1. Introduction

Tuberculosis (TB) is a bacterial infectious disease, caused by the obligate human pathogen, Mycobacterium tuberculosis (M tb). Tuberculosis remains a vital life-threatening diseases and posing the expanding global health crisis that mandates new therapeutic strategies. India contributes the highest number of new cases, accounting for 20% of the global burden, of which approximately 1.2 % occur in persons with HIV ^[1]. The bacterium is transmitted via the respiratory route as a highly infectious aerosol with varying outcomes occurring from this initial Mycobacterium tuberculosis exposure. However, the majority of individuals infected with M. tuberculosis have a non-contagious, clinically-latent infection with an absence of clinical symptoms ^[2]. Latently-infected individuals have a 5-10% risk of developing reactivation TB disease during their lifetime ^[3], often due to immunosuppressive circumstances, with HIV infection being the greatest identified cause ^{[4, 5]}. The history of TB is changed as the first drug with anti-mycobacterium activity was introduced. Streptomycin was exploited as a monotherapy and hence the first drug resistance got emerged ^[6]. Later on, several anti-mycobacterium drugs were discovered. However, the resistance against all of them got developed by the strain of M. tuberculosis. Therefore, the tuberculosis that is resistant to at least isoniazid and rifampicin, the most powerful first line anti TB drugs is called as Multi-Drug Resistance (MDR) strains. In addition to being MDR, the extensively drug resistance (XDR, resistance to MDR as well as to any member of the quinolone family and at least one of the second-line anti-TB drugs) was arisen subsequently by strains of M tb. Therefore, it is necessitated to understand the mechanism of MDR and develop the potent therapeutics which will not easily predispose to the resistance by strains of M tb. By virtue of the availability of the new information generated by decoding the complete genome sequence of M tb and major progresses in the molecular biology tools we shall understand the specific gene mutations which are responsible for drug resistance ^{[7, 8]}.

A number of molecular assays are available to detect the presence of M. tuberculosis and to assess drug resistance pattern ^[9]. Line probe assay is a method which relies on the hybridization of amplicons to probes on a nitrocellulose strip. The assay detects 99% and 88% RIF and INH resistant strains respectively in direct sputum samples or culture isolates ^[10]. Although, assay has advantage of short time analysis, it has some limitations which need to improve in nucleic acid amplification test. The limitation of assay is its inability to carry out species level identification of Mycobacterium and cannot detect novel mutations. Other major concern of the assay is possibility of cross contamination and mispairing of the DNA form another sources and may result in false positive results which cannot recommended as an accurate method.

The new method including PCR based sequencing has overcome the above limitations. This method has highest reproducibility and reliability as compared to line probe assay. This gives 100% specificity in the results and good interpretation helps in better understanding for the drug resistance and recommendation for the drug therapy. Polymerase Chain Reaction (PCR)-based sequencing is the key tool exploit for elucidating the genetic mechanisms of drug resistance in M.tb. However, the current knowledge of the techniques and diagnostic methods to screen gene mutations seems inadequate and hence there is a pressing need to improve the methodologies to explore the new candidate genes and detect novel mutations.

In the present study, we have focused on standardizing the rapid assay of PCR and sequencing for the detection of mutations in some of the candidate genes responsible for drug resistance in M. tb. The molecular aspects of drug resistance help to understand the molecular strategies to rapidly diagnose MDR-TB. The molecular aspects of drug resistance help to understand the molecular strategies to rapidly diagnose MDR-TB. We utilize PCR method for analyzing the drug sensitivity and mutation profile of these local M. tuberculosis isolates.

2. Materials and Methods

Sputum samples were received in a sterile container with prior consent of patient as per the guidelines of ethical committee of Jaslok Hospital and Research Center, Mumbai, India. Specimens were processed for acid fast staining to detect the presence of acid fast bacilli. Positive specimens were decontaminated by using equal amounts of N-acetyl-L-cysteine-sodium hydroxide solution (0.25g in 50 ml sodium hydroxide solution) and were incubated for 15 minutes. The solution was then diluted in phosphate buffer (pH 6.8) and centrifuged at 8,000rpm for 15min. The sediments obtained were stored in TE buffer (pH 8.0) at -20°C for subsequent PCR assay.

The genomic DNA was extracted from positive sputum samples by previously described simple boiling method ^[11]. The sediment obtained from decontamination procedure was washed three times in TE (Tris EDTA, pH 8.0) buffer by centrifugation at 12,000rpm for 10 minutes. The precipitation obtained was dissolved again in TE buffer(volume should be added according to the size of pellet)and boiled at 100°C for 15mins. The turbid supernatant containing DNA was used as a template for PCR amplification.

The extracted DNA was amplified with the oilgonuleotide primers (listed in Table 1). The presence of regulatory gene targets of M.tuberculosis was identified by Polymerase Chain.

Table 1. shows primer sequences for 1^st line drug resistant genes for M. tuberculosis

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Table 2. shows primer sequences for 2^nd line drug resistant genes for M. tuberculosis

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Reaction. Each PCR contained 10µl of DNA template (undiluted or threefold-concentrated extract). For amplification of all genes (Table 1 & Table 2), PCR was performed in a final volume of 25µl containing 10 mM Tris-HCl (pH 8.3), 25 mM MgCl2; 2mM deoxynucleoside triphosphate (Applied Biosystems); 10 pmoles of each primers (2µl), and 1.0 U of Taq DNA polymerase (Invitrogen, Paisley, UK)and PCR grade water to make final volume 25µl. The reaction mixture was then subjected to 40 cycles with run profiles: 95°C for 15min, 94°C for 1min, annealing according to genes (Table 1), and 72°C for 1min, followed by a final extension at 72°C for 10mins. All reactions were carried out on 2720 DNA Thermal Cycler (Applied Biosystems, USA). After PCR amplification, 10µl aliquot of the PCR product was electrophoresed for 1h through 2% agarose gel containing ethidium bromide dye, and the target band size of genes was visualized under UV illumination.

Before sequencing, PCR products were purified. Purified PCR products were used for pre-sequencing PCR with 5x sequencing buffer (Applied Biosystems, Warrington, UK), BigDye® Terminator v3.1 cycle (BDT) from Applied Biosystems, Foster city, CA, USA and 3.2 pmol/µl of either sense or antisense primer. PCR was carried out at 96°C for 1min. followed by 25 cycles of denaturation at 96°C for 10sec., annealing at 50°C for 5sec and elongation at 60°C for 4min. After pre-sequencing PCR, products were again cleaned and finally dissolved in Hi-Di^TM Formamide (Applied Biosystems, Foster city, CA, USA). Amplicons were then loaded onto Micro Amp™ Optical 96-Well Reaction Plate (Applied Biosytems, Foster city, USA) and subjected to cycle sequencing using an automated Genetic Analyzer 3100 (Applied Biosystems, Foster city, CA, USA) which utilizes ABI’s BigDye Terminator chemistry based on Sanger’s sequencing method. Both Sense and antisense strand sequencing were carried out to confirm all mutations reported in this study. All post run analyses were performed using ClustalW, BioEdit Sequence Alignment Editor. Each sequence was compared with the previously published of respective genes sequences of strain H37Rv in the NCBI database.

3. Results

A diverse set of MDR and XDR clinical isolates of M. tb was selected for the investigation of genetic mutations. First we screened for the known mutations in M. tb genome that are notorious to confer resistance to the antibiotics in the population of western India. Total 5 healthy individuals were recruited as a control in our study. We employed PCR and sequencing methods to find out the mutation and we observed that all the mutations were in the coding region of drug resistance genes. The mutation and percentage in no. of isolates is shown in Table 3.

In the PCR assay of all the 12 genes of clinical isolates were amplified by using primers pairs for the respective genes. The size of fragment of each gene is determined by using molecular marker 100 bp (Fermentas, Thermo Scientific, USA). Figure 1 and Figure 2 depict the amplification of first line drugs and second line drugs gene fragments respectively.

Figure 1. Amplification of first line drug gene

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Figure 2. Amplification of second line drug genes

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The first line drugs genes are rpoB, katG, ndh, inhA, kasA, rrs, rpsL,pncA and embB for the Rifampicin, Isoniazid, Streptomycin, Pyrazinamide and Ethambutanol respective antibiotics.

RMP is an important first-line drug for the treatment of TB. RMP interferes with RNA synthesis by binding to the β subunit of the RNA polymerase. The sequencing of the 436 bp central region of the rpoB gene revealed two point mutations at two different codons. Across the rpoB gene, 2 (10%) isolates showed a base substitution at codon 516, out of them D516V (Figure 3b) was observed in one drug resistant isolate and D516G (Figure 3c) in another causing GAC→GTC and GAC→GGC substitutions respectively. There was no mutation in normal individual (Figure 3a). Second point mutation at rpoB was found at codon 531, in 3(15%) isolates causing TCG→TTG substitution. It is designated as S531L (Figure 4b). The overall 5 mutations were observed in rpoB gene in our population.

Figure 3. Electropherogram showing mutation rpoB gene D516G/V

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Figure 4. Electropherogram showing mutation in rpoB gene S531L

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INH is a prodrug that is activated by the catalase peroxidase enzyme (KatG) encoded by the katG gene. Mutation in katG is the main mechanism of INH resistance. The 414 bp fragment of katG when subjected to sequencing, we found S315T (Figure 5b) missense mutation in 6 (30%) isolates causing Serine to Threonine amino acid change in the protein and AGC→ACC in the genome. There was no change in normal individual which is taken as positive control (Figure 5a). Interestingly, no mutations were observed in inhA, kasA and ndh promoter regions. In our population only katG shows mutation with high percentage.

Figure 5. Electrophoregram showing mutation in katG gene S315T

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SM is an aminoglycoside antibiotic that is active against a variety of bacterial species, including M. tuberculosis.SM inhibits protein synthesis by binding to the 30S subunit of bacterial ribosome, causing misreading of the mRNA message during translation. Mutations in rpsL and rrs are the major mechanism of SM resistance. For Streptomycin resistance, rpsL showed two substitutions at codon 43 and 88 causing AAG→AGG responsible to substitute Lysine by Arginine (Figure 6b) in 8 (40%) isolates. There was no change in normal individual (Figure 6a). The rrs gene did not reveal any reported alterations.

Figure 6. Electropherogram showing mutation in rpsL gene L43, 88R

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PZA is a prodrug that requires conversion to its active form pyrazinoic acid (POA), by the pyrazinamidase/ nicotinamidase enzyme encoded by the pncA gene of M. tuberculosis. The pncA mutations are highly diverse and scattered along the gene, which is unique to PZA resistance. The most frequent mutation at pncA195 was detected in 7 (35%) of those 40 isolates, causing CCT→CTT/CAT (Figure 7b) substitution.

Figure 7. Electropherogram showing mutation in pncA gene P195L

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EMB is a first line drugs that is used in combination along with RMP, INH and PZA to prevent the emergence of drug resistance. EMB interferes with the biosynthesis of cell wall arabinogalactan. Arabinosyl transferase encoded by embB, an enzyme involved in the synthesis of arabinogalactan, has been proposed as the target of EMB in M. tuberculosis and M. avium. In embB gene, overall, mutations were observed in 3 (15%) isolates. The 2 different types of mutations in embB were observed at embB306 causing base change at 2 triplet positions ATG →GTG (Figure 8b) and ATG→ATC/ATA (Figure 8c/d).

The second line drugs genes gyrA and gyrB for fluoroquinolones and thyA for para amino salicylic acid.

Figure 8. Electropherogram showing mutation in embB gene M306V/I

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In most bacterial species, FQs inhibit DNA gyrase (topoisomerase II) and topoisomerase IV, resulting in microbial death. DNA gyrase is a tetrameric A2B2 protein. M. tuberculosis has respectively gyrA and gyrB encoding the A and B subunits. A conserved region, the quinolone-resistance-determining region (QRDR) of gyrA and gyrB , has been found to be a most important area involved in the exhibition of FQ resistance in M. tuberculosis. In gyrA 2 (10%) isolates shows prominent mutation at codon 90 causing GCG to GTG (Figure 9b), all (100%) isolates were showing the mutation at codon 95 causing AGC to ACC (Figure 9c) and no mutation in gyrB gene. The gyrA gene mutation is high in our population.

Figure 9. Electropherogram showing mutation in gyrA gene A90V and S95T..

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PAS is recommended with the combination of isoniazid and streptomycin. However, its mechanism of action is not clearly elucidated but recently an association of p-amino salicylic acid resistance with mutations in the thyA gene that encodes thymidylate synthase A for thymine biosynthesis was shown. The common mutation in thyA gene is Thr202Ala was reported in many studies. No mutations in thyA gene were observed in our population.

Table 3. Mutations in the MDR and XDR genes in clinical isolates M. tuberculosis.

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The overall analysis of our mutation study reveals that alterations in rpoB, katG, rpsL, pncA and gyrA genes are with higher percentage (Table 3) in western part of India, which designates higher occurrence of this gene resistance in our population.

4. Discussion

Amongst the molecular methods available, the PCR directed DNA sequencing has proved its immense potential in the detection of alterations in the genome of M.tb. The precision and accuracy in exploring the genetic mutations is well exhibited by this method. The rapidly available information pertaining to alterations and drug susceptibility patterns reduces the spread of drug-resistant strains and even facilitate the rapid assortment of treatment regimens. Here, we employed PCR-combined DNA sequencing to detect drug resistance TB cases. In a cohort of 20 patients being investigated for tuberculosis, we have reported some of the mutations in the clinical isolates of M.tb.

Total 20 isolates from the western India showed a substitution(s) or a point mutation in genes of anti tubercular drugs. The majority of the mutations in the rpoB gene map to codon 516 and 531. Mani et al 2001 from India, found mutations most frequently at codon 531(56%) and codon 526 (19%) ^[12]. In a study from China, Lu et al., reported mutations at codon 531 (51.6% ^[13]. Abdelaal et al. in Egypt, also found frequencies of mutations at codons 531 (45%), and 516 (20%) ^[14]. However, the frequency of rpoB mutations in our population is found to be different from other studies reported across the globe. Collectively 10% and 15% of the clinical isolates of M.tb are found mutated at rpoB516 and rpoB531 respectively. It indicates that, these mutations at 531 and 516 are common with a slightly varied occurrence across the geographical regions. The genetic mutations in isoniazid (INH) genes confer the resistance to the drug and impact on the clinical presentation of the disease. One of the targets for activated INH is the protein encoded by the inhA locus. Ramaswamy and Musser, Basso and Blanchard, identified 6 point mutations in inhA gene (Ile16Thr, Ile21Thr, Ile21Val, Ile47Thr, Val78Ala and Ile95Pro) ^{[15, 16]}. Recently, in an Indian study, Vadwai et al found 21.4% resistance frequency in inhA gene ^[17]. The other mechanism for the INH resistance is in the protein encoded by ndh locus. Rozwarski et al and Miesel et al. 1998 found prominent 10 mutations in ndh gene at codon 110 and 268 ^{[18, 19]}. Lee et al and Piatek et al reveal 4 different substitutions in kasA at codon 66, 269, 312 and 413 ^{[20, 21]}. Nusrath et al 2008, observed no mutation at kasA locus in South India ^[22]. Though the occurrence of this mutation was high in their study, we did not find any mutations at any parts of these genes in western Indian scenario. The Ser315Thr substitution is estimated to occur in 30-60% of INH resistant isolates ^[23]. In 2006 population genetics for isoniazid was studied throughout the globe, they revealed 49% mutation in katG gene in India isolates ^[24]. A recently Indian study, by Anamika et al found that the most common mutation in INH isolates is katG gene at codon 315 with the frequency 82.29% ^[25]. However, 30% of katG resistant clinical isolates of M. tb showed the frequency of KatG315 (Ser to Thr) in our study.

Ramaswamy and Musser, 1998 reported point mutations in rrs and rpsL genes in 65–67% of STR resistant isolates. Mutation in rpsL gene at codon 43 and 88 are associated with STR and in rrs at position 491,512,516 was highly observed ^[15]. In contrast, in our study 40% of the clinical isolates showed substitutions in rpsL gene but no alteration was found in rrs gene. Jureen et al in their study from Sweden observed many mutations for pncA gene ^[26], and in 2009, S S Prabhu et al from India had found many mutations in pncA gene at various codons ^[27], but we observed overall 35% frequency of the mutation at codon 195 of pncA gene. Mita et al 2008 from India had found reported and some novel mutation in embB ^[28]. Shinnick et al 2009 isolate mutants containing point mutation with 55% frequency at embB codon 306 ^[29]. Of the mutants, our result shows 15% mutation at position 306 with polymorphism.

The most common single nucleotide mutation sites were codons 94, 91, and 90 in gyrA and, in total, the relative frequencies of these codons were 56.8%, 6.3% and 25.3% respectively ^[30]. In a study from china, Cheng et al 2003 reported in all 138 isolates point mutation at codon 95 and Lau et al found in one strain a novel mutation at codon 95 ^{[31, 32]}. Similarly, our study revealed 10% mutation frequency at codon 90 and in all 20(100%) isolates at codon 95 causing AGC to ACC (Ser to Thr). In gyrB gene the most common mutation was observed at codon 485 and 500 with 11.6% frequency in ofloxacin resistant strains ^[33]. However, no mutations were observed at gyrB in our study. The present study results clearly show that Thr202Ala in thyA is not involved in the development of resistance to PAS (Para amino salicyclic) in M. tuberculosis complex isolates. There was no mutation in thyA in our PAS resistant strains in our study ^[30].

5. Conclusion

The genetic mutations among the MDR and XDR genes are being observed in a diverse set of clinical isolates of Mycobacterium tuberculosis, resulting in development of resistance against the newer drugs and ultimately responsible for exacerbation of the disease. We found substitutions and missense mutations among the genes against first and second line drugs. Out of them, 5 (25%) in rpoB at codon 516 and 531, 6 (30%) in katG at codon 315, 8 (40%) in rpsL at codon 43 & 88, 3 (15%) in embB at codon 306 and 7 (35%) in pncA at codon 195. No mutations in inhA, ndh, kasA and rrs genes were obtained in our study. In the second line drugs, 2(10%) at codon 90 & 100% mutations at codon 95 in gyrA gene, no mutation in gyrB and thyA gene was observed. We cogitate that the high level of genetic alterations in Mycobacterium tuberculosis may be one of the reasons for the emergence of MDR and XDR more rapidly. More-detailed molecular and epidemiological studies with strictly defined patient groups may further elucidate this proposition and help to formulate the new potent drugs to conquer on the battle of MDR and XDR.

Acknowledgements

We acknowledged with thanks the management of Jaslok Hospital and Research Center, Mumbai, India for sanctioning and supporting financial assistance for this project. We are also thankful to Miss Priyal Chikliwala and Mrs. Naina Rane for technical help for this study.

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