Characterization of Genetic Variants Associated with Rifampicin Resistance Level in Mycobacterium tuberculosis Clinical Isolates Collected in Guangzhou Chest Hospital, China

H M Adnan Hameed; Cuiting Fang; Zhiyong Liu; Yanan Ju; Xingli Han; Yamin Gao; Shuai Wang; Gift Chiwala; Yaoju Tan; Ping Guan; Jinxing Hu; Xiaoli Xiong; Jiacong Peng; Yongping Lin; Muzammal Hussain; Nanshan Zhong; Dmitry A Maslov; Gregory M Cook; Jianxiong Liu; Tianyu Zhang

doi:10.2147/IDR.S375869

Characterization of Genetic Variants Associated with Rifampicin Resistance Level in Mycobacterium tuberculosis Clinical Isolates Collected in Guangzhou Chest Hospital, China

Infect Drug Resist. 2022 Sep 27:15:5655-5666. doi: 10.2147/IDR.S375869. eCollection 2022.

Authors

H M Adnan Hameed^#^{1

2

3

4}, Cuiting Fang^#^{1

2

3

4}, Zhiyong Liu^{1

2

3}, Yanan Ju^{1

2

3}, Xingli Han^{1

2

3

4}, Yamin Gao^{1

2

3

4}, Shuai Wang^{1

2

3

5}, Gift Chiwala^{1

2

3

4}, Yaoju Tan⁶, Ping Guan⁶, Jinxing Hu⁶, Xiaoli Xiong^{1

2

3}, Jiacong Peng^{3

7}, Yongping Lin^{3

7}, Muzammal Hussain⁴, Nanshan Zhong^{3

7

8}, Dmitry A Maslov⁹, Gregory M Cook^{10

11}, Jianxiong Liu⁶, Tianyu Zhang^{1

2

3

4}

Affiliations

¹ State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou, People's Republic of China.
² China-New Zealand Joint Laboratory of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou, People's Republic of China.
³ Guangdong-Hong Kong-Macau Joint Laboratory of Respiratory Infectious Diseases, Guangzhou, People's Republic of China.
⁴ University of Chinese Academy of Sciences (UCAS), Beijing, People's Republic of China.
⁵ National Clinical Research Center for Infectious Diseases, Guangdong Provincial Clinical Research Center for Tuberculosis, Shenzhen Third People's Hospital, Shenzhen, People's Republic of China.
⁶ State Key Laboratory of Respiratory Disease, Guangzhou Chest Hospital, Guangzhou, People's Republic of China.
⁷ State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, People's Republic of China.
⁸ Guangzhou National Laboratory, Guangzhou, People's Republic of China.
⁹ Laboratory of Bacterial Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia.
¹⁰ Department of Microbiology and Immunology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.
¹¹ Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand.

^# Contributed equally.

Abstract

Objective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF.

Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay.

Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to >64 µg/mL) of which 57 were MDR and 81 pre-XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426-452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284-1289 CCAGCT), (1295-1303 AATTCATGG), and insertion at (1300-1302 TTC) were detected within RRDR of three RIF^R strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIF^R strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively.

Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIF^R clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings.

Keywords: GeneXpert MTB/RIF; Mycobacterium tuberculosis; resistance-determining region; rifampicin-resistance; rpoB.

Grants and funding

This work was supported by the National Key R&D Program of China (2021YFA1300904), partially by the National Natural Science Foundation of China (NSFC 81973372), Joint Research the Russian Science Foundation (RSF)-NSFC Collaboration grant numbers 21-45-00018 (to D.M.), 82061138019 (to T.Z.), the Joint Research Health Research Council of New Zealand (HRC)-NSFC Collaboration grant numbers 20/1211 (to G.C.), 82061128001 (to T.Z.), supported by China-New Zealand Joint Laboratory on Biomedicine and Health, Department of Science and Technology of Guangdong Province (2019B110233003), the Chinese Academy of Sciences (154144KYSB20190005, YJKYYQ20210026), State Key Laboratory of Respiratory Disease (SKLRD-OP-202324 to Tianyu Zhang), and China Postdoctoral Fellowship, Guangdong province, Huangpu, GIBH-University of Chinese Academy of Sciences, President’s International Fellowship Initiative-CAS and State Key Laboratory of Respiratory Disease(SKLRD-Z-202301 to H.M. Adnan Hameed). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.