# Investigation of Ser315 Substitutions withinkatG Gene in Isoniazid-Resistant Clinical Isolates of Mycobacterium tuberculosis from South India

Authors: A. Nusrath Unissa; N. Selvakumar; Sujatha Narayanan; C. Suganthi; L. E. Hanna
Journal: BioMed Research International
 (2015)

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## Abstract

Mutation at codon 315 ofkatG 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.


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## Body

## 1. 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 ofM. tuberculosis resistant to INH are seen with increasing frequency (1 in 106) 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 resistance inM. 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 bykatG 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 [10–13]. Of these,inhA andahpC promoter mutations lead to low-level INH resistance, whereas mutation inkatG is responsible for high-level resistance. Zhang et al. (1992) demonstrated that mutation in thekatGgene, coding for KatG protein, is a major contributor to INH resistance inM. tuberculosis[10].The most prevalent mutation found to occur inkatGgene 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 μg/mL) [16]. When compared to other resistance-conferring mutations inkatG, 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.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 (INHR) isolates were quite distinct and had lower virulence and higher susceptibility to H2O2 [21–23]. Hence, it was of interest to analyze mutations inkatGgene encompassing codon 315 in INHR clinical isolates ofM. tuberculosis from south India by DNA sequencing method in the present study.


## 2. Methods

### 2.1. INH-Resistant Clinical Isolates ofM. tuberculosis

A total of 85INHR clinical isolates ofM. 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 thekatG 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 [18].


### 2.3. Amplification ofkatG Gene

Amplification was performed in the isolated genomic DNA using a reaction mixture containing 1μL 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 ofTaq 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 ofkatGgene 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 [24]. 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 4μL 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 forkatG is X68081.


## 2.1. INH-Resistant Clinical Isolates ofM. tuberculosis

A total of 85INHR clinical isolates ofM. 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 thekatG 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 [18].


## 2.3. Amplification ofkatG Gene

Amplification was performed in the isolated genomic DNA using a reaction mixture containing 1μL 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 ofTaq 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 ofkatGgene 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 [24]. 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 4μL 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 forkatG 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 ofM. tuberculosis, H37Rv, was used as the control. Of the 85 phenotypically INHR isolates included in the present study, 56 isolates (66%) were found to possess resistance conferring mutations to INH in thekatGgene upon genotyping. The remaining 29 phenotypically resistant isolates had no known mutations in thekatGgene. 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 thekatG gene in INHR 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 INHR 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 INHR strains circulating in counties like South Africa, Russia, Brazil, Lithuania, and Peru [24, 32–35].Table 1
Mutational profile of INH-resistant clinical isolates ofM. tuberculosisfrom south India.
Serial number
DST
katG
States
Serial number
DST
katG
States
1
MDR
ACC
TN
44
MDR
AAC
TN
2
MDR
ACC
TN
45
MDR
ACC
TN
3
MDR
ACC
TN
46
H
AAC
TN
4
H
TN
47
H
AAC
TN
5
H
TN
48
H
TN
6
H
TN
49
H
TN
7
H
ACC
TN
50
H
ACC
TN
8
MDR
TN
51
H
ACC
TN
9
MDR
ACC
TN
52
H
ACC
TN
10
MDR
ATC
TN
53
MDR
ATC
TN
11
MDR
ACC
TN
54
MDR
ACC
TN
12
MDR
ACC
TN
55
H
CGC
TN
13
H
TN
56
MDR
TN
14
H
TN
57
MDR
ACC
TN
15
MDR
ACC
TN
58
H
TN
16
MDR
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
85
H
ACC
K
43
MDR
ACC
TN
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 inkatG gene was slightly higher (70%) in MDR strains than in non-MDR (61%) INHR isolates. It has also been previously observed that S315T substitution is associated with MDR strains ofM.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 H37Rv [36]. This is in contrast to the classical notion that INHRM. tuberculosisisolates 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 ofkatGconfer INH resistance inM. tuberculosiswithout 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 INHRM. tuberculosisisolates from south India should be revisited.Further, the present finding goes in agreement with a study from eastern rural areas of China, which demonstrated thatINHR isolates were widely transmitted. Also the correlation of prevalence and transmission between INHR isolates especially with thekatG S315T substitution and MDR-TB was confirmed. The study also recognized thekatG S315T mutants among INHR strains, which could be seen as an unsafe factor for subsequent development of MDR-TB. Early detection of the patients with INHR 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 withkatG S315T substitution are hypothesized to have worse outcomes than those withinhA promoter mutations, especially when INH is used in the treatment [39].Thus, the present study shows a high prevalence of S315T substitution inkatG gene of INHR clinical isolates ofM. tuberculosis from south India. Also, this substitution is associated with high level resistance to INH and correlates with multidrug resistance.



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