonucleotide microarray hybridization patterns. J Clin Microbiol 44, 3752–3759.PubMedCrossRefGoogle Scholar
  27. 27.
    Caoili, J. C., Mayorova, A., Sikes, D., Hickman, L., Plikaytis, B. B., Shinnick, T. M. (2006) Evaluation of the TB-Biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis. J Clin Microbiol 44, 2378–2381.PubMedCrossRefGoogle Scholar
  28. 28.
    Aragon, L. M., Navarro, F., Heiser, V., Garrigo, M., Espanol, M., Coll, P. (2006) Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays. J Antimicrob Chemother 57, 825–831.PubMedCrossRefGoogle Scholar
  29. 29.
    Gryadunov, D., Mikhailovich, V., Lapa, S., Roudinskii, N., Donnikov, M., Pan'kov, S., Markova, O., Kuz'min, A., Chernousova, L., Skotnikova, O., Moroz, A., Zasedatelev, A., Mirzabekov, A. (2005) Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clin Microbiol Infect 11, 531–539.PubMedCrossRefGoogle Scholar
  30. 30.
    Tang, X., Morris, S. L., Langone, J. J., Bockstahler, L. E. (2005) Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance. J Microbiol Methods 63, 318–330.PubMedCrossRefGoogle Scholar
  31. 31.
    Yoshikawa, Y., Ichihara, T., Suzuki, Y. (2003) Detection of drug-resistant Mycobacterium tuberculosis isolates using DNA microarray. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi 14, 45–50.PubMedGoogle Scholar
  32. 32.
    Yue, J., Shi, W., Xie, J., Li, Y., Zeng, E., Liang, L., Wang, H. (2004) Detection of rifampin-resistant Mycobacterium tuberculosis strains by using a specialized oligonucleotide microarray. Diagn Microbiol Infect Dis 48, 47–54.PubMedCrossRefGoogle Scholar
  33. 33.
    Strizhkov, B. N., Drobyshev, A. L., Mikhailovich, V. M., Mirzabekov, A. D. (2000) PCR amplification on a microarray of gel-immobilized oligonucleotides: detection of bacterial toxin- and drug-resistant genes and their mutations. Biotechniques 29, 844–848, 850–842, 854 passim.PubMedGoogle Scholar
  34. 34.
    Tillib, S. V., Strizhkov, B. N., Mirzabekov, A. D. (2001) Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip. Anal Biochem 292, 155–160.PubMedCrossRefGoogle Scholar
  35. 35.
    Sivkov, A., Boldyrev, A. N., Azaev, M., Bodnev, S. A., Medvedeva, E. V., Baranova, O. I., Ivlev-Dantau, A. P., Blinova, L. N., Pasechnikov, A. D., Tat'kov, S. I. (2006) Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates. Mol Gen Mikrobiol Virusol 2, 20–25.Google Scholar
  36. 36.
    Cherkasova, E., Laassri, M., Chizhikov, V., Korotkova, E., Dragunsky, E., Agol, V. I., Chumakov, K. (2003) Microarray analysis of evolution of RNA viruses: evidence of circulation of virulent highly divergent vaccine-derived polioviruses. Proc Natl Acad Sci U S A 100, 9398–9403.PubMedCrossRefGoogle Scholar
  37. 37.
    Heller, M. J. (2002) DNA microarray technology: devices, systems, and applications. Annu Rev Biomed Eng 4, 129–153.PubMedCrossRefGoogle Scholar
  38. 38.
    Rudert, F. (2000) Genomics and proteomics tools for the clinic. Curr Opin Mol Ther 2, 633–642.PubMedGoogle Scholar
  39. 39.
    Cirino, P. C., Mayer, K. M., Umeno, D. (2003) Generating mutant libraries using error-prone PCR. Methods Mol Biol 231, 3–9.PubMedGoogle Scholar
  40. 40.
    Pritchard, L., Corne, D., Kell, D., Rowland, J., Winson, M. (2005) A general model of error-prone PCR. J Theor Biol 234, 497–509.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Dmitriy V. Volokhov
    • 1
  • Vladimir E. Chizhikov
    • 1
  • Steven Denkin
    • 1
  • Ying Zhang
    • 2
  1. 1.Center for Biologics Evaluation and ResearchU.S. Food and Drug AdministrationRockvilleUSA
  2. 2.Department of Molecular Microbiology and Immunology, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA

Personalised recommendations