Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis

G Model JIPH-878; No. of Pages 6

ARTICLE IN PRESS Journal of Infection and Public Health xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph

Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis Myo T. Zaw, Nor A. Emran, Zaw Lin ∗ Department of Pathobiological and Medical Diagnostics, Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Malaysia

a r t i c l e

i n f o

Article history: Received 25 July 2017 Received in revised form 5 March 2018 Accepted 8 April 2018 Keywords: Mutations Rifampicin-resistance determining region rpoB gene Rifampicin resistance Mycobacterium tuberculosis

a b s t r a c t Background: Rifampicin (RIF) plays a pivotal role in the treatment of tuberculosis due to its bactericidal effects. Because the action of RIF is on rpoB gene encoding RNA polymerase ␤ subunit, 95% of RIF resistant mutations are present in rpoB gene. The majority of the mutations in rpoB gene are found within an 81 bp RIF-resistance determining region (RRDR). Methodology: Literatures on RIF resistant mutations published between 2010 and 2016 were thoroughly reviewed. Results: The most commonly mutated codons in RRDR of rpoB gene are 531, 526 and 516. The possibilities of absence of mutation in RRDR of rpoB gene in MDR-TB isolates in few studies was due to existence of other rare rpoB mutations outside RRDR or different mechanism of rifampicin resistance. Conclusion: Molecular methods which can identify extensive mutations associated with multiple antituberculous drugs are in urgent need so that the research on drug resistant mutations should be extended. © 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mutations in rpoB gene associated with rifampicin resistance in M. tuberculosis isolates of different countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Two most common mutations in Brazil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Multiplex allele-specific polymerase chain reaction in India . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mutations at codon no. 490 in Vietnam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Different mutation patterns of rpoB gene in South Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Eastern Cape Province . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 KwaZulu-Natal Province . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mutation at codon 526 in Guizhou and Zhejiang, China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mutations outside RRDR of rpoB gene in Pakistan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Diversity of SNPs in rpoB gene of M. tuberculosis in Thailand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Materials and methods used in the studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mutation in rpoB gene as lineage specific polymorphism of Beijing genotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Authors’ contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author at: Department of Pathobiological and Medical Diagnostics, Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, 88400, Kota Kinabalu, Sabah, Malaysia. E-mail address: [email protected] (Z. Lin). https://doi.org/10.1016/j.jiph.2018.04.005 1876-0341/© 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Zaw MT, et al. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.04.005

G Model JIPH-878; No. of Pages 6

ARTICLE IN PRESS M.T. Zaw et al. / Journal of Infection and Public Health xxx (2018) xxx–xxx

2

Introduction

Multiplex allele-specific polymerase chain reaction in India

In 1980s, the duration of treatment of tuberculosis (TB) became shortened from 24 to six months because of the initiation of the short course regimen. However, adherence to treatment regimens was not fully accomplished due to relatively prolonged therapy [1]. Resistance of Mycobacterium tuberculosis (M. tuberculosis) to a single drug emerged and increased in different parts of the world [1]. In the early 1990s, multiple factors caused an emergence of multiple drug resistant tuberculosis (MDR-TB), known to be resistant to isoniazid and rifampicin which are the two most effective first line anti-tuberculous drugs (anti-TB drugs). In between 2002 and 2006, the MDR-TB was prevalent up to 22% among newly diagnosed cases and up to 60% among previously treated cases [1,2]. In 2002, extensively drug resistant tuberculosis (XDR-TB) was reported in 45 countries, which is defined to be resistant not only to isoniazid and rifampicin but also to at least one fluoroquinolone and to any of the following injectable second-line drugs: kanamycin, amikacin, or capreomycin [2]. Rifampicin (RIF) plays a pivotal role in the treatment of tuberculosis due to its bactericidal effects [3]. Although RIF resistant mutation is difficult to occur when compared with any other antiTB drugs, the rate of RIF resistance is increasing due to its wide usage [4]. Because the action of RIF is on rpoB gene encoding RNA polymerase ␤ subunit, more than 95% of RIF resistant mutations are associated with mutations in rpoB gene [5]. The majority of the mutations in rpoB gene are present within an 81 bp RIF-resistance determining region (RRDR), a mutation hot spot region. Among the different types of mutations, non-synonymous mutations are more common than insertion, deletions and frameshift mutations [6].

Polymerase chain reaction (PCR) followed by DNA-sequencing to find targeted SNP were the preferred method in the identification of RIF resistant mutations and the method was cost-effective and sensitive [7]. Multiplex allele-specific PCR (MAS-PCR) was the method which utilizes three sets of specific primers which were able to detect SNPs at most commonly mutated codons 531, 526 and 516. Seventy to over 90% of RIF resistant isolates harboured mutations in these codons [11]. Earlier detection of M. tuberculosis allows implementation of anti-TB treatment in time resulting in earlier control of the infection without wide dissemination [11]. The advantage of a genotypic method for the rapid detection of MDR-TB is faster than conventional methods. MAS-PCR was performed on 90 RIF resistant isolates and 37 RIF sensitive isolates from patients enroled between 2011 and 2013. rpoB 516, rpoB 526 and rpoB 531 mutations in RIF resistant clinical isolates were successfully detected by MAS-PCR assay [12]. Ser531Leu mutation was associated with RIF resistance in 49 isolates whereas His526Tyr SNP and Asp516Val SNP in 17 isolates and 5 isolates respectively [12]. Double mutations were observed in 15 isolates with Ser531Leu and His526Tyr SNPs while triple mutations were observed in 4 isolates with Ser531Leu, His526Tyr and Asp516Val SNPs in MAS-PCR [12]. Mutations outside the 81-bp RRDR have been observed in the study with ten Asn413His, six Asp 435Glu and eight Ala451Asp SNPs whereas mutations within RRDR, which were not able to be detectable by MAS-PCR were Arg511Cys, Val513Asp and Glu521Asp SNPs [12]. Mutations outside the RRDR have rarely been reported with less than 5% of RIF resistant isolates [13]. MIC determination to RIF was done for RIF resistance isolates in the study with the consequence of the finding that seven isolates exhibited high level resistance >128 mg/L. Most of the RIF resistant isolates showed intermediate level of MIC with 40 mg/L [12]. The result in the study was concordant with the finding SNPs at 531 and 526 were the most common mutations observed in other previous studies. Furthermore, MAS-PCR worked well with most common mutations in the rpoB RRDR in the study [12].

Mutations in rpoB gene associated with rifampicin resistance in M. tuberculosis isolates of different countries Two most common mutations in Brazil Although RIF has fast bactericidal action to tubercle bacilli and has been included in short-course regime, RIF resistance which emerges in the 1990s is a great threat in the control of TB. Mutations in the RRDR of rpoB gene contribute over 96% of RIF resistance in M. tuberculosis [7]. In the study in Brazil, Ser531Leu and Ser531Trp were the two most common mutations with the 58.5% and 20.8% of RIF resistant isolates [7]. The prevalence of Ser531Leu mutation was nearly the same as other studies in Brazil whereas Ser531Trp mutation was as common as the finding in Chile. The prevalence of this mutation in Chile and Brazil was thought to be a clonal expansion of the LAM9 lineage because this mutation was uncommon in the various regions of the world [7]. Twelve amino acids were surrounding the RIF binding pocket in which RNA polymerase active site is located. Mutation leading to change in one of the amino acids results in modification of active site. Mutation is associated with replacement of an amino acid having a compact side chain by an amino acid with a large side chain [8] and the consequence is inactivation of RNA polymerase resulting in RIF resistance. In the study, two of the isolates harboured the double mutations with one amino acid surrounding RIF binding pocket whereas another amino acid was outside the region of surrounding binding pocket [7]. The fitness cost of the first amino acid was believed to be compensated by the second amino acid. There were two mutations which were outside hotspot region in two isolates [7]. The two single nucleotide polymorphisms (SNPs) were rpoB Phe505Val and Ile572Val while Phe505Val was together with Asp516Phe in one isolate. However, two of MDR isolates did not carry any mutation in rpoB gene [7].

Mutations at codon no. 490 in Vietnam In the study, M. tuberculosis isolates were collected from TB patients in different regions of Vietnam between 2007 and 2009 [14]. Seventeen different types of mutations were observed in 74 isolates of RIF-resistant M. tuberculosis including 57 MDR strains. Single-nucleotide substitutions caused seven amino acid changes in most of the mutations. The common mutations were observed at three codons 531, 526 and 516 as concordant with some studies on RIF resistant markers [14]. Remarkably, occurrence of 4 substitutions at codon 531 was found in the study with Ser/Leu, Ser/Trp, Ser/Phe, Ser/Gln [14]. Similarly mutations at codon 526 were five in number and His/Leu, His/Asn, His/Arg, His/Tyr and His/Ser whereas the change in codon 516 was only one in seven isolates having Asp/Val alteration [14]. Strikingly, the mutation at codon 490 was more common than Asp516Val SNP with two substitutions Gln/His and Gln/Arg [14]. One double mutation was found in the study with Gln490Arg and Ser531Trp [14]. Mutiple mutations at each codon were the reason why there were 17 different mutations in the study although only seven codons harboured mutations. In addition 54 isolates of 57 MDR M. tuberculosis carried mutations in 81-bp hypervariable region of rpoB gene. Concordant with the other studies, there was no mutation in any RIF susceptible isolates [14]. In RIF-resistant isolates, mutations at rpoB codons 531, 526, and 516 are the most common worldwide. However, the frequencies of SNPs in these three codons were variable in different geographic regions [15]. Mutation in codon 531 was the predominant alter-

Please cite this article in press as: Zaw MT, et al. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.04.005

G Model JIPH-878; No. of Pages 6

ARTICLE IN PRESS M.T. Zaw et al. / Journal of Infection and Public Health xxx (2018) xxx–xxx

ation, a consistent finding with the other studies. Two mutations namely Leu530Met at codon 530 and a new amino acid change at SNP Ser531Trp were novel in the study [14]. Two amino acid changes at codon 490 were observed in 15 isolates and this mutation was outside the 81 bp RRDR of rpoB gene [14]. The other studies have reported the mutations outside RRDR at codon 490, codon 535, codon 504, codon 541, codon 553 and codon 572 [15]. No mutation was observed in the studied region of rpoB gene in three phenotypically RIF resistant isolates. Recent studies indicated that RIF resistant mutations were located outside the 81-bp rpoB region. Changes in genes which encode proteins involved in antibiotic permeation or metabolism result in drug resistance [16]. Taken together, these two factors suggested absence of mutations in three RIF resistant isolates. Different mutation patterns of rpoB gene in South Africa Eastern Cape Province In South Africa, MDR-TB and HIV infection caused one of the worst world epidemics during 2008 [26]. Eighty percent of TB cases in South Africa were present in the Eastern Cape of nine provinces in the country [17]. Because of the poverty, the transmission of TB was enhanced in the Eastern Cape Province [17]. Most people in the province assume TB was caused by strains that cannot be overcome by existing drugs [17]. MDR-TB cases in Eastern Cape Province isolated in 2012 and 2013 were detected for RIF resistant mutations and the results were totally different from the previous studies in the world. SNPs Tyr42Asp, Gly52Ala, His87Gly, Leu92Ser, Val441Gly, Leu450Ser, Leu457Pro were associated with RIF resistance indicating most of these were outside RRDR of rpoB gene and rare in the previous studies [17]. KwaZulu-Natal Province South Africa is included in the top ten drug resistant TB high burden countries in the world. The mechanisms associated with resistance to first-line drugs in MDR-TB and XDR-TB clinical isolates of KwaZulu-Natal, South Africa were studied [18]. The DNA sequences of rpoB, katG, inhA, pncA and embB genes of the isolates from patients who took treatment in Church of Scotland Hospital from 2005 to 2009 were studied [18]. Mutations associated with RIF resistance in MDR-TB isolates were Asp516Tyr, His526Leu, Ser531Leu, Leu533Pro, Pro535Thr, ILe572Met, Tyr645His [18]. For the 28 XDR-TB isolates, the main two mutations associated with RIF resistance were D516G and L533P combination in each isolate with the exception of three isolates having Asp516Gly SNP alone and one isolate carrying Ser531Leu [18]. Although both the MDR and XDR isolates had SNP at 516, different amino acid substitutions were observed in the study and the other similar study by Ioerger et al. [19] in which the whole genome sequencing approach was undertaken. MDR-RIF resistance mechanisms had great diversity when compared with XDR-RIF resistance. Emergence of the strains separately and independently acquired resistant mutations may be the reasons why mutations associated with the MDR and XDR isolates were different. This was discordant with the assumption that XDR-TB had the evolution from the MDR phenotype [20]. Mutation at codon 526 in Guizhou and Zhejiang, China One hundred M. tuberculosis isolates from Guizhou Province, China between 2007 and 2008 were investigated for rifampicin resistance by PCR-DNA sequencing [21]. Of 38 rifampicin resistant isolates, 13 non-synonymous (NS) mutations were observed at codon no. 531, 526, 516, 511, 522, 533, 550, 509 and 572 in terms of frequency [21]. One isolate had three mutations together whereas four isolates harboured two mutations. There were two

3

novel mutations observed in the study namely Val550Leu and Ser509Arg. Single base substitutions lead to all NS mutations in the study [21]. In the study in Guizhou, rpoB Ser531Leu was the most common SNP whereas mutations at codon 526 were the second most common SNP. The study compared the data from Guizhou with different TB high burden regions of China and nine other countries of Asia, America and Europe [21]. The outstanding observation is that mutation at codon 526 was highly common with more than 75% whereas SNP at codon 531 was less than 5% for RIF resistant isolates in Zhejiang, China [22]. The finding indicated that mutations in rpoB gene associated with RIF resistance were highly different even in the same country. However, SNP at 531 was still the most common in the other studied regions between 37% and 66% [21]. In concordant with the study in India where the RIF resistant mutations were studied by MAS-PCR [18], the third most common mutation was at codon 516 in rpoB gene [21]. Mutations outside RRDR of rpoB gene in Pakistan Drug resistance mostly developed by mutational changes than gene transfer from other bacteria. Drug resistance in M. tuberculosis isolates was thought to be due to spontaneous mutations [23]. The mutations in rpoB gene were different from one region of the world to another TB endemic region [24]. Mutations in rpoB gene were observed to be associated with a high-level of MIC to RIF in Asian countries [25]. Out of 63 resistant isolates among 1080 TB isolates in Pakistan, 24 isolates were found to be MDR-TB [26]. To identify mutations in the 81 hot spot region of the rpoB gene, PCR-DNA sequencing was done to 19 RIF-resistant isolates out of 24 MDR-TB isolates [26]. Eleven isolates carried SNP S531L at rpoB gene, the most common one in the study and the second common mutation occurred at codon 516 with two SNPs Asp516Val and Asp516Tyr. Ser512Ile, Leu533Pro were the other mutations found in this study whereas Tyr528Tyr was the synonymous mutation [26]. One MDR-TB isolate harboured the double mutations at codon 512 and 516 which was first described in Pakistan [26]. Similarly, the study in Punjab, Pakistan the Ser531Leu SNP and mutations at codon 516 with two SNPs were the most common rpoB NS mutations [27]. The SNP Ser512Ile was observed for the first time in MDR-TB isolates of Pakistan while it was found in the study in Poland [28]. Similarly Leu533Pro SNP was previously observed in Turkey [29].The isolate carrying double mutations at amino acid no. 512 and 516 was previously documented [30]. Five MDR-TB isolates did not carry any mutation in the 81 hotspot region of the rpoB gene, although these were phenotypically RIF resistance. The reason may be due to genotype variations which occurred worldwide or presence of the mutations outside the 81 bp region. The existence of other rare type of rpoB mutations or different mechanism of resistance to rifampicin is the possibility [31]. Diversity of SNPs in rpoB gene of M. tuberculosis in Thailand In the study, 39 M. tuberculosis isolates including three RIF susceptible isolates from Thailand were studied for mutations in rpoB gene associated with RIF resistance [32]. Similar to other previous studies from different region of the world Ser531Leu and His526Cys were the most prevalent SNPs in the study [32]. Rare type of mutation in the study was deletion of amino acid no. 523 which was observed in two isolates [32]. The other mutations at this amino acid were Gly523Ala and silent mutation which were indicated in three reports [33–35]. Four isolates with synonymous mutation at Gly536 was present in the study whereas nine silent mutations within the RRDR were reported in the previous study by Rama-

Please cite this article in press as: Zaw MT, et al. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.04.005

G Model JIPH-878; No. of Pages 6

ARTICLE IN PRESS M.T. Zaw et al. / Journal of Infection and Public Health xxx (2018) xxx–xxx

4

Table 1 List of countries in the studies reviewed and mutations within RRDR and outside RRDR of rpoB gene in M. tuberculosis. Country and year of publication

Mutations within RRDR

Mutations outside RRDR

Brazil (2015) [7] (section 2.1.) India (2015) [12] (section 2.2.) Vietnam (2011) [14] (section 2.3.)

Ser531Leu, Ser531Trp, Asp516Phe Ser531Leu, His526Tyr, Asp516Val Arg511Cys, Val 513 Asp, Glu521Asp Ser531Leu, Ser531Trp, Ser531Phe, Ser531Gln. His526Leu, His526Asn, His526Arg, His526Tyr and His526Ser Asp516Val, Leu530Met MDR-TB — Ser531Leu, His526Leu, Asp516Tyr, Leu533Pro, XDR-TB — Asp516Gly and Leu533Pro, S531L a Codon no. 531, 526, 516, 511, 522, 533,550, 509 and 572 Ser531Leu, Asp516Val and Asp516Tyr. Ser512Ile, Leu533Pro Ser531Leu and His526Cys, Gly523Ala Leu533Arg, Gly523Gly

Phe505Val and Ile572Val Asn413His, Asp 435Glu and Ala451Asp Gln490His and Gln490Arg

South Africa (2016) [18] (section 2.4.) China (2010) [21] (section 2.5.) Pakistan(2014) [26] (section 2.6.) Thailand (2014) [32] (section 2.7.) a

Pro535Thr,Ile572Met,Tyr645His Val550Leu Leu538Arg, Gly536Gly

Amino acids were not mentioned for all the mutations in the study in China.

soota et al. [33]. The remarkable two mutations Leu533Arg and Leu538Arg were found in the study, with the former appearing with His526Cys as double mutations in one isolate whereas the latter was outside the RRDR and reported previously in China [36]. These two mutations indicated the diversity of SNPs in rpoB gene of M. tuberculosis. The studies reviewed in this article, their year of publications and mutations within RRDR and outside RRDR of rpoB gene in M. tuberculosis were listed in Table 1.

A1075A and show RIF susceptible profile. rpoB A1075A was an polymorphism observed in whole genome sequencing studies without exhibiting RIF resistance and observed to be SNP for Beijing lineage. The polymorphism indicated that mutations in rpoB gene include lineage specific one outside RRDR. There were other lineage specific polymorphisms in other drug resistant genes associated with other antibiotics like Arg463Leu in katG for isoniazid, Glu92Asp in gidB for streptomycin, etc. [37].

Materials and methods used in the studies

Discussion

The studies were done on the sputum samples obtained between 2005 to 2013 and the time varies with the country of study [7,12,14,18,21,26,32]. The sputum samples were consecutive, random and selected ones which depend upon whether the patient has suffered from drug susceptible (DS), rifampicin resistant (RRTB), multi-drug resistant (MDR-TB) and extensively drug resistant (XDR-TB) [7,12,14,18,21,26,32]. Sample size determination is different from country to country depending on objectives. In India, samples were collected from out-patients after studying clinical symptoms, sputum smear results and chest X-ray for TB and from hospitalized patients according to the physician’s recommendation [12]. Sputum samples were obtained during routine work at the Central Laboratory of the State of Santa Catarina (SC), the reference laboratory for TB diagnosis in SC in South Africa [18]. Species identification was performed by morphological study of colonies on Lowenstein–Jensen (LJ) medium, acid-fast staining and biochemical tests. Drug susceptibility test (DST) was performed by using rifampicin concentration of 40 ␮g/mL to identify RIF resistant isolates [12]. The studies in Brazil, Vietnam, South Africa, China, and Pakistan were done by PCR, purification of PCR products and DNA sequencing [7,14,18,21,26] while in Thailand sequencing was done by pyrosequencing method [32]. However, in India, the study was done by MAS-PCR method targeting mutations at codons 531, 526 and 516 [12]. Quality control of the laboratory procedures was done by using M. tuberculosis reference strain H37Rv (ATCC 27294) [12].

Because RIF has fast bactericidal action to tubercle bacilli, it has been included in short-course regime [7]. However, the emergence of RIF resistance at the early 1990s results in problem in the control of TB. Drug resistance in tubercle bacilli mostly developed by mutational changes rather than genes transfer from other bacteria by mobile genetic elements. Drug resistance in MTB isolates was assumed to be due to spontaneous mutations [23]. RIF resistance is taken as a surrogate marker of drug resistant TB because emergence of rifampicin-resistance was relatively slow when compared to other antibiotics [32]. Mutations in amino acids which surround the RIF binding pocket containing active site of RNA polymerase results in modification of this active site with consequence of RIF resistance [8]. Earlier control of the infection needs earlier detection of M. tuberculosis and drug resistant M. tuberculosis with consequent initiation of anti-TB regime in time [11]. Genotypic methods are important for the rapid detection of MDR-TB. The common mutations associated with RIF resistance are alteration of amino acid at codons 531, 526 and 516 and this finding was repeatedly reported in previous studies from different locations of the world [11,12]. Leu533Arg and Leu538Arg were found in one study in Thailand [32] whereas Val550Leu and Ser509Arg were the two novel mutations of the China study [21]. These mutations indicated diversity of SNPs in rpoB gene of M. tuberculosis. NS mutations at codon 490 were observed in considerable number of isolates in Vietnam and this mutation was outside the hypervariable 81 bp RRDR [14]. The information indicated that there were many NS mutations outside RRDR in the other studies. Mutations in rpoB gene were observed to be well correlated with a high-level of MIC to RIF in Asian region [25]. The outstanding characteristics of mutations in rpoB gene associated with RIF resistance M. tuberculosis were mentioned in Table 2.

Mutation in rpoB gene as lineage specific polymorphism of Beijing genotype SNPs in rpoB gene were not only associated with RIF resistance in M. tuberculosis but also useful as lineage specific polymorphism especially for the Beijing genotype lineage, a common genotype associated with multiple drug resistance and prevalent in Asian region. In one study, two students from southeast Asian countries suffered pulmonary tuberculosis while they were studying in the same school in the United Kingdom. Study from epidemiological aspect showed no link between the two cases whereas drug resistant patterns of the isolates were different. However, genetically identical results were obtained by whole-genome sequencing with the finding the isolates were of Beijing lineage. The isolates were observed to have lineage specific polymorphisms including rpoB

Conclusions The common mutations at rpoB 531, rpoB 526 and rpoB 516 codons were successfully detected by MAS-PCR assay in RIF resistant clinical isolates although there were missing mutations even in RRDR of rpoB gene in South India [12]. GeneXpert and GenoType MTBDRplus molecular methods were used in detection of drug-resistant TB because of simplicity and rapidity [38]. Because of different mutations in genes associated with drug

Please cite this article in press as: Zaw MT, et al. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.04.005

G Model JIPH-878; No. of Pages 6

ARTICLE IN PRESS M.T. Zaw et al. / Journal of Infection and Public Health xxx (2018) xxx–xxx

5

Table 2 The outstanding characteristics of mutations of rpoB gene associated with RIF resistance in M. tuberculosis. Characteristics of mutations in rpoB gene associated with RIF resistance RIF-resistance determining region ranges from codon 507 to codon 533 (27 amino acids) [12,14,32]. (section 2.2., 2.3., 2.7.) The most commonly mutated codons of rpoB gene are 531, 526 and 516 in RRDR [11,12]. (section 2.2.) Multiple mutations at codon 531 and 526 were observed frequently in the studies [14]. (section 2.3.) There is no correlation with high level MIC and mutation at specific codon [12]. (section 2.2.) Mutations outside the RRDR have rarely been reported in <5% of RIF resistant isolates. One of the mutations is at codon 490 and two amino acid changes at codon 490 were observed in 15 isolates in Vietnam [14]. (section 2.3.) Studies in China, Thailand and Eastern Cape Province, South Africa indicated that diversities of rpoB gene mutations [18,21,32]. (section 2.4., 2.5., 2.7.1.) The rpoB A1075A was the synonymous mutation useful as lineage specific polymorphisms for Beijing lineage without exhibiting RIF resistance [37]. (section 3)

resistance, results were variable in sensitivity and specificity in different regions of the world. For the GeneXpert, mutations outside RRDR cannot be detected for RIF resistance. In addition, INH-monoresistant cases were not able to be identified by GeneXpert [39]. Detection of drug resistance is limited for fewer drugs and number of mutations in these molecular methods. Therefore, molecular methods which can identify extensive mutations associated with multiple anti-TB drugs are in urgent need so that the research on mutations associated with drug resistance should be extended [40]. Developments of rapid diagnostics which can include novel mutations are niche areas of TB research. Authors’ contribution ZL and MTZ designed and conceived the study. ZL, MTZ and NAE wrote the manuscript. All the authors read and approved the final manuscript. Funding No funding sources. Competing interests None declared. Ethical approval Not required. Acknowledgements We would like to thank Professor Dr. Zainal Arifin Mustapha, Dean, Faculty of Medicine and Health Sciences, University Malaysia Sabah, for the continuous support throughout the project. References [1] Sandgren A, Strong M, Muthukrishnan P, Weiner BK, Church GM, Murray MB. Tuberculosis drug resistance mutation database. PLoS Med 2009;6(2):e1000002, http://dx.doi.org/10.1371/journal.pmed.1000002. [2] World Health Organization. Antituberculosis drug resistance in the world fourth global report; 2008. Available: http://www.who.int/tb/publications/ 2008/drs report4 26feb08.pdf. [3] Rattan A, Kalia A, Ahmad N. Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives. Emerg Infect Dis 1998;4:195–209. [4] Long R. Drug-resistant tuberculosis. Can Med Assoc J 2000;163:425–8. [5] Lai C, Xu J, Tozawa Y, Okamoto-Hosoya Y, Yao X, Ochi K. Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans. Microbiology 2002;148:3365–73. [6] Rienthong D, Rienthong S, Boonin C, Worsawad S, Kasetjaroen Y. Rapid detection for early appearance of rifampin and isoniazid resistance in Mycobacterium tuberculosis. Siriraj Med J 2009;61:49–55. [7] Prim RI, Marcos MA, Senna SG, Nogueira CL, Figueiredo ACC, de Oliveira JG. Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil. Mem Inst Oswaldo Cruz 2015;110(5):618–23.

[8] Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 2001;104:901–12. [11] Mokrousov I, Otten T, Narvskaya O. Allele specific rpoBPCR assays for detection of Rifampin-resistant Mycobacterium tuberculosis in sputum smears. Antimicrob Agents Chemother 2003;47:2231–5. [12] Thirumurugana R, Kathirvelb M, Vallayyachari K, Surendar K, Samrota AV, Muthaiah M. Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chainreaction in Puducherry, South India. J Infect Public Health 2015;8:619–25. [13] Heep M, Rieger U, Beck D, Lehn N. Mutations in the beginning of the rpoB gene can induce resistance to rifampin both in Helicobacter pylori and Mycobacterium tuberculosis. Antimicrob Agents Chemother 2000;44:1075–7. [14] Minh NN, Bac NV, Son NT, Lien VTK, Ha CH, Cuong NH. Molecular characteristics of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Vietnam. J Clin Microbiol 2011;50(3):598–601. [15] Cavusoglu C, Hilmioglu S, Guneri S, Bilgic A. Characterization of rpoB mutations in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Turkey by DNA sequencing and line probe assay. J Clin Microbiol 2002;40:4435–8. [16] Kapur V, Li L, Iordanescu S, Hamrick MR, Wanger A, Kreiswirth BN, et al. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J Clin Microbiol 1994;32:1095–8. [17] Bhembe NL, Nwodo UU, Govender S, Hayes C, Ndip RN, Okoh AI. Molecular detection and characterization of resistant genes in Mycobacterium tuberculosis complex from DNA isolated from tuberculosis patients in the Eastern Cape province South Africa. BMC Infect Dis 2014;14:479 http://www.com/14712334/14/479. [18] Dookie N, Sturm AW, Moodley P. Mechanisms of first-line antimicrobial resistance in multi-drug and extensively drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa. BMC Infect Dis 2016;16:609. [19] Ioerger TR, Koo S, No EG, Chen X, Larsen MH, Jacobs WR, et al. Genome analysis of multi- and extensively-drug-resistant tuberculosis from KwaZulu-Natal, South Africa. PLoS One 2009;4:2–11. [20] Van Embden JDA, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993;31:406–9. [21] Chen L, Gan X, Li N, Wang J, Li K, Zhang H. rpoB gene mutation profile in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Guizhou, one of the highest incidence rate regions in China. J Antimicrob Chemother 2010, http://dx.doi.org/10.1093/jac/dkq102. [22] Sheng J, Li J, Sheng G, Yu H, Huang H, Cao H, et al. Characterization of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis from eastern China. J Appl Microbiol 2008;105:904–11. [23] Parsons LM, Driscoll JR, Taber HW, Salfinger M. Drug resistance in tuberculosis. Infect Dis Clin North Am 1997;11:905–28. [24] Adikaram CP, Perera J, Wijesundera SS. Geographical profile of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates in Sri Lanka. Microb Drug Resist 2012;18:525–30. [25] Bahrmand AR, Titov LP, Tasbiti AH, Yari S, Graviss EA. High-level rifampin resistance correlates with multiple mutations in the rpoB gene of pulmonary tuberculosis isolates from the Afghanistan border of Iran. J Clin Microbiol 2009;47:2744–50. [26] Qazi O, Rahman H, Tahir Z, Qasim M, Khan S, Anjum AA, et al. Mutation pattern in rifampicin resistance determining region of gene in multidrugresistant Mycobacterium tuberculosis isolates from Pakistan. Int J Mycobacteriol 2014;3:173–7. [27] Sadiq NK, Stefan N, Muhammad G, Mazhar Q, Sima S, Zahid SM, et al. Molecular characterization of multidrug resistant isolates of Mycobacterium tuberculosis from patients in Punjab, Pakistan. Pak J Zool 2013;45:93–100. [28] Sajduda A, Brzostek A, Poplawska M, Augustynowicz-Kopec E, Zwolska Z, Niemann S, et al. Molecular characterization of rifampin- and isoniazidresistant Mycobacterium tuberculosis strains isolated in Poland. J Clin Microbiol 2012;42:2425–31. [29] Cavusoglu C, Hilmioglu S, Guneri S, Bilgic A. Characterization of rpoB mutations in rifampin-resistant clinical isolates of Mycobacterium tuberculosis

Please cite this article in press as: Zaw MT, et al. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.04.005

G Model JIPH-878; No. of Pages 6

ARTICLE IN PRESS M.T. Zaw et al. / Journal of Infection and Public Health xxx (2018) xxx–xxx

6

[30]

[31]

[32]

[33]

[34]

from Turkey by DNA sequencing and line probe assay. J Clin Microbiol 2002;40:4435–8. Tan Y, Hu Z, Zhao Y, Cai X, Luo C, Zuo C, et al. The beginning of the rpoB gene in addition to the rifampin resistance determination region might be needed for identifying rifampin/rifabutin cross-resistance in multidrugresistant Mycobacterium tuberculosis isolates from Southern China. J Clin Microbiol 2012;50:81–5. Heep M, Brandstatter B, Rieger U, Lehn N, Richter E, Rusch-Gerdes S, et al. Frequency of rpoB mutations inside and outside the cluster region in rifampin-resistant clinical Mycobacterium tuberculosis isolates. J Clin Microbiol 2001;39:107–10. Htike KPM, Pitaksajjakul P, Tipkrua N, Wongwit W, Jintaridh P, Ramasoota P. Novel mutation detection in rpoB of rifampicin-resistant Mycobacterium tuberculosis using pyrosequencing. Southeast Asian J Trop Med Public Health 2014;45(4):843–52. Ramasoota P, Pitaksajjakul P, Phatihattakorn W, Pransujarit V, Boonyasopun J. Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains from Thailand and its evolutionary implication. Southeast Asian J Trop Med Public Health 2006;37:136–47. Bostanabad S, Fateh A, Seyedi K, Abdolrahimi F, Karimi A, Tasbiti AH, et al. Frequency and molecular characterization of rifampicin-resistance in the rpoB region of multiple drug resistance (MDR) isolates from tuberculosis patients in southern endemic region of Iran. Iran J Biotechnol 2007;5:212–8.

[35] Bahrmand AR, Titov LP, Tasbiti AH, Yari S, Graviss EA. High-level rifampicin resistance correlates with multiple mutations in the rpoB gene of pulmonary tuberculosis isolates from the Afghanistan border of Iran. J Clin Microbiol 2009;47:2744–50. [36] Yue J, Shi W, Xie J, Li Y, Zeng F, Wang H. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China. J Clin Microbiol 2003;41:2209–12. [37] Torok ME, Reuter S, Bryant J, Koser CU, Stinchecombe SV, Nazareth B, et al. Rapid whole-genome sequencing for investigation of a suspected tuberculosis outbreak. J Clin Microbiol 2013;51(2):611–4. [38] Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363(11):1005–15. [39] Jacobson KR, Theron D, Victor TC, Streicher EM, Warren RM, Murray MB. Treatment outcomes of isoniazid-resistant tuberculosis patients, Western Cape Province, South Africa. Clin Infect Dis 2011;53:369–72. [40] Torres JN, Paul LV, Rodwell TC, Victor TC, Amallraja AM, Elghraoui A, et al. Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates. Emerg Microbes Infect 2015;4:e42, http://dx.doi.org/10.1038/emi.2015.42.

Please cite this article in press as: Zaw MT, et al. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.04.005