QUANTITATIVE ANALYSIS OF GYRA AND GYRB KNOWN MUTATIONS IN DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS TREATED WITH OFLOXACIN
Objective: The aim of the study is to measure the minimum inhibitory concentrations (MICs) of ofloxacin antibiotic from gyrA and gyrB mutations present in fluoroquinolones (FQs) resistant strains of Mycobacterium tuberculosis (MTB) and to further concentrate the potential association between gene mutations and phenotypic resistance based on their MICs.
Methods: Different levels of ofloxacin MICs levels were detected in 31 archived multi drug-resistant MTB isolates showing gyrA mutations in codon at A90V, S91P, D94A, D94N/Y, D94 G, and D94H and two gyrB probes (N538D and E540V). The MIC determinations were made using the 1% proportion method.
Results: Genotypic assay detected 32 mutations in the gyrA (n=29) and gyrB (n=3) genes among the 31 FQs resistant isolates. Most frequently seen in gyrA mutations at codon D94G (n=16; 50%), these mutations had a clearly elevated MIC level from 2 to 16 μg/ml, that was phenotypically resistant. The A90V mutation region consistently exhibited the lowest levels of ofloxacin resistance, with three out of eight (37.50%) of these isolates had a MIC of <2 μg/ml. In addition, a further strain of S91P mutations for MIC was determined to be less than the critical concentration (CC). These low levels of resistance have been detected in a phenotypic manner which is noticeable in the study. Furthermore, fewer mutations in codons at D94A, D94N/Y were identified. Three wild-type absent isolates from gyrB QRDR were identified and the MIC of those strains for ofloxacin was lower than the critical cutoff value.
Conclusion: Based on the results, it is shown that different resistance mutations were associated with different levels of MICs and the current concentration for MGIT will be lowered from 2 μg/ml to 1 μg/ml for the ofloxacin drug.
2. World Health Organization. Technical Report on Critical Concentrations for Drug Susceptibility Testing of Medicines Used in the Treatment of Drug-Resistant Tuberculosis. Geneva: World Health Organization; 2018. Available from: https://www.apps.who.int/iris/ handle/10665/260470. [Last accessed on 2020 May 15].
3. World Health Organization. The Use of Molecular Line Probe Assays for the Detection of Resistance to Second-Line Anti-Tuberculosis Drugs: Policy Guidance. Geneva: World Health Organization; 2016. Available from: https://www.apps.who.int/iris/handle/10665/246131. [Last accessed on 2020 May 28].
4. Palomino JC, Martin A. Drug resistance mechanisms in Mycobacterium tuberculosis. Antibiotics (Basel) 2014;3:317-40.
5. Bernard C, Veziris N, Brossier F, Sougakoff W, Jarlier V, Robert J, et al. Molecular diagnosis of fluoroquinolone resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2015;59:1519-24.
6. Maruri F, Sterling TR, Kaiga AW, Blackman A, van der Heijden YF, Mayer C, et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 2012;67:819-31.
7. Kambli P, Ajbani K, Sadani M, Nikam C, Shetty A, Udwadia Z, et al. Correlating minimum inhibitory concentrations of ofloxacin and moxifloxacin with gyrA mutations using the genotype MTBDRsl assay. Tuberculosis (Edinb) 2015;95:137-41.
8. Willby M, Sikes RD, Malik S, Metchock B, Posey JE. Correlation between GyrA substitutions and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2015;59:5427-34.
9. HAIN Life Science. Geno Type MTBDRsl VER 2.0 Instructions for Use. Document IFU-317A-01. Nehren, Germany: HAIN Life Science. Available from: http://www.hain-lifescience.de/en/instructions-for-use. html.
10. Gardee Y, Dreyer AW, Koornhof HJ, Omar SV, da Silva P, Bhyat Z, et al. Evaluation of the Geno Type MTBDRsl Version 2.0 assay for second-line drug resistance detection of Mycobacterium tuberculosis isolates in South Africa. J Clin Microbiol 2017;55:791-800.
11. Canetti G, Froman S, Grosset J, Hauduroy P, Langerova M, Mahler HT, et al. Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull World Health Organ 1963;29:565-78.
12. Angeby KA, Jureen P, Giske CG, Chryssanthou E, Sturegård E, Nordvall M, et al. Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data. J Antimicrob Chemother 2010;65:946-52.
13. Farhat MR, Jacobson KR, Franke MF, Kaur D, Sloutsky A, Mitnick CD, et al. Gyrase mutations are associated with variable levels of fluoroquinolone resistance in Mycobacterium tuberculosis. J Clin Microbiol 2016;54:727-33.
14. Rufai SB, Singh J, Kumar P, Mathur P, Singh S. Association of gyrA and rrs gene mutations detected by MTBDRsl V1 on Mycobacterium tuberculosis strains of diverse genetic background from India. Sci Rep 2018;8:9295.
15. Tagliani E, Cabibbe AM, Miotto P, Borroni E, Toro JC, Mansjö M, et al. Diagnostic performance of the new version (v2.0) of Geno Type MTBDRsl assay for detection of resistance to fluoroquinolones and second-line injectable drugs: A multicenter study. J Clin Microbiol 2015;53:2961-9.
16. Ruesen C, Riza AL, Florescu A, Chaidir L, Editoiu C, Aalders N, et al. Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania. Sci Rep 2018;8:9676.
17. Disratthakit A, Prammananan T, Tribuddharat C, Thaipisuttikul I, Doi N, Leechawengwongs M, et al. Role of gyrB mutations in pre-extensively and extensively drug-resistant tuberculosis in Thai clinical isolates. Antimicrob Agents Chemother 2016;60:5189-97.
18. von Groll A, Martin A, Jureen P, Hoffner S, Vandamme P, Portaels F, et al. Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in gyrA and gyrB. Antimicrob Agents Chemother 2009;53:4498-500.
19. Rigouts L, Coeck N, Gumusboga M, de Rijk WB, Aung KJ, Hossain MA, et al. Specific gyrA gene mutations predict poor treatment outcome in MDR-TB. J Antimicrob Chemother 2016;71:314-23.
20. Cambau E, Viveiros M, Machado D, Raskine L, Ritter C, Tortoli E, et al. Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study. J Antimicrob Chemother 2015;70:686-96.
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