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Identification of single nucleotide polymorphism in rpoB gene among Mycobacterium tuberculosis isolates from Pakistan.

Byline: Memona Yasmin Nosheen Mujtaba and Rubina T. Siddiqui

Abstract

The emergence of multidrug resistant strains of Mycobacterium tuberculosis is a threat to tuberculosis control programs. The most effective strategy to control the spread of these strains is to use an accurate and rapid drug susceptibility test. Resistance to rifampicin one of the most potent first line anti-TB drugs is found to be associated with mutations in hot-spot" region of RNA polymerase gene (rpoB) are geographically distributed. M. tuberculosis isolates from various regions of Pakistan were collected and mutations were detected by reverse hybridization using in-house line probe assay. The rpoB gene from 45 isolates was cloned and sequenced to validate the results obtained by line probe assay. The most common mutations were found to be in codon 531 (56.45%) followed by codon 526 (9.68%) 516 (4.84%) and 518 (3.22%). Certain silent mutations and several novel mutations outside the hot-spot" region were also found. Copyright 2014 Friends Science Publishers

Keywords: Multiple drug resistance; Mutations; Rifampicin resistance; Line probe assay

Introduction

Tuberculosis though curable is still one of the leading causes of mortality all over the world. The severity of the problem is reflected by the fact that over one third of human population is infected with tuberculosis. The incidence of tuberculosis is particularly high in South East Asian countries which harbor about 40% of global TB cases. In Pakistan the magnitude of TB burden in terms of the number of cases per 100000 populations is found to be 231that rank Pakistan 5th amongst 22 high TB burden countries (WHO 2012). The problem of TB is compounded by high rate of multiple drug resistance (resistance to at least rifampicin and isoniazid the two most potent first line anti tuberculosis drugs). High rate of MDR is a continuous challenge to tuberculosis control program not only indeveloping but also in developed countries. A laboratorybased data has suggested that the incidence of MDR tuberculosis has increased from 14% to 47% in Pakistan (Tanveer et al. 2008). Such an alarmingly high rate of MDR in communities poses continuous public health threat by hampering the efficacy of WHO recommended short course treatment regimen.Rifampicin is a key component of WHO recommended short course chemotherapy. Patients in whom resistance to rifampicin develops show poor prognosis (Fischl et al. 1992). It has been observed that in more than 90% cases rifampicin resistance is found to be coupled with isoniazid resistance (Drobniewski and Wilson1998). Thus rifampicin resistance can be used as a surrogate marker for the detection of MDR-TB (Hashmi et al. 2013). Rifampicin binds with AY subunit of DNA-dependent RNA polymerase and interferes with the transcription and elongation of RNA. Rifampicin resistance occurs by single step high level mutations with a frequency of 10-9. Genetic basis of the resistance to rifampicin lies in rifampicin resistance determining region (RRDR) of rpoB gene where mutations occur in distinct 81 bp hot-spot" region. This consists of 27 amino acids encompassing the codon 507 to533 (Drobniewski and Wilson 1998). Several groups of workers from different parts of the world have characterized the mutations associated with rifampicin resistance in RRDR of rpoB gene (Donnabella et al. 1994; Hirano et al.1999; Valim et al. 2000; Mani et al. 2001).Determining the mutation spectrum of rpoB gene among M. tuberculosis strains prevalent in Pakistan is important because it would not only help to identify new strains specific to this region of the world but will also be useful in designing an in-house molecular diagnostic assay for rapid screening of MDR strains. The study is of particular importance in the context when commercially available line probe assay based diagnostic kits are being introduced in public sector for rapid MDR screening. Since type and frequency of mutations in RRDR of rpoB gene are known to bevaried geographically hence molecular tests based on mutations specific to this geographical setting will be more accurate to screen MDR cases (Jou et al. 2005; ). So the present study was aimed to screen mutations in rpoBgene by DNA sequencing and an in-house developed reverse hybridization line probe assay.

Materials and MethodsCollection of SpecimensA total of 141 samples 97 cultures while 44 clinical (bloodand sputum) specimens were included in this study. Culture isolates were obtained from different cities of Pakistan including Lahore (31 isolates) Peshawar (31 isolates)Karachi (11 isolates) and Faisalabad (10 isolates) and aregiven in Table 1. All the 44 clinical specimens were collected from Faisalabad and its periphery which included38 blood and 6 sputum samples. Clinical specimens were confirmed as M. tuberculosis after amplification of IS6110element using specific primers.Preparation of Genomic DNA DNA from M. tuberculosis cultures on LJ slants was extracted by CTAB method (van Embden et al. 1993)while the extraction of DNA from clinical samples was performed using SDS/proteinase K method(Hill et al. 1972)Sputum samples were decontaminated by NaOH/N-acetyl- L-cystein solution before extraction of DNA. About 200 ngof DNA from culture isolates while 5 L of DNA from clinical specimens was used as target for the amplificationof hotspot region of rpoB gene by polymerase chainreactions.Amplification of RRDR of rpoB Gene Amplification of RRDR of rpoB gene of M. tuberculosis was achieved by nested PCR using LiPA OP1 (outer forward primer) 5'-GAGAATTCGGTCGGCGAGCTGATCC-3' LiPA OP2 (outer reverse primer) 5'- CGAAGCTTGACCCGCGCGTACACC-3'; and LiPA IP1(inner forward primer) 5'- GGTCGGCATGTCGCGGATGG-3' LiPA IP2 (inner reverse primer) 5'-GCACGTCGCGGACCTCCAGC-3'. Inner primer LiPA IP2 was biotinylated at 3' end (Innogenetics Zwijndrecht Belgium). The outer and inner pair of primers generated a fragment of 395 bp and 257 bp respectively. The PCR was performed in a final volume of25 L containing 1X PCR buffer 1.5 mM MgCl2 0.2 mM each dNTPs (Fermentas Cat.# R0181) and 1U of Taq DNA polymerase (Fermentas Cat.# EP0402). Outer pair of primers was used in the first round of PCR cycle with 10 pM of each primer. The cycling parameters involved were denaturation at 95C for 5 min followed by 30 cycles of95C for 60 sec 58C for 30 sec and 72C for 30 sec. The second round of PCR was performed using inner set of primers with 20 pM of each primer and 1L of PCR product from the first round of PCR cycle. The cycling parameters

Table 1: Mutations in rpoB gene found by DNA sequencing and its comparison with in-house line probe assay

###Mutations

###Mutation detected by detected by in-Isolate Origin

###sequencing###house line probe

###Remarks

###assay

0607 Faisalabad (531)TCGTTG (531)TCGTTG

###Outside the

0017###Karachi (506)TTCATC###No mutation

###hotspot

7###Karachi (531)TCGTTG###(531)TCGTTG

###Outside the

22###Karachi###(569)ATCAAC###No mutation

###hotspot

###Outside the

160###Lahore###(477)GACGAT###No mutation

###hotspot

###Outside the

1188###Lahore###(494)AACAGC###No mutation

###hotspot

###Outside the

145###Lahore###(520)CCGCCA###No mutation

###hotspot

640###Lahore###(531) TCGTTG (531)TCGTTG

###(531) TCGTTG###Outside the

1000###Lahore###(531)TCGTTG

###(548)CGCTGC###hotspot

###Outside the

1249###Lahore###(538) TCTTGT###No mutation

###hotspot

###Not

###(571)CTGCCG

1048###Lahore###(531) TCG###included in

###(531) TCGTGG

###LiPA

###Not

###( 511) CTGCGG (511)CTG--CGG

P95###Peshawar

###(516)GACTAC (516)GAC

###included in

###LiPA

P81###Peshawar###(516) GACGTC (516)GACGTC

###Outside the

###(521) CTG CCG###hotspot

P79###Peshawar###(531) TCGTTG (531)TCGTTG###Not

###(503)AAGAAA###included in

###LiPA

P73###Peshawar###(526) CACTAC (526)CACTAC

P49###Peshawar###(531) TCGTTG (531)TCGTTG

P65###Peshawar###(531) TCGTTG (531)TCGTTG

P67###Peshawar###(531) TCGTTG (531)TCGTTG

P98###Peshawar###(531) TCGTTG (531)TCGTTG

P78###Peshawar###(531) TCGTTG (531)TCGTTG

###Outside the

###(531) TCGTTG

P96###Peshawar

###(554) CACCAT

###(531)TCGTTG###hotspot

involved were; denaturation at 95C for 5 min followed by30 cycles of 95C for 60 sec 65C for 30 sec and 72C for30 sec. The reaction was incubated at 72C for 7 min to allow final extension.

Detection of Mutations in RRDR ofrpoBGene by Sequencing

For 45 culture isolates 257 bp amplified PCR product was cloned in a T/A cloning vector from Fermentas (pTZ57R/T). The clones were commercially sequenced by M13 forward and reverse primers (Macrogen Korea). Sequence information was stored assembled and analyzed using the Laser gene sequence analysis package (DNAStar Inc. Madison WI USA). The sequencing data was aligned and the mutations were analyzed in RRDR of rpoB gene.

Determination of Mutations in RRDR of rpoB Gene byIn-house Reverse Hybridization Line Probe Assay

To screen the mutations associated with rifampicin resistance in RRDR of rpoB gene of M. tuberculosis an in- house reverse hybridization line probe assay was used. Briefly 10 L of each PCR product was heat denatured after diluting in 140 L of hybridization solution (5XSSPE/0.5%SDS) and applied to nylon filters at right angle to the oligonucleotides using Miniblotter45. Hybridization was carried out at 45C for 30 min. To remove unbound PCR products filters were washed briefly with 2XSSPE/0.1%SDS at room temperature and then with 2XSSPE/0.1%SDS twice at 55C for 10 min. Detection of hybridization signals was carried out using Biotin Chromogenic Detection kit (Fermentas Cat.# K0662).

Results

Sequencing analysis of rpoB gene

Among randomly selected 45 sequenced isolates 24(52.17%) showed no mutation while 21 (46.67%) isolateswere found to have 26 point mutations of 14 different types(Table 1). Among these isolates 17 had single mutation 3had double mutations and only one had triple mutations.Codon 531 was found to be highly mutated as 11 isolates possessed mutation 531 (TCGTTG) while one isolate exhibited 531 (TCGTGG). Other mutations in RRDR region of rpoB gene of M. tuberculosis found in the present study by sequencing include rpoB 511 (CTGCCG) rpoB516 (GACGTC) rpoB 516 (GACTAC) rpoB 521 (CTGCCG) and rpoB 526 (CACTAC). Mutations incodon rpoB 477 (GACGAT) rpoB 503 (AAGAAA) and rpoB 520 (CCGCCA) were silent mutations. Novel mutations found in the present study included rpoB 494 (AACAGC) rpoB 503 (AAGAAA) rpoB 505(TTCTCC) rpoB 506 (GACATC) rpoB 538(TCTTGT) rpoB 548 (CGCTGC) rpoB 554 (CACCAT) rpoB 569 (ATCAAC) and rpoB 571 (CTGCCG). Two isolates had double mutations of which one had mutation in codon 531 (TCGTTG) along with a novel mutation 548 (CGCTGC) while second had a novel mutation in codon 554 (CACCAT) that coexisted with 531 (TCGTTG) mutation. Another isolate had mutation in codon 511 (CTGCCG) and 516 (GACTAC). One isolate that contained triple mutations had mutations in codon 531 (TCGTTG) and 521 (CTGCCG). This particular isolate also had a novel mutation 503 (AAGAAA) that resides outside the RRDR of rpoB gene (Table 2).

The Detection of Common Mutations by In-House LineProbe Assay

Results of in-house line probe assay were in agreement with those obtained by DNA sequencing (Table 2).

Table 2: Mutation profile in rpoB gene in Mycobacterium tuberculosis isolates. Shown are the nucleotide mutations with frequency (%) and the resulting change in amino acids

###Mutation

Codon###Nucleotide###Amino acid###Type of

###mutation###change###mutation

###frequency

###(%)

477###GAGGAT###GluAsp###Substitution###1.61

494###AACAGC###AsnSer###Substitution###1.61

503###AAGAAA###LysLys###Silent###1.61

505###TTCTCC###PheSer###Substitution###1.61

506###TTCATC###PheIle###Substitution###1.61

511###CTGCCG###LeuPro###Substitution

###8.06

511###CTGCGG###LeuArg###Substitution

515###ATG###Metunknown###unknown###1.61

516###GACTAC###AspTyr###Substitution

516###GACGTC###AspVal###Substitution###4.84

516###GAC CAG###Asp Gln###Deletion

518###AAC###Asn###Deletion###3.22

520###CCGCCA###ProPro###Silent###1.61

521###CTGCCG###LeuPro###Substitution###1.61

526###CACTGC###HisCys###Substitution

526###CACCGC###HisArg###Substitution

###9.68

526###CACTAC###HisTyr###Substitution

526###CACGAC###HisAsp###Substitution

526###CAC###Hisunknown###Unknown

531###TCGTTG###SerLeu###Substitution###56.45

531###TCGTGG###SerTrp###Substitution

538###CTGGTG###LeuVal###Substitution###1.61

548###CGCTGC###ArgCys###Substitution###1.61

554###CACCAT###HisHis###Silent###1.61

Hence the sensitivity and specificity of in-house line probe assay was found to be 100% in comparison to sequencing.Mutations In the present study the mutations were mostly located at codon at codon 531526516 and 511of rpoB gene. At codon 531 two different substitution mutations were observed while 6 different substitution mutations were observed in codon 526. On the other hand codon 511 showed 5 different substitution mutations and codon 516 showed 2 substitution and one deletion mutations.

Use of Line Probe Assay for Screening Mutations inClinical Specimens

Forty-four clinical samples were screened for the mutations in RRDR of rpoB gene. Out of 44 specimens only two isolates showed the presence of mutation in RRDR of rpoB gene which were found to be in codon 511 (CTGCCG) and codon 531 (TCGTTG). Specimen that had mutation in codon 531 also gave positive signal with wild type probe showing the presence of mixed infection.

Discussion

About 50% (24 out of 45) of sequenced isolates had wild type rpoB gene sequence while 21 samples showed 14 different point mutations in rpoB gene in 26 isolates.

Mutations in codon 503 520 and 554 were silent mutations as they did not change the encoded amino acid. An interesting finding in this study was the presence of multiple mutations in some resistant isolates where mutation in codon 531 was found to be coexisting with some novel mutations. Mutations at codon 511 and 516 also coexisted in one isolate. Presence of multiple mutations in the rifampicin resistant isolates is not uncommon and is also reported previously (Moghazeh et al. 1996; Mani et al.2001; Huitric et al. 2006). Certain novel mutations in codons rpoB 477 rpoB 494 rpoB 505 rpoB 506 rpoB 521 rpoB 538 and rpoB 548 were also found in the present study. A mutation that resides in codon outside the RRDR of rpoB gene is not a new phenomenon. Several other studies (Mani et al. 2001; Cavusoglu et al. 2002) reportedthe novel mutations outside the RRDR of rpoB gene.It is remarkable that all the mutations that reside in RRDR of rpoB gene were correctly identified by the in- house assay resulting in 100% concordance between the in- house assay results and the results of DNA sequence analysis of rpoB gene. The isolate having double mutation (in codon 511 and 516) was also detected by in-house assay adding to the sensitivity of this assay.The overall mutation spectrum was quite varied with the most frequently mutated codon found to be 531 followed by 526 511 and 516 with frequencies of 56.45%9.68% 8.06 % and 4.84 respectively. Contribution of the mutations that reside inside the RRDR was 87.09%. Dominance of the substitution mutation at codon 531 followed by 526 and 516 was also observed in other studies (Ramaswamy and Musser 1998; Lingala et al. 2010). The increased prevalence of mutations at codon 531 is becauseof the molecular and physiological processes that result inthe development of the resistance without decrease in fitness of these strains (Arnold et al. 2005; Jenkins et al.2009).A low mutation frequency (4.84%) at codon 516 observed in the present investigation is contrasting to the results of Hungarian study (Bartfai et al. 2001) and the study conducted on Asian isolates (Hirano et al. 1999) where mutations at codon 516 were found in 37.9% and13.3% isolates respectively. These comparative studies indicate that the frequency of mutations in codon 531526 16 and 511 is subjected to strong geographical variations.The frequency of mutations in RRDR of rpoB gene in clinical samples was 4.54% which is similar to the incidence of drug resistance reported from Pakistan (WHO2012). One patient was found to be infected with two strains of M. tuberculosis but it is difficult to determine the relativeabundance of the subpopulations in the sample as PCR cannot discriminate between the templates from the wild and the mutant organism. This is one of the major drawbacks of the molecular assay for the diagnosis of MDR-TB while dealing with the clinical isolates which notonly affects the sensitivity of the molecular assayparticularly in a situation where percentage of the resistant population is low but also is an obstacle for the successful therapy. Therefore molecular tests are preferred to be used in conjunction with the conventional tests.The degree of rifampicin resistance in M. tuberculosis isolates strongly correlates with the site and type of mutation in rpoB gene (Zaczek et al. 2009). Lower resistance is attributed by the mutations at codon 522 518516 (Williams et al. 1998) and 511 (Moghazeh et al. 1996; Bodmer et al. 1995) while mutations at codon 531 (Huang et al. 2002) and 526 are usually known to cause high level of resistance (Hwang et al. 2003). This shows that the position of mutation in the allele at molecular level is critically affecting the complex drug target interaction thatleads to the varied levels of drug resistance. Pakistan issharing its geographical boundaries with the countries like India Afghanistan and Iran where tuberculosis is highly endemic. Prevalence of M. tuberculosis isolates in Pakistan harboring mutations associated with high level of drug resistance is critical as similar mutation profiles are observed in these countries. Lingala et al. (2010) showed that the majority of strains circulating in this particular geographical setting are highly resistant to rifampicin. One of the possible explanations of such similar trends is historical connections between these countries and this probably represents prevalent strains of this region.Our data serves as the baseline information regarding the geographical and temporal trends in the prevalence of specific mutations as well as for geographical differences in mutation frequencies for the development of new diagnostic tool. This data may also enable to feature effective and better policies in this particular geographical setting for the control of tuberculosis. However level of resistance by different novel mutations identified in the present study demands further reseach work respective fields.

Acknowledgement

We are thankful to Dr. Sabira Tehsin National TB Reference Laboratory Islamabad; Dr. Shahid A. Abbasi Armed Forces Institute of Pathology Rawalpindi and Dr. Rizwan Iqbal Pakistan Medical Research Council Lahore for providing M. tuberculosis isolates.

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Author:Memona Yasmin; Nosheen Mujtaba; Rubina T. Siddiqui
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Oct 31, 2014
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