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Tuberc Respir Dis > Volume 81(3); 2018 > Article
Yun, Lee, Yoo, Cho, Park, Kook, and Lee: Patterns of rpoC Mutations in Drug-Resistant Mycobacterium tuberculosis Isolated from Patients in South Korea

Abstract

Background

Rifampicin (RFP) is one of the principal first-line drugs used in combination chemotherapies against Mycobacterium tuberculosis, and its use has greatly shortened the duration of chemotherapy for the successful treatment of drug-susceptible tuberculosis. Compensatory mutations have been identified in rpoC that restore the fitness of RFP-resistant M. tuberculosis strains with mutations in rpoB. To investigate rpoC mutation patterns, we analyzed 93 clinical M. tuberculosis isolates from patients in South Korea.

Methods

Drug-resistant mycobacterial isolates were cultured to determine their susceptibility to anti-tubercular agents. Mutations in rpoC were identified by sequencing and compared with the relevant wild-type DNA sequence.

Results

In total, 93 M. tuberculosis clinical isolates were successfully cultured and tested for drug susceptibilities. They included 75 drug-resistant tuberculosis species, of which 66 were RFP-resistant strains. rpoC mutations were found in 24 of the 66 RFP-resistant isolates (36.4%). Fifteen different types of mutations, including single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%), were identified, and 12 of these mutations are reported for the first time in this study. The most frequent mutation involved a substitution at codon 452 (nt 1356) resulting in amino acid change F452L.

Conclusion

Fifteen different types of mutations were identified and were predominantly single-nucleotide substitutions (91.7%). Mutations were found only in dual isoniazid- and RFP-resistant isolates of M. tuberculosis. No mutations were identified in any of the drug-susceptible strains.

Introduction

The worldwide emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) threatens global efforts to control tuberculosis1,2. Isoniazid (INH) and rifampicin (RFP) are the main components of first-line anti-tuberculosis treatment and are effective drugs for the treatment of tuberculosis3,4,5. MDR-TB is resistant to INH and RFP, and the emergence of MDR-TB is placing an increasing burden on South Korea4,5,6.
Mycobacterium tuberculosis can acquire resistance to RFP through mutations in rpoB, which encodes the β subunit of RNA polymerase7,8,9. Mutations in rpoC, which encodes the β′ subunit of RNA polymerase, have also been associated with increased in vitro fitness and were overrepresented in MDR-TB isolates from countries with high MDR-TB burdens10,11. One study showed that M. tuberculosis isolates harbouring an rpoB mutation also carried nonsynonymous mutations in rpoC 10.
In this study, we investigated rpoC mutation patterns in drug-resistant and susceptible M. tuberculosis isolates from patients in South Korea to determine the epidemiological relevance of rpoC nonsynonymous mutations in a high-incidence setting of MDR- and XDR-TB.

Materials and methods

1. Mycobacterial isolates and susceptibility testing

Ninety-three M. tuberculosis isolates with clinically observed drug resistance were collected at National Masan Hospital and Pusan National University College of Medicine in South Korea and were cultured to determine their susceptibility to anti-tubercular agents (Table 1). Each isolate was cultured on Löwenstein-Jensen medium at 37℃ for 3-4 weeks and tested for resistance to critical concentrations of capreomycin (40 µg/mL), ethambutol (2.0 µg/mL), INH (0.2 µg/mL), kanamycin (40 µg/mL), ofloxacin (2 µg/mL), streptomycin (4 µg/mL), pyrazinamide (100 µg/mL, Wayne's pyrazinamidase assay12), and RFP (40 µg/mL). M. tuberculosis H37Rv (American Type Culture Collection [ATCC] 27294) was used as a positive control in all of the experiments. The following drug-resistant profiles were defined: MDR, resistance to both RFP and INH; MDR-plus, resistance to any of the second-line injectable drugs (INH+RFP+Inj.D) or to any fluoroquinolone drugs (INH+RFP+FQ); XDR, extensively drug-resistant; DR, drug resistance other than MDR (including MDR-plus and XDR); and S, susceptibility to all of the drugs. Sixty-six M. tuberculosis isolates were RFP resistant (Table 1).
This study was approved by the institutional review board (IRB) at the International Tuberculosis Research Centre, and all subjects signed an informed consent form.

2. Polymerase chain reaction and sequencing of rpoC

The rpoC region (1,730 bp) was amplified by polymerase chain reaction (PCR) using the GeneAmp PCR System 9600 (PerkinElmer, Foster City, CA, USA) with primers 5′-CGAAAACCTCTACCGCGAAC-3′ and 5′-CACGGAAGGAGGACTTGACC-3′10.
Briefly, the PCR parameters were 5 minutes at 95℃, followed by 40 cycles of 45 seconds at 94℃, 45 seconds at 60℃, and 60 seconds at 72℃, with a final extension step at 72℃ for 10 minutes. The PCR products were purified using a QIAEX II Gel Extraction Kit (Qiagen Inc., Mainz, Germany) according to the manufacturer's instructions and sequenced using a BigDye Terminator cycle sequencing kit with AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA) using primers 5′-CGAAAACCTCTACCGCGAAC-3′ and 5′-CACGGAAGGAGGACTTGACC-3′10. The nucleotide sequences were analyzed using BioEdit software version 5.0.9.1 (Ibis Biosciences, Carlsbad, CA, USA), Chromas version 2.33 (Technelysium, Brisbane, QLD, Australia, http://www.technelysium.com.au/chromas.html), and the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information, Bethesda, MD, USA, http://blast.ncbi.nlm.nih.gov/Blast.cgi). Mutations in the rpoC-encoding regions were defined as any nucleotide changes that led to translational changes in RpoC compared with the RFP-susceptible strain, H37Rv (ATCC 27294).

Results

Ninety-three clinical isolates were included in this study, and all were from South Korean patients. Drug susceptibility testing identified 75 multidrug-resistant isolates: 20 were categorized as MDR-TB, seven were categorized as MDR-plus, 36 were categorized as XDR-TB, and 12 were categorized as DR-TB. Moreover, 66 of the cultured M. tuberculosis isolates were found to be RFP-resistant. Eighteen isolates were categorized as S (Table 1).
rpoC PCR products were amplified from 93 isolates and sequenced. Fifteen different types of mutations were identified in 24 isolates (24/93, 25.8%), all of which were resistant to both INH and RFP. The rpoC mutation rate of the MDR- and XDR-TB isolates was 37.0% (10/27) and 38.9% (14/36), respectively (Table 2).
Single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%) in the rpoC region were identified, but no deletion nor insertion mutations were detected in any of the isolates. No mutations were identified in the rpoC region of any drug-susceptible strains. A mutation at codon 452 (nt 1356) was the most common mutation (7/24, 29.2%) and a mutation at codon 531 (nt 1594), which is the most frequently mutated nucleotide in rpoB , was also detected in these isolates (Supplementary Table S1)7,8,9. Twelve different mutation sites (at codon 281 [nt 843], 416 [nt 1249], 434 [nt 1302], 446 [nt 1338], 561 [nt 1683], 575 [nt 1726], 581 [nt 1745], 728 [nt 2186], 747 [nt 2242], 801 [nt 2403], 812 [nt 2437], and 813 [nt 2441]) are reported for the first time in this study10,11; these new mutations are shown in Table 3.

Discussion

RFP is one of the principal first-line drugs used in combination chemotherapy for tuberculosis, and RFP resistance is a valuable surrogate marker of MDR-TB. Over 90% of RFP-resistant clinical M. tuberculosis isolates possess genetic alterations in rpoB2,8.
rpoC encodes the β′ subunit of RNA polymerase, and Comas et al.11 suggested that the acquisition of particular mutations in rpoC by RFP-resistant M. tuberculosis strains leads to the emergence of MDR strains with high fitness over time. Additionally, de Vos et al.10 showed that nonsynonymous mutations in the rpoC region were prevalent among RFP-resistant isolates in a South African high-burden setting; these mutations were strongly associated with the transmission of RFP-resistant strains.
rpoC mutations have not been studied in South Korea; thus, we investigated the rpoC mutation patterns in drug-resistant and susceptible M. tuberculosis isolates from patients in South Korea. Fifteen different types of mutations were identified, 12 of which were reported for the first time in this study (Table 3)10,11. A mutation at codon 452 was the most common mutation (7/24, 29.2%), and a mutation at codon 531, which is the most frequently mutated nucleotide in rpoB, was also detected in these isolates (Supplementary Table S1)7,8,9.
Mutations were found only in the MDR-TB isolates and no rpoC mutations were identified in any of the drug-susceptible strains (Tables 2, 3).
Therefore, we suggest that rpoC mutations could be used DNA-based diagnosis for detection of INH and RFP drug resistance; however, more extensive studies on larger collections of isolates are needed.
In summary, 15 different types of mutations were identified. Substitutions in a single nucleotide were the most common mutation found (22/24, 91.7%), and mutations were found only in dual INH- and RFP-resistant isolates in this study.

Acknowledgments

This work was supported by a research grant from the Jeju National University Hospital Research Fund of Jeju National University in 2013.

Notes

Authors' Contributions: Conceptualization: Lee KH. Methodology: Yun YJ, Lee JS, Yoo JC, Cho E, Park D. Formal analysis: Lee KH, Yun YJ, Lee JS, Yoo JC, Park D. Data curation: Yun YJ, Lee JS, Yoo JC, Lee KH, Cho E, Park D. Validation: Lee KH, Yun YJ, Lee JS, Yoo JC, Kook YH. Investigation: Lee KH. Writing-original draft preparation: Yun YJ, Lee KH. Writing-review and editing: Yun YJ, Lee JS, Yoo JC, Lee KH. Approval of final manuscript: all authors.

Conflicts of Interest: No potential conflict of interest relevant to this article was reported.

Supplementary Material

Supplementary material can be found in the journal homepage (http://www.e-trd.org).
Supplementary Table S1. Mutations detected in the rpoB gene of 80 isolates.

Supplementary Table S1

Mutations detected in the rpoB gene of 80 isolates
trd-81-222-s001.pdf

References

1. Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, et al. Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis. Lancet 2010;375:1830-1843. PMID: 20488523.
crossref pmid
2. World Health Organization. Global tuberculosis report 2015. Geneva: World Health Organization; 2015.

3. Abate D, Tedla Y, Meressa D, Ameni G. Isoniazid and rifampicin resistance mutations and their effect on second-line anti-tuberculosis treatment. Int J Tuberc Lung Dis 2014;18:946-951. PMID: 25199009.
crossref pmid
4. Jeon D. Medical management of drug-resistant tuberculosis. Tuberc Respir Dis 2015;78:168-174.
crossref
5. Park JS. Issues related to the updated 2014 Korean guidelines for tuberculosis. Tuberc Respir Dis 2016;79:1-4.
crossref
6. Islam T, Hiatt T, Hennig C, Nishikiori N. Drug-resistant tuberculosis in the WHO Western Pacific Region. Western Pac Surveill Response J 2014;5:34-46. PMID: 25685604.
crossref pmid pmc
7. Yun YJ, Lee KH, Haihua L, Ryu YJ, Kim BJ, Lee YH, et al. Detection and identification of Mycobacterium tuberculosis in joint biopsy specimens by rpoB PCR cloning and sequencing. J Clin Microbiol 2005;43:174-178. PMID: 15634968.
crossref pmid pmc
8. Yue J, Shi W, Xie J, Li Y, Zeng E, Wang H. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China. J Clin Microbiol 2003;41:2209-2212. PMID: 12734282.
crossref pmid pmc
9. 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;40:4435-4438. PMID: 12454132.
crossref pmid pmc
10. de Vos M, Muller B, Borrell S, Black PA, van Helden PD, Warren RM, et al. Putative compensatory mutations in the rpoC gene of rifampin-resistant Mycobacterium tuberculosis are associated with ongoing transmission. Antimicrob Agents Chemother 2013;57:827-832. PMID: 23208709.
crossref pmid pmc
11. Comas I, Borrell S, Roetzer A, Rose G, Malla B, Kato-Maeda M, et al. Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes. Nat Genet 2011;44:106-110. PMID: 22179134.
crossref pmid pmc pdf
12. Wayne LG. Simple pyrazinamidase and urease tests for routine identification of mycobacteria. Am Rev Respir Dis 1974;109:147-151. PMID: 4203284.
pmid
Table 1

Drug resistance profiles of 93 Mycobacterium tuberculosis isolates

SM: streptomycin; INH: isoniazid; RFP: rifampicin; CPM: capreomycin; KM: kanamycin; OFX: ofloxacin; MFX: moxifloxacin; PZA: pyrazinamide; EMB: ethambutol; XDR: extensively drug-resistant; MDR: multidrug-resistant; S: susceptibility to all of the drugs; DR: drug resistance other than MDR (including MDR-plus and XDR); MDR-plus: INH+RFP+fluoroquinolone or INH+RFP+injectable drugs.

No. Drug resistance Drug resistance profile No. Drug resistance Drug resistance profile
1 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 48 None detected S
2 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 49 CPM DR
3 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR 50 INH, RFP, OFX, MFX, PZA MDR
4 INH, RFP, CPM, KM, PZA MDR 51 INH, RFP, KM, OFX, MFX, PZA XDR
5 SM, INH, RFP, EMB, OFX, MFX, PZA MDR 52 INH, RFP MDR
6 INH, RFP, OFX, MFX MDR 53 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR
7 SM, INH, RFP, EMB, CPM, KM, MFX, PZA XDR 54 SM, INH, RFP, EMB, OFX, PZA XDR
8 SM, INH, RFP, MFX, PZA MDR 55 SM, INH, RFP, OFX, MFX, PZA XDR
9 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 56 SM, INH, RFP, CPM, OFX, MFX, PZA XDR
10 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 57 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR
11 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR 58 INH, RFP, CPM, OFX, MFX, PZA XDR
12 SM, INH, RFP, KM, OFX, MFX, PZA XDR 59 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR
13 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 60 RFP, CPM DR
14 SM, INH, RFP, EMB, CPM, KM, OFX, MFX XDR 61 INH, RFP MDR
15 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 62 INH DR
16 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 63 INH, RFP, LEV, OFX, MFX, MDR-plus
17 SM, INH, RFP, CPM, KM, PZA MDR 64 INH, RFP, LEV, OFX, MFX, KM, AMK, CPM XDR
18 INH, RFP, OFX, MFX, PZA MDR 65 INH, RFP, LEV, OFX, MFX, KM, AMK, CPM XDR
19 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 66 INH, RFP, OFX, KM XDR
20 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 67 RFP DR
21 INH, RFP, CPM, KM, MFX, PZA XDR 68 INH, RFP, MDR
22 None detected S 69 INH, RFP, CPM MDR-plus
23 None detected S 70 None detected S
24 None detected S 71 INH, RFP, LEV, OFX MDR-plus
25 None detected S 72 INH, RFP MDR
26 None detected S 73 INH, RFP MDR
27 SM, INH, CPM, PZA DR 74 INH, RFP MDR
28 INH DR 75 INH, RFP MDR
29 None detected S 76 RFP, CPM DR
30 None detected S 77 None detected DR
31 None detected S 78 INH, RFP, LEV, OFX, MFX, KM, AMK, CPM XDR
32 SM, INH, RFP, EMB, KM, OFX, MFX, PZA XDR 79 INH, RFP, LEV, OFX MDR-plus
33 None detected S 80 INH, RFP, LEV, OFX MDR-plus
34 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 81 CPM DR
35 None detected S 82 INH, RFP, LEV, OFX MDR-plus
36 None detected S 83 None detected S
37 INH DR 84 INH, RFP MDR
38 SM, INH, RFP, EMB, CPM, KM, MFX, PZA XDR 85 None detected S
39 INH, RFP, EMB, OFX, MXF, PZA MDR 86 CPM DR
40 INH, RFP, CPM, KM, PZA MDR 87 CPM DR
41 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR 88 None detected S
42 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 89 INH, RFP MDR
43 SM, INH, RFP, EMB, CPM, KM, PZA MDR 90 INH, RFP, MFX, CPM XDR
44 SM, INH, RFP, CPM, KM, MFX, PZA XDR 91 INH, RFP, CPM MDR-plus
45 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 92 None detected S
46 SM, INH, RFP, CPM, KM, OFX, MFX XDR 93 None detected S
47 SM, INH, RFP, OFX, MFX, PZA MDR
Table 2

Isolates with rpoC mutations (n=24)

Isolates with mutations in rpoC are resistant to both INH and RFP.

SM: streptomycin; INH: isoniazid; RFP: rifampicin; CPM: capreomycin; KM: kanamycin; OFX: ofloxacin; MFX: moxifloxacin; PZA: pyrazinamide; EMB: ethambutol; XDR: extensively drug-resistant; MDR-plus: INH+RFP+fluoroquinolone or INH+RFP+injectable drugs.

No. Drug resistance Drug resistance profile No. Drug resistance Drug resistance profile
2 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 50 INH, RFP, OFX, MFX, PZA MDR
8 SM, INH, RFP, MFX, PZA MDR 53 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR
9 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 54 SM, INH, RFP, EMB, OFX, PZA XDR
11 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR 57 SM, INH, RFP, EMB, CPM, KM, OFX, MFX, PZA XDR
13 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 63 INH, RFP, LEV, OFX, MFX, MDR-plus
16 SM, INH, RFP, CPM, KM, OFX, MFX, PZA XDR 65 INH, RFP, LEV, OFX, MFX, KM, AMK, CPM XDR
17 SM, INH, RFP, CPM, KM, PZA MDR 71 INH, RFP, LEV, OFX MDR-plus
20 INH, RFP, CPM, KM, OFX, MFX, PZA XDR 73 INH, RFP MDR
32 SM, INH, RFP, EMB, KM, OFX, MFX, PZA XDR 74 INH, RFP MDR
38 SM, INH, RFP, EMB, CPM, KM, MFX, PZA XDR 79 INH, RFP, LEV, OFX MDR-plus
44 SM, INH, RFP, CPM, KM, MFX, PZA XDR 80 INH, RFP, LEV, OFX MDR-plus
46 SM, INH, RFP, CPM, KM, OFX, MFX XDR 89 INH, RFP MDR
Table 3

Mutations detected in the rpoC gene of 93 Mycobacterium tuberculosis isolates

Values are presented as number (%).

*New mutation not reported in previous studies. Isolates with mutations in rpoC are resistant to both INH and RFP.

MDR-TB: multidrug-resistant tuberculosis; XDR-TB: extensively drug-resistant tuberculosis; MDR-plus: INH+RFP+fluoroquinolone or INH+RFP+injectable drugs; DR: drug resistance other than MDR (including MDR-plus and XDR); S: susceptibility to all of the drugs; INH: isoniazid; RFP: rifampicin.

Nucleotide change (nucleotide No.) Translational change (codon No.) Cultured isolates (n=93) MDR-TB isolates (n=20) XDR-TB isolates (n=36) MDR-plus isolates (n=7) DR isolates (n=12) S isolates (n=18) No.
Substitution
843 A>G I281V* 3 (3.23) 2 (2.15) - 1 (1.08) - - 71, 73, 89
1249 A>G N416S* 1 (1.08) - 1 (1.08) - - - 20
1302 C>A P434T* 1 (1.08) - - 1 (1.08) - - 79
1338 C>A L446M* 1 (1.08) - - 1 (1.08) - - 63
1356 T>C F452L 7 (7.53) 1 (1.08) 6 (6.45) - - - 2, 11, 13, 17, 44, 53, 57
1450 T>C V483A 1 (1.08) - 1 (1.08) - - - 38
1450 T>G V483G 1 (1.08) - 1 (1.08) - - - 46
1683 T>C S561P* 1 (1.08) - 1 (1.08) - - - 54
1726 C>T A575V* 1 (1.08) - 1 (1.08) - - - 16
2186 C>T G728G* 1 (1.08) 1 (1.08) - - - - 74
2242 A>G D747G* 2 (2.15) 1 (1.08) 1 (1.08) - - - 8, 65
2437 C>T T812I* 1 (1.08) 1 (1.08) - - - - 50
2441 G>C Q813H* 1 (1.08) - 1 (1.08) - - - 32
Multi-site mutations -
1683 T>C, 1745 G>A S561P*, M581I* 1 (1.08) M581I* - 1 (1.08) - - - 9
1302, 1303 CC>GT 2403A>T P434V, T801S* 1 (1.08) - - 1 (1.08) - - 80


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