Research Article | Open Access
Investigation of Ser315 Substitutions within katG Gene in Isoniazid-Resistant Clinical Isolates of Mycobacterium tuberculosis from South India
Mutation at codon 315 of katG gene is the major cause for isoniazid (INH) resistance in Mycobacterium tuberculosis (M. tuberculosis). Substitution at codon 315 of katG gene was analyzed in 85 phenotypically resistant isolates collected from various parts of southern India by direct sequencing method. The obtained results were interpreted in the context of minimum inhibitory concentration (MIC) of INH. Of the 85 phenotypically resistant isolates, 56 (66%) were also correlated by the presence of resistance mutations in the katG gene; 47 of these isolates had ACC, 6 had AAC, 2 had ATC, and one had CGC codon. The frequency of Ser315 substitution in katG gene was found to be higher (70%) amongst multidrug-resistant (MDR) strains than among non-MDR (61%) INH-resistant isolates. Further, the frequency of mutations was found to be greater (74%) in isolates with higher MIC values in contrast to those isolates with low MIC values (58%). Therefore, the study identified high prevalence of Ser315Thr substitution in katG gene of INH-resistant isolates from south India. Also, isolates harboring this substitution were found to be associated with multidrug and high level INH resistance.
Although tuberculosis (TB) is a preventable, treatable, and curable disease, it still remains as a major public health problem. The period of 6–9 months needed to treat the disease is too long, and the treatment is often associated with significant toxicity. These factors make patient compliance to therapy very difficult, leading to the emergence and selection of drug-resistant TB bacteria . The emergence of multidrug-resistant (MDR) TB, defined as strains which are resistant to two most potent anti-TB drugs, namely, isoniazid (INH) and rifampicin (RIF), and XDR-TB, defined as MDR-TB strains that are resistant to second-line TB drugs, that is, fluoroquinolones and at least one of the injectable aminoglycosides (capreomycin, kanamycin) or amikacin, has worsened the situation further . Globally, 450,000 people developed MDR-TB in 2012; more than half of these cases were from India, China, and the Russian Federation. It is estimated that about 9.6% of MDR-TB cases had XDR-TB. About 170,000 MDR-TB deaths are estimated to have occurred in 2012 .
Resistance against all known anti-TB drugs has been reported. However, isolates of M. tuberculosis resistant to INH are seen with increasing frequency (1 in 106) as compared to isolates resistant to other drugs . Also, resistance to INH, alone or in combination with other drugs, is now the second most common cause for resistance. Globally, it has been estimated that INH resistance was found in 10.3% of new cases and 27.7% of treated cases . An earlier study based on susceptibility testing from south India also reported high resistance to INH in 15.4% of cases compared to 4.4% cases of RIF resistance .
INH has been used extensively as the frontline anti-TB drug and a drug of choice for chemoprophylaxis, acting as a principle component in the current six-month short course chemotherapy regimen. It has long been recognized that INH resistance in M. tuberculosis correlates with the loss of catalase and peroxidase (CP) activity in resistant strains [7–9]. In other words, INH resistance is often accompanied by loss and/or reduction of CP or KatG activity coded by katG gene. In fact, INH is a prodrug that requires cellular activation by KatG protein to its active form, before it can exert its toxic effect on the bacillus . There has been considerable interest to understand the molecular basis of INH resistance. Mutations in several genes (katG, inhA, ahpC, kasA, and others) have been associated with INH resistance [10–13]. Of these, inhA and ahpC promoter mutations lead to low-level INH resistance, whereas mutation in katG is responsible for high-level resistance. Zhang et al. (1992) demonstrated that mutation in the katG gene, coding for KatG protein, is a major contributor to INH resistance in M. tuberculosis .
The most prevalent mutation found to occur in katG gene is AGC to ACC at codon 315 , or Ser315Thr (S315T) substitution. The appearance of this mutation was most frequent amongst MDR strains . However, this mutation was also reported to be associated with intermediate or high levels of resistance to INH (1 to 10 μg/mL) . When compared to other resistance-conferring mutations in katG, S315T substitution was found to result in near-normal CP activities. Also it maintains the levels of virulence and confers resistance to INH simultaneously . Therefore, it may be considered as a reliable biomarker for the detection of INH resistance.
There are very few studies [18–20] on molecular characterization of INH resistance from India and there is paucity of data for south Indian isolates. Previous studies have clearly shown that the south Indian INH-resistant () isolates were quite distinct and had lower virulence and higher susceptibility to H2O2 [21–23]. Hence, it was of interest to analyze mutations in katG gene encompassing codon 315 in clinical isolates of M. tuberculosis from south India by DNA sequencing method in the present study.
2.1. INH-Resistant Clinical Isolates of M. tuberculosis
A total of 85 clinical isolates of M. tuberculosis were randomly selected from south Indian TB patients (20–60 years old) belonging to both the sexes from 2006 to 2009 at the National Institute for Research in Tuberculosis (NIRT), Chennai, India. Of the 85 isolates collected, 67 were from Tamil Nadu, 14 were from Andhra Pradesh, 3 were from Karnataka, and one was from Kerala. Drug susceptibility testing (DST) was performed on the cultures; they were coded and subjected to DNA sequencing for identification of mutations in the katG gene.
2.2. DNA Extraction from INH-Resistant Strains
Genomic DNA was prepared from the resistant strains using sodium chloride and cetyltrimethylammonium bromide method as described previously .
2.3. Amplification of katG Gene
Amplification was performed in the isolated genomic DNA using a reaction mixture containing 1 μL of forward and reverse primers (10 pmol) each, 6 μL of deoxyribonucleoside triphosphates (dNTPs) mix (2.5 mM), 2.5 μL of 10X PCR buffer, 10–50 ng of template genomic DNA, and 1 U of Taq DNA polymerase (Amersham Biosciences, UK). The amplification was performed in a thermal controller (MJ Research, USA) with 30 cycles (1 minute (min) at 95°C, 30 seconds at 63°C, and 1 min at 72°C, followed by a final extension step at 72°C for 10 min). The primers of katG gene forward sequence (5′-A A A C A G C G G C G C T G G A T C G T 3′) and reverse sequence (5′-G T T G T C C C A T T T C G T C G G G G 3′) were used to generate a 209 bp fragment encompassing the S315T codon . The amplicons were purified using GFX COLUMN (Amersham Biosciences, UK) according to the manufacturer’s instructions.
2.4. DNA Sequencing
Sequencing of the amplicon was carried out using an automated DNA sequencer (ABI Prism 310 Genetic Analyzer-Applied Biosystems, USA), using the above-mentioned primers and the BigDye terminator sequencing kit (Applied Biosystems). To 4 μL of the terminator ready reaction mix, l μL of the amplified fragment (2-3 ng) and 1 μL of the primer (1 pmol/μL) were added and the volume was made up to 20 μL using deionised water. The reaction mixture was subjected to cycle sequencing. The samples were vortexed and spun, then heated at 95°C for 2 min, and immediately chilled on ice. They were vortexed and spun again and placed on ice and loaded onto the DNA sequencer. We refer to supporting documents for the figures (see Supplementary Material available online at http://dx.doi.org/10.1155/2014/257983). The sequence obtained was compared with sequences available in EMBOSS database via http://www.ebi.ac.uk/emboss/align/ using the alignment tool. The GenBank accession number for katG is X68081.
3. Results and Discussion
On the basis of DST results, 46 strains were found to have a minimum inhibitory concentration (MIC) of 1 mg/liter (L), 17 had an MIC of 5 mg/L, and the remaining strains had MICs of >5 mg/L. Amongst the 85 isolates, 51% were MDR cases, and 48% were found to be INH monoresistant cases with or without resistance to other TB drugs (data not shown). A laboratory reference strain of M. tuberculosis, H37Rv, was used as the control. Of the 85 phenotypically isolates included in the present study, 56 isolates (66%) were found to possess resistance conferring mutations to INH in the katG gene upon genotyping. The remaining 29 phenotypically resistant isolates had no known mutations in the katG gene. Of the 56 isolates with mutations, 47 had ACC (Thr), 6 had AAC (Asn), two had ATC (Ile), and one had CGC (Arg) substitution at codon 315 (Table 1). This observation suggests a high frequency distribution of S315 substitutions in south Indian isolates, which is consistent with the global pattern ; therefore, this region may be regarded as a hot spot region. The predominance of 315 mutations in the katG gene in clinical isolates has been documented in several studies, with observed frequencies ranging between 30% and >90%. In countries such as Scotland, Spain, Italy, and Uruguay [25–28] the prevalence of strains with S315T substitution was found to be relatively low (20–50%). Studies from countries like Netherlands, India, Africa, China, and Dubai have reported moderate levels of prevalence of the 315 substitutions, ranging between 50 and 70% [16, 18, 19, 29–31]. The S315T substitutions have been reported to be highly prevalent (60 to >90%) among strains circulating in counties like South Africa, Russia, Brazil, Lithuania, and Peru [24, 32–35].
|Strains with MIC of 1 mg/liter (number 1–28, 68–85), 5 mg/liter (number 29–45), and >5 mg/liter (number 46–67). Tamil Nadu cultures (number 1–67) and other states cultures (number 68–85). AP: Andhra Pradesh, KA: Karnataka, K: Kerala, H: isoniazid, and MDR: multidrug resistant.|
In this study, the frequency of S315T substitution in katG gene was slightly higher (70%) in MDR strains than in non-MDR (61%) isolates. It has also been previously observed that S315T substitution is associated with MDR strains of M. tuberculosis. Further, the mutation has important implications for the transmission and control of MDR-TB . We also observed a strong correlation between the MICs of INH and occurrence of S315T substitutions in our isolates with 74% of them showing high MIC values (≥5 mg/L) and were associated with S315T substitution, while only 58% of isolates with low MIC value of 1 mg/L had this mutation. These findings are in agreement with the observations made earlier by van Soolingen et al. .
In one our previous study, we compared virulence using five clinical mutants of KatG and found that the virulence of S315 mutants from south India was comparable to that of wild type H37Rv . This is in contrast to the classical notion that M. tuberculosis isolates from south India are inherently less virulent than fully susceptible organisms [22, 23]. Our present findings on the high prevalence of S315 mutants in south Indian isolates suggest their wider transmission. Moreover, Cohen et al. (2004) provided ecological evidence to support the theory that mutations at position 315 of katG confer INH resistance in M. tuberculosis without diminishing virulence or transmissibility . Also, in general, the concept of virulence is multifactorial and comprises several genes and factors. The functional loss of a single gene (katG) due to point mutation that is AGC to ACC may lead resistance to INH but need not necessarily compromise virulence. Therefore, the classical concept of less virulent nature of M. tuberculosis isolates from south India should be revisited.
Further, the present finding goes in agreement with a study from eastern rural areas of China, which demonstrated that isolates were widely transmitted. Also the correlation of prevalence and transmission between isolates especially with the katG S315T substitution and MDR-TB was confirmed. The study also recognized the katG S315T mutants among strains, which could be seen as an unsafe factor for subsequent development of MDR-TB. Early detection of the patients with strains would facilitate the modification of treatment regimens and appropriate infection control measures can be taken in time to reduce the risk of further development and transmission of MDR-TB . Additionally, the impact of INH resistance upon treatment outcomes is a matter of great concern. Patients with katG S315T substitution are hypothesized to have worse outcomes than those with inhA promoter mutations, especially when INH is used in the treatment .
Thus, the present study shows a high prevalence of S315T substitution in katG gene of clinical isolates of M. tuberculosis from south India. Also, this substitution is associated with high level resistance to INH and correlates with multidrug resistance.
Conflict of Interests
There is no conflict of interests amongst the authors.
Dr. Ameeruddin Nusrath Unissa receives financial support for postdoctoral fellowship from the Indian Council of Medical Research.
The fig-1 shows the amplicon of 209 bp of katG gene in 2-13 lanes from clinical samples and a control at 14th lane. Whereas the Fig. 2 and Fig. 3 represents electropherogram showing the sequences of 209 bp segment of katG gene with the wild type codon (AGC) and the mutant codon (ACC).
- R. Prasad, “MDR TB: current status,” Indian Journal of Tuberculosis, vol. 52, pp. 121–131, 2005.
- Centers for Disease Control and Prevention, “Revised definition of extensively drug-resistant tuberculosis,” Morbidity and Mortality Weekly Report, vol. 55, no. 43, p. 1176, 2006.
- “World Health Organization: Media centre,” Fact sheet No. 104, 2013.
- J. B. Nachega and R. E. Chaisson, “Tuberculosis drug resistance: a global threat,” Clinical Infectious Diseases, vol. 36, no. 1, pp. S24–S30, 2003.
- World Health Organization, International Union against Tuberculosis and Lung Disease, Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002–2007, Fourth Global Report, World Health Organization, 2008.
- C. N. Paramasivan, K. Bhaskaran, and V. Venkataraman, “Surveillance of drug resistance in tuberculosis in the state of Tamil Nadu,” Indian Journal of Tuberculosis, vol. 47, pp. 27–33, 2000.
- F. G. Winder, P. Brennan, and C. Ratledge, “Synthesis of fatty acids by extracts of mycobacteria and the absence of inhibition by isoniazid,” Biochemical Journal, vol. 93, no. 3, pp. 635–640, 1964.
- G. Middlebrook, “Isoniazid-resistance and catalase activity of tubercle bacilli; a preliminary report,” American Review of Tuberculosis, vol. 69, pp. 471–472, 1954.
- G. Middlebrook and M. L. Cohn, “Some observations on the pathogenicity of isoniazid-resistant variants of tubercle bacilli,” Science, vol. 118, no. 3063, pp. 297–299, 1953.
- Y. Zhang, B. Heym, B. Allen, D. Young, and S. Cole, “The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis,” Nature, vol. 358, no. 6387, pp. 591–593, 1992.
- A. Banerjee, E. Dubnau, A. Quemard et al., “inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis,” Science, vol. 263, no. 5144, pp. 227–230, 1994.
- K. Mdluli, R. A. Slayden, Y. Zhu et al., “Inhibition of a Mycobacterium tuberculosisβ-Ketoacyl ACP synthase by isoniazid,” Science, vol. 280, no. 5369, pp. 1607–1610, 1998.
- S. Sreevatsan, X. Pan, Y. Zhang, V. Deretic, and J. M. Musser, “Analysis of the oxyR-ahpC region in isoniazid-resistant and - susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities,” Antimicrobial Agents and Chemotherapy, vol. 41, no. 3, pp. 600–606, 1997.
- S. Ramaswamy and J. M. Musser, “Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update,” Tubercle and Lung Disease, vol. 79, no. 1, pp. 3–29, 1998.
- H. J. Marttila, H. Soini, E. Eerola et al., “A Ser315Thr substitution in KatG is predominant in genetically heterogeneous multidrug-resistant Mycobacterium tuberculosis isolates originating from the St. Petersburg area in Russia,” Antimicrobial Agents and Chemotherapy, vol. 42, no. 9, pp. 2443–2445, 1998.
- D. van Soolingen, P. E. W. de Haas, H. R. van Doorn, E. Kuijper, H. Rinder, and M. W. Borgdorff, “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,” The Journal of Infectious Diseases, vol. 182, no. 6, pp. 1788–1790, 2000.
- A. S. Pym, B. Saint-Joanis, and S. T. Cole, “Effect of katG mutations on the virulence of Mycobacterium tuberculosis and the implication for transmission in humans,” Infection and Immunity, vol. 70, no. 9, pp. 4955–4960, 2002.
- N. Siddiqi, M. Shamim, N. K. Jain et al., “Molecular genetic analysis of multi-drug resistance in Indian isolates of Mycobacterium tuberculosis,” Memorias do Instituto Oswaldo Cruz, vol. 93, no. 5, pp. 589–594, 1998.
- A. N. Unissa, N. Selvakumar, S. Narayanan, and P. R. Narayanan, “Molecular analysis of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from India,” International Journal of Antimicrobial Agents, vol. 31, no. 1, pp. 71–75, 2008.
- A. N. Unissa, S. Narayanan, C. Suganthi, and N. Selvakumar, “Detection of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from India using Ser315Thr marker by comparison of molecular methods,” International Journal of Molecular and Clinical Microbiology, vol. 1, pp. 52–59, 2011.
- D. A. Mitchison, J. B. Selkon, and J. Lloyd, “Virulence in the guinea-pig, susceptibility to hydrogen peroxide, and catalase activity of isoniazid-sensitive tubercle bacilli from South Indian and British Patients,” The Journal of Pathology and Bacteriology, vol. 86, pp. 377–386, 1963.
- T. V. Subbaiah, D. A. Mitchison, and J. B. Selkon, “The susceptibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacilli,” Tubercle, vol. 41, no. 5, pp. 323–333, 1960.
- Tuberculosis Chemotherapy Centre Madras, “A concurrent comparison of isoniazid plus PAS with three regimens of isoniazid alone in the domiciliary treatment of pulmonary tuberculosis in South India,” Bulletin of the World Health Organization, vol. 23, no. 4-5, pp. 535–585, 1963.
- P. Kiepiela, K. S. Bishop, A. N. Smith, L. Roux, and D. F. York, “Genomic mutations in the katG, inhA and aphC genes are useful far the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa,” Tubercle and Lung Disease, vol. 80, no. 1, pp. 47–56, 2000.
- Z. Fang, C. Doig, A. Rayner, D. T. Kenna, B. Watt, and K. J. Forbes, “Molecular evidence for heterogeneity of the multiple-drug-resistant Mycobacterium tuberculosis population in Scotland (1990 to 1997),” Journal of Clinical Microbiology, vol. 37, no. 4, pp. 998–1003, 1999.
- S. Samper, M. J. Iglesias, M. J. Rabanaque et al., “Systematic molecular characterization of multidrug-resistant Mycobacterium tuberculosis complex isolates from Spain,” Journal of Clinical Microbiology, vol. 43, no. 3, pp. 1220–1227, 2005.
- L. Rindi, L. Bianchi, E. Tortoli, N. Lari, D. Bonanni, and C. Garzelli, “Mutations responsible for Mycobacterium tuberculosis isoniazid resistance in Italy,” International Journal of Tuberculosis and Lung Disease, vol. 9, no. 1, pp. 94–97, 2005.
- G. Varela, S. González, P. Gadea et al., “Prevalence and dissemination of the Ser315Thr substitution within the KatG enzyme in isoniazid-resistant strains of Mycobacterium tuberculosis isolated in Uruguay,” Journal of Medical Microbiology, vol. 57, no. 12, pp. 1518–1522, 2008.
- W. H. Haas, K. Schilke, J. Brand et al., “Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa,” Antimicrobial Agents and Chemotherapy, vol. 41, no. 7, pp. 1601–1603, 1997.
- M. Zhang, J. Yue, Y.-P. Yang et al., “Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China,” Journal of Clinical Microbiology, vol. 43, no. 11, pp. 5477–5482, 2005.
- S. Ahmad, E. Fares, G. F. Araj, T. D. Chugh, and A. S. Mustafa, “Prevalence of S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Dubai and Beirut,” International Journal of Tuberculosis and Lung Disease, vol. 6, no. 10, pp. 920–926, 2002.
- I. Mokrousov, O. Narvskaya, T. Otten, E. Limeschenko, L. Steklova, and B. Vyshnevskiy, “High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001,” Antimicrobial Agents and Chemotherapy, vol. 46, no. 5, pp. 1417–1424, 2002.
- M. S. N. Silva, S. G. Senna, M. O. Ribeiro et al., “Mutations in katG, inhA, and ahpC genes of Brazilian isoniazid-resistant isolates of Mycobacterium tuberculosis,” Journal of Clinical Microbiology, vol. 41, no. 9, pp. 4471–4474, 2003.
- D. Bakonyte, A. Baranauskaite, J. Cicenaite, A. Sosnovskaja, and P. Stakenas, “Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical isolates in Lithuania,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 6, pp. 2009–2011, 2003.
- P. Escalante, S. Ramaswamy, H. Sanabria et al., “Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru,” Tubercle and Lung Disease, vol. 79, no. 2, pp. 111–118, 1998.
- A. N. Unissa, S. Narayanan, and N. Selvakumar, “Virulence in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from south India,” International Journal of Molecular and Clinical Microbiology, vol. 1, pp. 87–96, 2011.
- T. Cohen, M. C. Becerra, and M. B. Murray, “Isoniazid resistance and the future of drug-resistant tuberculosis,” Microbial Drug Resistance, vol. 10, no. 4, pp. 280–285, 2004.
- Y. Hu, S. Hoffner, W. Jiang, W. Wang, and B. Xu, “Extensive transmission of isoniazid resistant M. tuberculosis and its association with increased multidrug-resistant TB in two rural counties of Eastern China: a molecular epidemiological study,” BMC Infectious Diseases, vol. 10, article 43, 2010.
- K. R. Jacobson, D. Theron, T. C. Victor, E. M. Streicher, R. M. Warren, and M. B. Murray, “Treatment outcomes of isoniazid-resistant tuberculosis patients, Western Cape Province, South Africa,” Clinical Infectious Diseases, vol. 53, no. 4, pp. 369–372, 2011.
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