Isoniazid MIC and KatG Gene Mutations among Mycobacterium tuberculosis Isolates in Northwest of Iran

Document Type : Original Article


1 Tuberculosis and Lung Research Centre and Paramedical Faculty, Tabriz University of Medical Sciences, Tabriz, Iran

2 Biotechnology Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran

3 Infectious and Tropical Disease Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran

4 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

5 Microbiology department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran


Isoniazid (INH) is one of the main first line drugs used in treatment of tuberculosis and development of resistance against this compound can result in serious problems in treatment procedures. Resistance to INH is mediated mainly by mutation in KatG gene that is coded for the catalase enzyme. The proportional method for detection of INH-resistance is time consuming due to the slow growth rate of Mycobacterium tuberculosis. In this study, we used PCR-RFLP approach for screening of common mutations in KatG gene for detection of INH resistance, and compared the results to minimal inhibitory concentration (MIC) in M. tuberculosis isolates.
Materials and Methods
Fifty M. tuberculosis isolates were subjected to study of which, 25 strains were INH-resistant and 25 strains were INH-sensitive.
Of 25 INH-resistant strains, the mutation was identified in 56% and 20% in the KatG315 and KatG463 loci, respectively. In 24% of INH-resistant strains, no mutation was observed in the studied loci. INH MIC was <0.2 pg/ml in all sensitive strains whereas among 25 INH -resistant isolates, INH MIC was higer than 0.2 pg/ml ranged from 0.2 to 3.2 pg/ml.
Our findings revealed that PCR-RFLP is capable to identify INH-resistance in more than 76% of INH- resistant M. tuberculosis strains, and could be used for rapid identification of INH resistance. High levels of INH MIC were observed in the strains which had mutation in the KatG gene in position 315.


1. Simon S, Listiawan I. Drug resistance in Mycobacterium tuberculosis: a molecular perspective. J Indonesian Med Assoc 2003; 4:26-35.
2. Abbadi SH, Sameaa GA, Morlock G, Cooksey RC. Molecular identification of mutations associated with anti-tuberculosis drug resistance among strains of Mycobacterium tuberculosis. Int J Infect Dis 2009; 13:673-678.
3. Abe C, Kobayashi I, Mitarai S, Wada M, Kawabe T , Takashima T, et al. Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to Isoniazid in Japan. J Clin Microbiol 2008; 46:2263-2268.
4. Mathuria JP, Nath G, Samaria JK, Anupurba S. Molecular characterization of INH-resistant Mycobacterium tuberculosis isolates by PCR-RFLP and multiplex-PCR in North India. Infect Genet Evol 2009; 9:1352-1355.
5. Ghebremichael S, Petersson R, Koivula T, Pennhag A, Romanus V, Berggren I, et al. Molecular epidemiology of drug resistant tuberculosis in Sweden. Microbes Infect 2008; 10: 699-705.
6. Timmins GS, Deretic V. Mechanisms of action of isoniazid. Mol microbial 2006; 65:1220-1227.
7. Ramaswamy SV, Reich R, Dou SJ, Jasperse L, Pan X, Wanger A, et al. Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2003; 47:1241-12450.
8. Hristea A, Otelea D, Paraschiv S, Macri A, Baicus C, Moldovan O, et al. Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR. Indian J Med Microbiol 2010; 28:211-216
9. Narvskaya O, Otten T, Limeschenco E, Sapozhnikova N, Graschenkova O, Steklova L, et al. 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 2002; 21:596-602.
10- Kim SY, Park YJ, Kim WI, Lee SH, Chang CL, Kang SJ, et al. Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea. Diagn Microbiol Infect Dis 2003; 47:497-502.
11.Guo H, Seet Q, Denkin S, Parsons L, Zhang Y. Molecular characterization of isoniazid-resistant isolates of Mycobacterium tuberculosis from the USA. J Medical Microbiol 2006; 55:1527-1531.
12.Goodwin A. Mycobacterium tuberculosis and other Nontuberculosis mycobacteria. In: Mahon CR, Lehman DC, Manuselis G.Textbook of Diagnostic Microbiology, 3rd ed. New York: Saunders Elsevier; 2007.p.673-716.
13.Marttila HJ, Soini H, Huovinen P, Viljanen MK. katG mutations in isoniazid-resistant Mycobacterium tuberculosis isolates recovered from Finnish patients. Antimicrob Agents Chemother 1996; 40:2187-2189.
14.Poojary A, Nataraj G, Kanade S, Metha P, Baveja S. Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis: it's utility in resource poor settings. Indian J Med Microbiol 2006; 24:268-272.
15.Murray PR, Rosenthal KS, Pfalle MA. Medical Microbilogy. 5th ed. USA: Elsevier Mosby; 2005.p.297-310.
16.Leung ET, Kam KM, Chiu A, Ho PL, Seto WH, Yuen KY, et al. Detection of KatG Ser315Thr substitution in respiratory specimens from patients with isoniasid-resistant Mycobacterium tuberculosis using PCR-RFLP. J Med Microbiol 2003; 52, 999- 1003.
17.Marttila HJ, Soini H, Huovinen P, Viljanen MK. KatG mutations in isoniazid-resistant Mycobacterium tuberculosis isolates recovered from Finnish patients. Antimicrob Agents Chemother 1996; 40:2187-2189.
18.Velayati AA, Masjedi MR, Farnia P, Tabarsi P, Ghanavi J, Ziazarifi AH, et al. Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran. Chest 2009; 136:420-425.
19.Zaker Bostanabad S, Titov LP, Bahremand R. Frequency and molecular characterization of isoniazid resistance in the KatG region of MDR isolates from tuberculosis patients in southern endemic border of Iran. Infect Genet Evol 2008; 8:15-29. 
20.Mohajeri P, Tavakoli A, Moghim S. Detection of mutation at codon 315 KatG gene as a gene marker associated with isoniazid resistance, in Mycobacterium tuberculosis strains isolated from patients in Isfahan and Tehran by PCR-RFLP method. J Zanjan Unive Med Sci Health Services 2009; 17:29-40.
21.Cavusoglu C, Turhan A, Akinci P, Soyler I. Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates. J Clin Microbiol 2006; 44:2338-2342.
22.Aktas E, Durmaz R, Yang D, Yang Z. Molecular characterization of isoniazid and rifampin resistance of Mycobacterium tuberculosis clinical isolates from Malatya, Turkey. Microb Drug Resist 2005; 11:94-99.
23.Ahmad S, Mokaddas E. Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East. Int J Antimicrob Agents 2004; 23:473-479.
24.Ahmad S, Fares E, Araj GF, Chaugh TD, Mustafa AS. Prevalence of S315T mutation within the KatG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Dubai and Beirut. Int J Tuberc Lung Dis 2002; 6:920-926.
25.Valvatne H, Syre H, Kross M, Ruth Stavrum R, Phyu S, Grewal M. Isoniazid and rifampicin resistance- associated mutations in Mycobacterium tuberculosis isolates from Yangon, Myanmar: implications for rapid molecular testing. J Antimicrob Chemother 2009; 64:694-701.
26.Musser JM. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 1995; 8:496-514.
27. Zhang M, Yue J, Yang Y, Zhang H, Lei J, Jin R.,Zhang X, et al. Detection of mutation associated with Isoniazid resistance in Mycobacterium tuberculosis isolates from China. J Clin Microbiol 2005; 43:5477-82.
28.Haas WH, Schilke K, Brand J, Amthor B, Weyer K, Fourie PB, et al. Molecular analysis of KatG gene mutations in strains of Mycobacterium tuberculosis complex from Africa. J Antimicrob Chemother 1997; 41:1601-1603.
29.Telenti A, Honore N, Bernasconi C, March J, Ortega A, Heym B, et al. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J Clin Microbiol 1997; 35: 719-23.
30.Dalla Costa ER, Ribeiro MO, Silva MS, Arnold LS, Rostirolla DC, Cafrune PI, et al. Correlations of mutations in the KatG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America. BMC Microbiol 2009; 9:39.
31.van Soolingen D, de Haas PE, van Doom HR, Kuijper E, Rinder H, Borgdorff MW. Mutations at amino acid position 315 of the KatG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands. J Infect Dis 2000; 182:1788-1790.