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.


Introduction
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 [1]. 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 [2]. 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 [3].
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 10 6 ) as compared to isolates resistant to other drugs [4]. 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 [5]. 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 [6].
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 2 BioMed Research International resistance in M. tuberculosis correlates with the loss of catalase and peroxidase (CP) activity in resistant strains [7][8][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 [7]. 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][11][12][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 [10].
The most prevalent mutation found to occur in katG gene is AGC to ACC at codon 315 [14], or Ser315Thr (S315T) substitution. The appearance of this mutation was most frequent amongst MDR strains [15]. However, this mutation was also reported to be associated with intermediate or high levels of resistance to INH (1 to 10 g/mL) [16]. 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 [17]. Therefore, it may be considered as a reliable biomarker for the detection of INH resistance.

INH-Resistant Clinical Isolates of M. tuberculosis.
A total of 85 INH R 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.

DNA Extraction from INH-Resistant Strains.
Genomic DNA was prepared from the resistant strains using sodium chloride and cetyltrimethylammonium bromide method as described previously [18].

Amplification of katG Gene.
Amplification was performed in the isolated genomic DNA using a reaction mixture containing 1 L of forward and reverse primers (10 pmol) each, 6 L of deoxyribonucleoside triphosphates (dNTPs) mix (2.5 mM), 2.5 L 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 - were used to generate a 209 bp fragment encompassing the S315T codon [24]. The amplicons were purified using GFX COLUMN (Amersham Biosciences, UK) according to the manufacturer's instructions.

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 L of the terminator ready reaction mix, l L of the amplified fragment (2-3 ng) and 1 L of the primer (1 pmol/ L) were added and the volume was made up to 20 L 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.

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, H 37 Rv, was used as the control. Of the 85 phenotypically INH R 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 [14]; therefore, this region may be regarded as a hot spot region. The predominance of 315 mutations in the katG gene in INH R clinical isolates has been documented in several  TN  59  MDR  TN  17  MDR  AAC  TN  60  MDR  ACC  TN  18  H  TN  61  H  ACC  TN  19  H  AAC  TN  62  H  ACC  TN  20  MDR  ACC  TN  63  H  TN  21  MDR  ACC  TN  64  MDR  ACC  TN  22  H  TN  65  MDR  ACC  TN  23  MDR  ACC  TN  66  MDR  ACC  TN  24  MDR  TN  67  MDR  ACC  TN  25  H  ACC  TN  68  MDR  ACC  AP  26  H  ACC  TN  69  H  AP  27  H  ACC  TN  70  H  ACC  AP  28  MDR  TN  71  MDR  AP  29  H  TN  72  MDR  AP  30  MDR  TN  73  MDR  ACC  AP  31  H  TN  74  MDR  AP  32  MDR  AAC  TN  75  MDR  AP  33  H  ACC  TN  76  MDR  ACC  AP  34  H  ACC  TN  77  MDR  AP  35  H  ACC  TN  78  MDR  ACC  AP  36  H  ACC  TN  79  MDR  ACC  AP  37  MDR  ACC  TN  80  H  ACC  AP  38  MDR  ACC  TN  81  H  ACC  AP  39  H  ACC  TN  82  H  KA  40  H  ACC  TN  83  H  KA  41  MDR  TN  84  H  ACC  KA  42  H  ACC  TN [24,[32][33][34][35].
In this study, the frequency of S315T substitution in katG gene was slightly higher (70%) in MDR strains than in non-MDR (61%) INH R 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 [17]. 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. [16].
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 H 37 Rv [36]. This is in contrast to the classical notion that INH R 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 [37]. 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 INH R 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 INH R isolates were widely transmitted. Also the correlation of prevalence and transmission between INH R isolates especially with the katG S315T substitution and MDR-TB was confirmed. The study also recognized the katG S315T mutants among INH R strains, which could be seen as an unsafe factor for subsequent development of MDR-TB. Early detection of the patients with INH R 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 [38]. 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 [39].
Thus, the present study shows a high prevalence of S315T substitution in katG gene of INH R 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.