AiM  Vol.2 No.3 , September 2012
Molecular Profiling of Drug Resistant Isolates of Mycobacterium tuberculosis in North India
Abstract: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem because treatment is complicated, cure rates are well below those for drug susceptible tuberculosis (TB), and patients may remain infectious for months or years despite receiving the best available therapy. To gain a better understanding of MDR-TB, we characterized isolates recovered from 69 patients with MDR-TB, by use of IS6110 restriction fragment-length polymorphism (RFLP) analysis; spacer oligonucleotide genotyping (i.e. spoligotyping). Clinical isolates from patients with tuberculosis have been considered to contain clonally expanded Mycobacterium tuberculosis (MTB) strain. Over the years, the identification method based on IS6110 insertion sequences has been established as the standard for typing strains of MTB. IS6110 RFLP fingerprinting is very convincing when it is applied to classify MTB isolates harboring a large number of IS6110 in their chromosomes. Therefore, in the present study we have characterized the isolates from the patients suffering from MDR TB, on the basis of conserved Variable Number Tandem Repeats (VNTR), Direct Repeats (DR) and Insertion Sequences (IS) IS6110 elements. The polymorphic data showed significant level of dissimilarities among all the MDR isolates of MTB. Comparative studies with the DR and VNTR data substantiate that polymorphism occur among MDR-TB cases as shown by the number of repeats present in different clinical isolates.
Cite this paper: D. K. Tripath, K. Srivastava, S. Kant and K. K. Srivastava, "Molecular Profiling of Drug Resistant Isolates of Mycobacterium tuberculosis in North India," Advances in Microbiology, Vol. 2 No. 3, 2012, pp. 317-326. doi: 10.4236/aim.2012.23038.

[1]   D. van Soolingen, P. E. W. de Hass, P. W. M. Hermans, P. M. Groenen and J. D. van Embden, “Comparison of Various Repetitive DNA Elements as Genetic Markers for Strain Differentiation and Epidemiology of M. tuberculosis,” Journal of Clinical Microbiology, Vol. 31, No. 8, 1993, pp. 1987-1995.

[2]   P. Venkataraman and Paramasivan, “Drug Resistance in Tuberculosis and Issues Related to Multidrug Resistance in Planning for TB Control in India,” The Health Administrator, Special Issue on Tuberculosis, Vol. 15, 2002, pp. 127-136.

[3]   Centers for Disease Control and Prevention (CDC), “Extensively Drug-Resistant Tuberculosis-United States”, 1993-2006.

[4]   D. A. Mitchison, “Drug Resistance in Tuberculosis,” European Respiratory Journal, Vol. 25, No. 2, 2005, pp. 376-379. doi:10.1183/09031936.05.00075704

[5]   I. Mokrousov, O. Narvskaya, E. Limeschenko, T. Otten and B. Vyshnevskiy, “Detection of Ethambutol Resistant M. tuberculosis Isolates by Multiplex Allele Specific PCR Assay Targeting embB 306 Mutations,” Journal of Clinical Microbiology, Vol. 40, No. 5, 2002, pp. 1617-1620. doi:10.1128/JCM.40.5.1617-1620.2002

[6]   I. Mokrousov, T. Otten and M. Filipenko, “Detection of Isoniazid Resistant M. tuberculosis Isolates by Multiplex Allele Specific PCR Assay Targeting katG Codons 351 Variation,” Journal of Clinical Microbiology, Vol. 40, No. 7, 2002, pp. 2509-2512. doi:10.1128/JCM.40.7.2509-2512.2002

[7]   A. Van Rie, A. Warren and R. I. Mshanga, “Analysis for a Limited Number of Gene Codons Can Predict Drug Resistance of M. tuberculosis in a High-Incidence Community,” Journal of Clinical Microbiology, Vol. 39, No. 2, 2001, pp. 636-641. doi:10.1128/JCM.39.2.636-641.2001

[8]   L. M. Parsons, M. Salfinger and A. Coleridge, “Phenotypic and Molecular Characterization of M. tuberculosis Isolates to both Isoniazid and Ethambutol,” Antimicrobial Agents and Chemotherapy, Vol. 49, No. 6, 2005, pp. 2218-2225. doi:10.1128/AAC.49.6.2218-2225.2005

[9]   Z. Yang, R. Durmaz and D. Yang, “Simultaneous Detection of Isoniazid, Rifampicin, and Ethambutol by a Single Multiplex Allele Specific Polymerase Chain Reaction (PCR) Assay,” Diagnostic Microbiology & Infectious Disease, Vol. 53, No. 3, 2005, pp. 201-208. doi:10.1016/j.diagmicrobio.2005.06.007

[10]   A. Hristea, D. Otelea, S. Paraschiv, A. Macri, C. Baicus, O. Moldovan, M. Tinischi, V. Arama and A. Streinu-Cercel, “Detection of M. tuberculosis Resistance Mutations to Rifampin and Isoniazid by Real-Time PCR,” Indian Journal of Medical Microbiology, Vol. 28, No. 3, 2010, pp. 211-216. doi:10.4103/0255-0857.66474

[11]   I. Mokrousov, T. Otten, B. Vyshnevskiy and O. Narvskaya, “Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant M. tuberculosis in Sputum Smear,” Antimicrobial Agents and Chemotherapy, Vol. 47, No. 7, 2003, pp. 2231-2235. doi:10.1128/AAC.47.7.2231-2235.2003

[12]   G. Canetti, W. Fox, A. Khomenko, H. T. Mahler, N. K. Menon, D. A. Mitchison, N. Rist and N. A. Smelev, “Advances in Techniques of Testing Mycobacterial Drug Sensitivity and the Use of Sensitivity Tests in Tuberculosis Control Programmes,” Bulletin of the World Health Organization, Vol. 41, No. 1, 1969, pp. 21-43.

[13]   T. H. Weniger, J. Krawczyk, P. H. Supply, S. Niemann and D. Harmsen, “MIRU-VNTRplus: A Web Tool for Polyphasic Genotyping of M. tuberculosis Complex Bacteria,” Nucleic Acids Research, Vol. 38, Supplement 2, 2010, pp. 1-6. doi:10.1093/nar/gkq351

[14]   R. Johnson, M. E. Streicher, E. G. Louw, R. M. Warren, P. D. van Helden and T. C. Victor, “Drug Resistance in M. tuberculosis,” Current Issues in Molecular Biology, Vol. 8, No. 2, 2006, pp. 97-112.

[15]   B. Madison, B. Robinson-Dunn and I. George, “Multicenter Evaluation of Ethambutol Susceptibility Testing of M. tuberculosis by Agar Proportion 15 and Radiometric Methods,” Journal of Clinical Microbiology, Vol. 40, No. 11, 2002, pp. 3976-3979. doi:10.1128/JCM.40.11.3976-3979.2002

[16]   H. Rinder, K. T. Mieskes and T. Loscher, “Heteroresistance in Mycobacterium tuberculosis,” The International Journal of Tuberculosis and Lung Disease, Vol. 5, No. 4, 2001, pp. 339-345.

[17]   V. Nikolayevskyy, Y. Balabanova, T. Simak, N. Malomanova, I. Fedorin and F. Drobniewski, “Performance of the Genotype(R) MTBDR Plus Assay in the Diagnosis of Tuberculosis and Drug Resistance in Samara, Russian Federation,” BMC Clinical Pathology, Vol. 10, 2009, pp. 2-22. doi:10.1186/1472-6890-9-2

[18]   G. C. Baldeviano-Vidalon, N. Quispe-Torres and C. Bonilla-Asald, “Multiple Infection with Resistant and Sensitive M. tuberculosis Isolates during Treatment of Pulmonary Tuberculosis Patients,” The International Journal of Tuberculosis and Lung Disease, Vol. 9, No. 10, 2005, pp. 1155-1160.

[19]   S. A. Watterson, S. M. Wilson, M. D. Yates and F. A. Drobniewski, “Comparison of Three 18 Molecular Assays for Rapid Detection of Rifampin Resistance in M. tuberculosis,” Journal of Clinical Microbiology, Vol. 36, No. 7, 1998, pp. 1969-1973.

[20]   C. Dye, S. Scheele, P. Dolin, V. Pathania and M. C. Raviglione, “Global Burden of Tuberculosis: Estimated Incidence, Prevalence and Mortality by Country,” Journal of the American Medical Association, Vol. 282, No. 7, 1999, pp. 677-686.

[21]   WHO, “Anti-Tuberculosis Drug Resistance WHO/IUALD Global Project on Anti-Tuberculosis Drug Resistance Surveillance,” Geneva, 1997, WHO/TB/97.229.

[22]   C. Dye, Z. Fengzeng, S. Scheele and B.Williams, “Evaluating the Impact of Tuberculosis Control: Number of Deaths Prevented by Short-Course Chemotherapy in China,” International Journal of Epidemiology, Vol. 29, No. 3, 2000, pp. 558-564. doi:10.1093/ije/29.3.558

[23]   WHO, “Global Tuberculosis Control Surveillance: Planning, Financing,” Geneva, 2009, pp. 1-303.

[24]   WHO, “Global Tuberculosis Control,” Geneva, Switzerland, 2003.

[25]   WHO, “Anti Tuberculosis Drug Resistance in the World, Prevalence and Trends,” Geneva, Switzerland, 2000.

[26]   WHO, “Drug-Resistant Isolates of TB Increasing Worldwide,” WHO 19, Geneva, 2000.

[27]   L. Herrera-León, T. Molina, P. Saíz, J. A. Sáez-Nieto and M. S. Jimenez, “New Multiplex PCR for Rapid Detection of Isoniazid-Resistant M. tuberculosis Clinical Isolates,” Antimicrobial Agents and Chemotherapy, Vol. 49, No. 1, 2005, pp. 144-147. doi:10.1128/AAC.49.1.144-147.2005

[28]   C. Abe, I. Kobayashi and S. Mitarai, “Biological and Molecular Characteristics of M. tuberculosis Clinical Isolates with Low-Level Resistance to Isoniazid in Japan,” Journal of Clinical Microbiology, Vol. 46, No. 7, 2008, pp. 2263-2268. doi:10.1128/JCM.00561-08

[29]   S. V. Ramaswamy and J. M. Musser, “Molecular Genetic Basis of Antimicrobial Agent Resistance in M. tuberculosis: 1998 Update,” Tubercle and Lung Disease, Vol. 79, No. 1, 1998, pp. 3-29. doi:10.1054/tuld.1998.0002

[30]   C. Plinke, H. S. Cox, S. Kalon, D. Doshetov. Rüsch-Gerdes and S. Niemann S, “Tuberculosis Ethambutol Resistance: Concordance between Phenotypic and Genotypic Test Results,” Tuberculosis, Vol. 89, No. 6, 2009, pp. 448-452. doi:10.1016/

[31]   J. I. Sekiguchi, T. Miyoshi-Akiyama, E. Augustynowicz-Kopec, “Detection of Multidrug Resistance in Mycobacterium tuberculosis,” Journal of Clinical Microbiology, Vol. 45, No. 1, 2007, pp. 179-192. doi:10.1128/JCM.00750-06

[32]   N. Gonzalez, M. J., Torres, J. Aznar and J. C. Palomares, “Molecular Analysis of Rifampin and Isoniazid Resistance of M. tuberculosis Clinical Isolates in Seville, Spain,” Tubercle and Lung Disease, Vol. 79, No. 3, 1999, pp. 187-190. doi:10.1054/tuld.1998.0195

[33]   J. Wang, R. M. Burger and K. Drlica, “Role of Superoxide in Catalase-Peroxidase-Mediated Isoniazid Action against Mycobacteria,” Antimicrobial Agents and Chemotherapy, Vol, 42, No. 3, 1998, pp. 709-711.

[34]   S. V. Ramaswamy, A. G. Amin, S. Goksel, C. E. Stager, S. J. Dou and H. L. Sahli, “Molecular Genetic Analysis of Nucleotide Polymorphisms Associated with Ethambutol Resistance in Human Isolates of M. tuberculosis,” Antimicrobial Agents and Chemotherapy, Vol. 44, No. 2, 2000, pp. 326-336. doi:10.1128/AAC.44.2.326-336.2000

[35]   P. T. Kent and G. P. Kubica, “Public Health Mycobacteriology: A Guide for the Level III Laboratory. US Department of Health and Drug Resistance Pattern of M. tuberculosis Isolated from Patients Attending a Referral Hospital Wadud et al. Bangladesh,” Journal of Medical Microbiology, Vol. 17, 2000, pp. 1132-1140

[36]   J. P. Hosek, P. Svastova, M. Moravkova, I. Pavlik and M. Bartos, “Methods of Mycobacterial DNA Isolation from Different Biological Material: A Review,” Veterinary Medicine, Vol. 51, No. 5, 2006, pp. 180-192.

[37]   E. Mazars, S. Lesjean, A. L. Banuls, M. Gilbert, V. Vincent, B. Gicquel, M. Tibayrenc, C. Locht and P. H. Supply, “High-Resolution Minisatellite-Based Typing as a Portable Approach to Global Analysis of M. tuberculosis Molecular Epidemiology,” Proceedings of the National Academy of Sciences, Vol. 98, No. 4, 2001, pp. 1901-1906. doi:10.1073/pnas.98.4.1901

[38]   E. B. Hill, L. G. Wayne and M. Gross, “Purification of Mycobacterial Deoxyribonucleic Acid,” Journal of Bacteriology, Vol. 112, No. 3, 1972, pp. 1033-1039.

[39]   N. Smittipat and P. Palittapongarnpim, “Identification of Possible Loci of Variable Number of Tandem Repeats in M tuberculosis,” Tubercle and Lung Disease, Vol. 80, No. 2, 2000, pp. 69-74. doi:10.1054/tuld.2000.0236

[40]   I. J. Wiid, C. Werely, N. Beyers, P. Donald, P. D. van Helden, “Oligonucleotide (GTG)5 as a marker for M. tuberculosis Strain Identification.” Journal of Clinical Microbiology, Vol. 32, No. 5, 1994, pp. 1318-1321.

[41]   P. Palittapongampim, S. Chomyc, A. Fanning and D. Kunimoto, “DNA Fragment Length Polymorphism Analysis of M. tuberculosis Isolates by Arbitrarily Primed Polymerase Chain Reaction,” The Journal of Infectious Diseases, Vol. 167, No. 4, 1993, pp. 975-978. doi:10.1093/infdis/167.4.975

[42]   R. C. Chan, M. M. M. Hui, E. W. Chan, et al., “Genetic and Phenotypic Characterization of Drug-Resistant M. tuberculosis Isolates in Hong Kong,” Journal of Antimicrobial Chemotherapy, Vol. 59, No. 5, 2007, pp. 866-873.

[43]   P. M. Gronen, A. E. Bunschoten, D. Van Soolingen and J. D. A. van Embden, “Nature of DNA Polymorphism in the Direct Repeat Cluster of M. tuberculosis: Application for Strain Differentiation by a Novel Typing Method,” Molecular Microbiology, Vol. 10, No. 5, 1993 pp. 1057-1065. doi:10.1111/j.1365-2958.1993.tb00976.x

[44]   R. M. Warren, N. C. Gey van Pittius, M. Barnard, A. Hesseling, E. Engelke, M. de Kock, M. C. Gutierrez, G. K. Chege, T. C. Victor, E. G. Hoal and P. D. van Helden “Differentiation of M. tuberculosis Complex by PCR Amplification of Genomic Regions of Difference,” The International Journal of Tuberculosis and Lung Disease, Vol. 10, No. 7, 2006, pp. 818-822.

[45]   WHO, TB Country Profile-Jordan, 2010.

[46]   H. Van Deutekom, P. H. Supply, P. E. W. Haas, E. Wilery, S. P. Hoijng, C. Locht, R. A. Coutinho and D. van Soolingen, “Molecular Typing of M. tuberculosis by Mycobacterial Interspersed Repetitive Unit Variable-Number Tandem Repeat Analysis, a More Accurate method for Identifying Epidemiological Links between Patients with Tuberculosis,” Journal of Clinical Microbiology, Vol. 43, No. 9, 2005, pp. 4473-4479. doi:10.1128/JCM.43.9.4473-4479.2005

[47]   N. Thorne, A. Underwood, S. Gharbia and C. Arnold, “Evolutionary Clues from Comparative Analysis of M. tuberculosis Variable-Number Tandem Repeat Sequences within Genetic Families,” Infection, Genetics and Evolution, Vol. 7, No. 2, 2005, pp. 239-246. doi:10.1016/j.meegid.2006.09.006

[48]   ICDDR’B, Health and Science Bulletin, Vol. 1, 2002, pp. 6-10.

[49]   D. Lina and P. M. Sweta, “Drug Resistance in Tuberculosis,” Bombey Hospital Journal, 1999.

[50]   L. Sandman, N. W. Schluger, A. L. Davidow and S. Bonk, “Risk Factors for Rifampin-Monoresistant Tuberculosis: A Case-Control Study,” American Journal of Respiratory and Critical Care Medicine, Vol. 159, No. 2, 1999, pp. 468-472.

[51]   A. Hristea, D. Otelea, S. Paraschiv, A. Macri, C. Baicus, O. Moldovan, M. Tinischi, V. Arama and A. Streinu-Cercel, “Detection of M. tuberculosis Resistance Mutations to Rifampin and Isoniazid by Real-Time PCR,” Indian Association of Medical Microbiologists, Vol. 28, No. 3, 2010, pp. 211-216. doi:10.4103/0255-0857.66474

[52]   F. A. Post, P. A. Willcox, B. Mathema, L. M. Steyn, K. Shean, S. V. Ramaswamy, E. A. Graviss, E. Shashkina, B. N. Kreiswirth and G. Kaplan, “Genetic Polymorphism in M. tuberculosis Isolates from Patients with Chronic Multidrug-Resistant Tuberculosis,” The Journal of Infectious Diseases, Vol. 190, No. 1, 2004, pp. 99-106. doi:10.1086/421501

[53]   D. Van Soolingen, P. W. M. Hermans, P. E. W. de Haas, D. R. Soll and J. D. van Embden, “Occurrence and Stability of Insertion Sequences in Mycobacterium Tuberculosis Complex Strains: Evaluation of an Insertion Sequence-Dependent Polymorphism as a Tool in the Epidemiology of Tuberculosis,” Journal of Clinical Microbiology, Vol. 29, No. 11, 1991, pp. 2578-2586.

[54]   S. M. M. Roring, D. Brittain, A. E. Bunschoten, M. S. Hughes, R. A. Skuce, J. D. A. Van Embden and S. D. Neil, “Spacer Oligotyping of Mycobacterium bovis Isolates Compared to Typing by Restriction Fragment Length Polymorphism Analysis Using PGRS, DR and IS6110,” Veterinary Microbiology, Vol. 61, No. 1-2, 1998, pp. 111-120. doi:10.1016/S0378-1135(98)00178-3

[55]   K. L. Horan, R. Freeman., K. Weigel, M. Semret, S. Pfaller, T. C. Covert, D. Van Soolingen, S. C. Leao, M. A. Behr and G. A. Cangelosi, “Novel Genetic Polymorphisms That Further Delineate the Phylogeny of the Mycobacterium Tuberculosis Complex,” Journal of Bacteriology, Vol. 188, No. 12, 2006, pp. 4271-4287.

[56]   D. Thierry, M. D. Cave, K. D. Eisenach, J. T. Crawford, J. H. Bates and B. Gicquel, “IS6110, and IS-like Element of M. tuberculosis Complex,” Nucleic Acids Research, Vol. 18, No. 1, 1990, pp.188-192. doi:10.1093/nar/18.1.188

[57]   P. B. Bifani, M. Mathema, S. Campo, B. Moghazeh, E. Nivin, J. Shashkina, S. S. Driscoll, R. Munsiff, R. Frothingham and B. N. Kreiswirth, “Molecular Identification of Streptomycin Monoresistant M. tuberculosis Related to Multidrug-Resistant W Strain,” Emerging Infectious Diseases, Vol. 7, No. 5, 2001, pp. 842-848. doi:10.3201/eid0705.010512