PCR-Based Methodology for Detecting Multidrug-Resistant Strains of Mycobacterium tuberculosis Beijing Family Circulating in Russia

  • I. Mokrousov
  • T. Otten
  • A. Vyazovaya
  • E. Limeschenko
  • M. L. Filipenko
  • C. Sola
  • N. Rastogi
  • L. Steklova
  • B. Vyshnevskiy
  • O. Narvskaya
Article

Abstract

The Beijing genotype of Mycobacterium tuberculosis has been identified in 40–50% of the clinical isolates studied in Russia during the last decade. This genotype has been reported to be associated with multiple drug resistance and possesses some significant pathogenic properties. Therefore, early identification of such strains is of extreme importance in the timely detection of drug resistance. The present study was performed on 354 strains isolated in Russia from 1996 to 2002 and previously characterised by IS6110-restriction fragment length polymorphism (RFLP) typing and spoligotyping. These strains included 198 Beijing family strains and 156 strains of other genotypes (IS6110-RFLP profiles). A subsequent polymerase chain reaction (PCR) analysis with IS6110-derived outwardly oriented primers (IS6110-PCR) easily discriminated the Beijing strains from non-Beijing strains. The multiplex allele-specific (MAS)-PCR assays were further used to detect mutations in katG315 and rpoB531, associated with resistance to isoniazid and rifampin, respectively. The katG315 and rpoB531 mutations were found to be more prevalent among Beijing (96.8% and 77.3%) than among non-Beijing strains (85.7% and 28%). Consequently, we propose a two-step methodology based on routine PCR and simple agarose gel electrophoresis in order to detect (i) a Beijing family strain using IS6110-PCR, and, (ii) its possible resistance to the major anti-tuberculosis drugs using specific MAS-PCR assays.

References

  1. 1.
    Sreevatsan S, Pan X, Stockbauer K, Connell N, Kreiswirth B, Whittam T, Musser JM (1997) Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionary recent global dissemination. Proc Natl Acad Sci USA 97:9869–9874CrossRefGoogle Scholar
  2. 2.
    Embden JDA van, Gorkom T van, Kremer K, Jansen T, der Zeijst BAM van, Schouls LM (2000) Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria. J Bacteriol 182:2393–2401CrossRefPubMedGoogle Scholar
  3. 3.
    Sola C, Filliol I, Legrand E, Mokrousov I, Rastogi N (2001) Mycobacterium tuberculosis phylogeny reconstruction based on combined numerical analysis with IS1081, IS6110, VNTR, and DR-based spoligotyping suggests the existence of two new phylogeographical clades. J Mol Evol 53:680–689CrossRefPubMedGoogle Scholar
  4. 4.
    Soolingen D van, Qian L, Haas PEW de, Douglas JT, Traore H, Portaels F, Quing Z, Enkhasaikan D, Nymadawa P, Embden JDA van (1995) Predominance of a single genotype of Mycobacterium tuberculosis in countries of East Asia. J Clin Microbiol 33:3234–3238PubMedGoogle Scholar
  5. 5.
    Sola C, Filliol I, Guttieres MC, Mokrousov I, Vincent V, Rastogi N (2001) Spoligotype database of Mycobacterium tuberculosis: biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives. Emerg Infect Dis 7:390–396PubMedGoogle Scholar
  6. 6.
    Bifani PJ, Mathema B, Kurepina NE, Kreiswirth BN (2002) Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol 10:45–52CrossRefPubMedGoogle Scholar
  7. 7.
    Bifani PJ, Mathema B, Liu Z, Moghazeh SL, Shopsin B, Tempalski B, Driscol J, Frothingham R, Musser JM, Alcabes P, Kreiswirth BN (1999) Identification of a W variant outbreak of Mycobacterium tuberculosis via population-based molecular epidemiology. JAMA 282:2321–2327CrossRefPubMedGoogle Scholar
  8. 8.
    Plikaytis BB, Marden JL, Crawford JT, Woodley CL, Butler WR, Shinnick TM (1994) Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis. J Clin Microbiol 32:1542–1546PubMedGoogle Scholar
  9. 9.
    Fang Z, Kenna DT, Doig C, Smittipat DN, Palittapongarnpim P, Watt B, Forbes KJ (2001) Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates. J Bacteriol 183:5279–5284CrossRefPubMedGoogle Scholar
  10. 10.
    Fang Z, Forbes KJ (1997) A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome. J Clin Microbiol 35:479–481PubMedGoogle Scholar
  11. 11.
    Sampson SL, Warren RM, Richardson M, Spuy GD van der, Helden P van (1999) Disruption of coding regions by IS6110 insertion in Mycobacterium tuberculosis. Tuberc Lung Dis 79:349–359CrossRefGoogle Scholar
  12. 12.
    Jansen R, Embden JDA van, Gaastra W, Schouls LM (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43:1565–1575CrossRefPubMedGoogle Scholar
  13. 13.
    Glynn JR, Whiteley J, Bifani PJ, Kremer K, Soolingen D van (2002) Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 8:843–849PubMedGoogle Scholar
  14. 14.
    Bifani PJ, Mathema B, Campo M, Moghazeh S, Nivin B, Shashkina E, Driscoll J, Munsiff SS, Frothingham R, Kreiswirth BN (2001) Molecular identification of streptomycin monoresistant Mycobacterium tuberculosis related to multidrug-resistant W strain. Emerg Infect Dis 7:842–848PubMedGoogle Scholar
  15. 15.
    Mokrousov I, Narvskaya O, Limeschenko E, Otten T, Vyshnevskyi B (2002) Novel IS6110 insertion sites in the direct repeat locus of Mycobacterium tuberculosis clinical strains from the St. Petersburg area of Russia, and evolutionary and epidemiological considerations. J Clin Microbiol 40:1504–1507CrossRefPubMedGoogle Scholar
  16. 16.
    Narvskaya O, Mokrousov I, Otten TF, Vyshnevskiy BI (1999) Genetic marking of polyresistant Mycobacterium tuberculosis strains isolated in the north-west of Russia. Probl Tuberk N3:39–41Google Scholar
  17. 17.
    Mokrousov I, Narvskaya O, Otten T, Limeschenko E, Steklova L, Vyshnevskyi B (2002) High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001. Antimicrob Agents Chemother 46:1417–1424CrossRefPubMedGoogle Scholar
  18. 18.
    Ramaswamy SV, Musser JM (1998) Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 79:3–29CrossRefPubMedGoogle Scholar
  19. 19.
    Victor TC, Helden PD van, Warren R (2002) Prediction of drug resistance in M. tuberculosis: molecular mechanisms, tools and applications. IUBMB Life 53:231–237PubMedGoogle Scholar
  20. 20.
    Mokrousov I, Filliol I, Legrand E, Sola C, Otten T, Vyshnevskaya E, Limeschenko E, Vyshnevskiy B, Narvskaya O, Rastogi N (2002) Molecular characterization of multiple-drug-resistant Mycobacterium tuberculosis isolates from north-western Russia and analysis of rifampin resistance using RNA/RNA mismatch analysis as compared to the line-probe assay and sequencing of the rpoB gene. Res Microbiol 153:213–219CrossRefPubMedGoogle Scholar
  21. 21.
    Narvskaya O, Mokrousov I, Limeschenko E, Otten T, Steklova L, Graschenkova O, Vishnevsky B (2000) Molecular characterization of Mycobacterium tuberculosis strains from north-west region of Russia. EpiNorth 2:22–24Google Scholar
  22. 22.
    Marttila HJ, Soini H, Eerola E, Vyshnevskaya E, Vyshnevskiy BI, Otten TF, Vasilyef AV, Viljanen M (1998) A Ser315Thr substitution in KatG is predominant in genetically heterogeneous multidrug-resistant Mycobacterium tuberculosis isolates originating from the St. Petersburg area in Russia. Antimicrob Agents Chemother 42:2443–2445PubMedGoogle Scholar
  23. 23.
    Toungoussova OS, Sandven P, Mariandyshev AO, Nizovtseva NI, Bjune G, Caugant DA (2002) Spread of drug-resistant Mycobacterium tuberculosis strains of the Beijing genotype in the Archangel Oblast, Russia. J Clin Microbiol 40:1930–1937CrossRefPubMedGoogle Scholar
  24. 24.
    Generozov EV, Akopian TA, Govorun VM, Chernoussova LN, Larionova EE, Savinkova SN, Smirnova TG, Golyshevskaia VI, Khomenko AG (2000) Molecular characteristics of multiresistant clinical strains of Mycobacterium tuberculosis isolated in Russia. Mol Genet Mikrobiol Virusol N1:11–17Google Scholar
  25. 25.
    Narvskaya O, Otten T, Limeschenko E, Sapozhnikova N, Graschenkova O, Steklova L, Nikonova A, Filipenko ML, Mokrousov I, Vyshnevskiy B (2002) Nosocomial outbreak of multidrug-resistant tuberculosis caused by a strain of Mycobacterium tuberculosis W-Beijing family in St. Petersburg, Russia. Eur J Clin Microbiol Infect Dis 21:596–602CrossRefPubMedGoogle Scholar
  26. 26.
    Mokrousov I, Narvskaya O, Otten T, Vyazovaya A, Limeschenko E, Steklova L, Vyshnevskyi B (2002) Phylogenetic reconstruction within Mycobacterium tuberculosis Beijing genotype in northwestern Russia. Res Microbiol 153:629–637CrossRefPubMedGoogle Scholar
  27. 27.
    World Health Organization (1993) Treatment of tuberculosis. Guidelines for national programs. WHO, GenevaGoogle Scholar
  28. 28.
    Viljanen MK, Vyshnevskiy BI, Otten TF, Vyshnevskaya E, Marjamäki M, Soini H, Laippala PJ, Vasilyef AV (1998) Survey of drug-resistant tuberculosis in northwestern Russia from 1984 through 1994. Eur J Clin Microbiol Infect Dis 17:177–183PubMedGoogle Scholar
  29. 29.
    World Health Organization (1998) Laboratory services in tuberculosis control. Part III. Culture. Document no. WHO/TB/98.258. WHO, GenevaGoogle Scholar
  30. 30.
    Dragon EA, Spadoro JP, Madej R (1993) Quality control of polymerase chain reaction. In: Persing DH, Smith TF, Tenover FC, White TJ (eds) Diagnostic molecular microbiology. American Society for Microbiology, Washington DC, pp 160–168Google Scholar
  31. 31.
    Mokrousov I, Otten T, Filipenko M, Vyazovaya A, Chrapov E, Limeschenko E, Steklova L, Vyshnevskiy B, Narvskaya O (2002) Detection of isoniazid-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting katG codon 315 variation. J Clin Microbiol 40:2509–2512CrossRefPubMedGoogle Scholar
  32. 32.
    Kamerbeek J, Schouls L, Kolk A, Agterveld M van, Soolingen D van, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, Embden JDA van (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35:907–914PubMedGoogle Scholar
  33. 33.
    Embden JDA van, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnick TM, Small PM (1993) Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 31:406–409PubMedGoogle Scholar
  34. 34.
    Friedman CR, Stoeckle M, Johnson WD Jr, Riley LW (1995) Double-repetitive-element PCR method for subtyping Mycobacterium tuberculosis clinical isolates. J Clin Microbiol 33:1383–1384PubMedGoogle Scholar
  35. 35.
    Ross BC, Dwyer B (1993) Rapid, simple method for typing isolates of Mycobacterium tuberculosis by using the polymerase chain reaction. J Clin Microbiol 31:329–334PubMedGoogle Scholar
  36. 36.
    Krüüner A, Hoffner SE, Sillastu H, Danilovits M, Levina K, Svenson SB, Ghebremichael S, Koivula T, Källenius G (2001) Spread of drug-resistant pulmonary tuberculosis in Estonia. J Clin Microbiol 39:3339–3345CrossRefPubMedGoogle Scholar
  37. 37.
    Crevel R van, Nelwan RH, Lenne W de, Veeraragu Y, der Zanden AG van, Amin Z, Meer JW van der, Soolingen D van (2001) Mycobacterium tuberculosis Beijing genotype strains associated with febrile response to treatment. Emerg Infect Dis 7:880–883PubMedGoogle Scholar
  38. 38.
    Zhang M, Cong J, Yang Z, Samten B, Barnes PF (1999) Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J Infect Dis 179:1213–1217CrossRefPubMedGoogle Scholar
  39. 39.
    Chernoussova LN, Andreevskaya SN, Smirnova TG, Katulina NI, Shudrova MA (2001) Genotyping of mycobacteria recovered from the tuberculosis patients from a penitentiary hospital. Probl Tuberk N7:60–62Google Scholar
  40. 40.
    Portaels F, Rigouts L, Bastian I (1999) Addressing multidrug-resistant tuberculosis in penitentiary hospitals and in the general population of the former Soviet Union. Int J Tuber Lung Dis 3:582–588Google Scholar
  41. 41.
    Pfyffer GE, Strassle A, Gorkom T van, Portaels F, Rigouts L, Mathieu C, Mirzoev F, Traore H, Embden JDA van (2001) Multidrug-resistant tuberculosis in prison inmates, Azerbaijan. Emerg Infect Dis 7:855–861PubMedGoogle Scholar
  42. 42.
    Qian L, Abe C, Lin TP, Yu MC, Cho SN, Wang S, Douglas JT (2002) rpoB genotypes of Mycobacterium tuberculosis Beijing family isolates from East Asian countries. J Clin Microbiol 40:1091–1094CrossRefPubMedGoogle Scholar
  43. 43.
    Rie A van, Warren R, Mshanga I, Jordaan AM, Spuy GD van der, Richardson M, Simpson J, Gie RP, Enarson DA, Beyers N, Helden PD van, Victor TC (2001) Analysis for a limited number of gene codons can predict drug resistance of Mycobacterium tuberculosis in a high-incidence community. J Clin Microbiol 39:636–641CrossRefPubMedGoogle Scholar
  44. 44.
    Mokrousov I, Otten T, Vyshnevskiy B, Narvskaya O (2002) Detection of embB306 mutations in ethambutol-susceptible Mycobacterium tuberculosis clinical isolates from northwestern Russia: implications for genotypic resistance testing. J Clin Microbiol 40:3810–3813CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • I. Mokrousov
    • 1
  • T. Otten
    • 2
  • A. Vyazovaya
    • 1
  • E. Limeschenko
    • 1
  • M. L. Filipenko
    • 3
  • C. Sola
    • 4
  • N. Rastogi
    • 4
  • L. Steklova
    • 5
  • B. Vyshnevskiy
    • 2
  • O. Narvskaya
    • 1
  1. 1.Laboratory of Molecular MicrobiologySt. Petersburg Pasteur InstituteSt. PetersburgRussia
  2. 2.Laboratory of Microbiology of TuberculosisResearch Institute of PhthisiopulmonologySt. PetersburgRussia
  3. 3.Novosibirsk Institute of Bioorganic ChemistryNovosibirskRussia
  4. 4.Institut Pasteur de GuadeloupePointe-à-Pitre French West Indies
  5. 5.City Anti-Tuberculosis DispensarySt. PetersburgRussia

Personalised recommendations