Characterization of Mutations Linked with Second Line Anti-TB Drug Resistance in Pakistan

Riffat Jabeen, Memona Yasmin, Hafiza Rabia Dar, Rubina Tabassum Siddiqui, Inaam Ullah


Background: The incidence of multiple drug resistance tuberculosis is on the rise worldwide and Pakistan is one of 30 high TB burden countries. Resistance to second line drugs especially fluoroquinolones is being reported by many laboratories. This is increasing the gravity of the situation resulting in extensively drug resistant cases, which is difficult to treat, and has more side effects.

Methods: One hundred and thirty-three (133) clinical isolates of M. tuberculosis, collected by convenience sampling, were characterized for mutations in eth-A, gyrA, msh-A, rrs genes, and the promoter region of inh-A gene that confer resistance to second line anti-TB drugs. The mutations were detected by allele-specific-PCR and PCR amplification followed by SSCP and DNA sequencing.

Results: Mutations in gyrA gene at codon 91, 94 and 95 were found in 4 (3.0%) M. tuberculosis isolates. Mutations in rrs gene were found in 17 (12.8%) isolates, ten (7.5%) isolates had mutation at A1401G position, 5 (3.76%) isolates at C1402T position and 3 (2.25%) isolates had G1484T mutation. For resistance to ethionamide, none of the isolates showed mutation in eth-A gene. In promoter region of inh-A gene, mutations were detected at -C15T, -A112G, -C110T in two samples. Two mutations, A312T and A332G, were found in msh-A gene in one sample. Collectively, 24 (18%) isolates were found to harbor mutations associated with second line anti TB drug resistance.

Conclusion: Our work revealed high frequency of mutations (18%) associated with resistance against second line anti-TB drugs. This situation can lead to increase in XDR-TB cases. We, therefore, recommend improved diagnostic and drug sensitivity testing, better prescription, and development of superior drugs to control tuberculosis.   

Keywords: Antibiotic resistance; Mycobacterium tuberculosis; Second line anti-TB drugs

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WHO. Global tuberculosis report. World Health Organization. (2020).

WHO, WHO. Global tuberculosis report. World Health Organization. (2018).

Brief Report on World TB Day 2020 in Pakistan,

van der Heijden YF, Maruri F, Blackman A, Holt E, Warkentin J, et al. Fluoroquinolone exposure prior to tuberculosis diagnosis is associated with an increased risk of death. The International Journal of Tuberculosis and Lung Disease, (2012); 16(9): 1162-1167.

Von Groll A, Martin A, Jureen P, Hoffner S, Vandamme P, et al. Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in gyrA and gyrB. Antimicrobial agents and chemotherapy, (2009); 53(10): 4498-4500.

Baulard AR, Betts JC, Engohang-Ndong J, Quan S, McAdam RA, et al. Activation of the pro-drug ethionamide is regulated in mycobacteria. Journal of Biological Chemistry, (2000); 275(36): 28326-28331.

DeBarber AE, Mdluli K, Bosman M, Bekker L-G, Barry CE. Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences, (2000); 97(17): 9677-9682.

Morlock GP, Metchock B, Sikes D, Crawford JT, Cooksey RC. ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrobial agents and chemotherapy, (2003); 47(12): 3799-3805.

Georghiou SB, Magana M, Garfein RS, Catanzaro DG, Catanzaro A, et al. Evaluation of genetic mutations associated with Mycobacterium tuberculosis resistance to amikacin, kanamycin and capreomycin: a systematic review. PloS one, (2012); 7(3): e33275.

Somerville W, Thibert L, Schwartzman K, Behr MA. Extraction of Mycobacterium tuberculosis DNA: a question of containment. Journal of clinical microbiology, (2005); 43(6): 2996-2997.

Bozeman L, Burman W, Metchock B, Welch L, Weiner M, et al. Fluoroquinolone susceptibility among Mycobacterium tuberculosis isolates from the United States and Canada. Clinical infectious diseases, (2005); 40(3): 386-391.

Giannoni F, Iona E, Sementilli F, Brunori L, Pardini M, et al. Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy, (2005); 49(7): 2928-2933.

Jabeen K, Shakoor S, Chishti S, Ayaz A, Hasan R. Fluoroquinolone-resistant Mycobacterium tuberculosis, Pakistan, 2005–2009. Emerging infectious diseases, (2011); 17(3): 566.

Choi S-H, Kim EY, Kim Y-J. Systemic use of fluoroquinolone in children. Korean journal of pediatrics, (2013); 56(5): 196.

Singhal R, Reynolds PR, Marola JL, Epperson LE, Arora J, et al. Sequence analysis of fluoroquinolone resistance-associated genes gyrA and gyrB in clinical Mycobacterium tuberculosis isolates from patients suspected of having multidrug-resistant tuberculosis in New Delhi, India. Journal of clinical microbiology, (2016); 54(9): 2298-2305.

Huang T-S, Kunin CM, Shin-Jung Lee S, Chen Y-S, Tu H-Z, et al. Trends in fluoroquinolone resistance of Mycobacterium tuberculosis complex in a Taiwanese medical centre: 1995–2003. Journal of Antimicrobial Chemotherapy, (2005); 56(6): 1058-1062.

Lakshmi R, Kumar V, Baskaran M, Sundar S, Rahman F, et al. Pattern of ethionamide susceptibility and its association with isoniazid resistance among previously treated tuberculosis patients from India. Brazilian Journal of Infectious Diseases, (2011); 15(6): 619-620.

Ongaya V, Githui W, Meme H, Kiiyukia C, Juma E. High ethionamide resistance in Mycobacterium tuberculosis strains isolated in Kenya. African Journal of Health Sciences, (2012); 20(1-2): 37-41.

Leung K, Yip C, Yeung Y, Wong K, Chan W, et al. Usefulness of resistant gene markers for predicting treatment outcome on second‐line anti‐tuberculosis drugs. Journal of applied microbiology, (2010); 109(6): 2087-2094.

Jugheli L, Bzekalava N, de Rijk P, Fissette K, Portaels F, et al. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrobial agents and chemotherapy, (2009); 53(12): 5064-5068.

Alangaden GJ, Bone SA. The clinical use of fluoroquinolones for the treatment of mycobacterial diseases. Clinical infectious diseases, (1997); 25(5): 1213-1221.

Maus CE, Plikaytis BB, Shinnick TM. Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy, (2005); 49(8): 3192-3197.

Taniguchi H, Chang B, Abe C, Nikaido Y, Mizuguchi Y, et al. Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system. Journal of bacteriology, (1997); 179(15): 4795-4801.

Suzuki Y, Katsukawa C, Tamaru A, Abe C, Makino M, et al. Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene. Journal of clinical microbiology, (1998); 36(5): 1220-1225.


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