PCR with hydrolysis probes for detection of fluoroquinolone resistance mutations in Mycobacterium tuberculosis

Early diagnostics of resistance to fluoroquinolones facilitates early start of adequate therapy and increases the chance of a favorable outcome of the tuberculosis. Application of genetic methods permits to obtain within 1–2 days the results of Mycobacterium tuberculosis resistance detection to anti-tuberculosis drugs, unlike the classical methods requiring up to 1−2 month.

The aim of the study is to develop a method for the M. tuberculosis identification and detection of point mutations in codons 90, 91, 94 of the gyrA gene associated with the resistance to fluoroquinolones.

There were 88 cultures of mycobacteria studied: M. tuberculosis (n = 81), M. tuberculosis H37Rv, M. chelonae (n = 1), M. gordonae (n = 1), M. fortuitum (n = 1), and other three isolates of non-tuberculosis mycobacteria isolated from patients in the Republican Scientific and Practical Center for Pulmonology and Phthisiatry. The types of mutations in the gyrA gene were studied by the standard GenoTypeMTBDRsl method (HAIN, Germany), Sanger sequencing, and by the developed real-time PCR method. Based on the analysis of mutations in the gyrA gene in 78 isolates of M. tuberculosis, the dominant mutations were found to be mutations Asp94Gly and Ala90Val, which were identified in 21 and 27 isolates correspondingly: they accounted for 64 % of all mutations. M. tuberculosis also harbored mutations p.ASP94ALA and p.ASP94TYR/HIS in 6 (8.0 %) and 9 (12.0 %) isolates, respectively. One strain harbored a mutation at triplet 88 and one strain had a double mutation (p.ALA90VAL and p.ASP94GLY). The developed real-time PCR method demonstrated a high frequency of coincidence of results with the phenotypic determination of resistance to ofloxacin and the results testing by the standard GenoTypeMTBDRsl method and sequencing.

The developed method is accomplished to identify M. tuberculosis, and discriminate mutations p.ALA90VAL, p.SER91PRO, p.ASP94ALA, p.ASP94TYR/HIS, p.ASP94GLY, p.ASP94ASN providing diagnostics of resistance to fluoroquinolones.

1. Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in gyrA and gyrB / A. Von Groll [et al.] // Antimicrob. Agents Chemother. – 2009. – Vol. 53, N 10. – P. 4498–4500. https://doi.org/10.1128/AAC.00287-09

2. Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project / M. Zignol [et al.] // Lancet Infect. Dis. – 2016. – Vol. 16, N 10. – P. 1185–1192. https://doi.org/10.1016/S1473-3099(16)30190-6

3. Zhang, Y. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015 / Y. Zhang, W.-W. Yew // Int. J. Tuberc. Lung Dis. – 2015. – Vol. 19, N 11. – P. 1276–1289. https://doi.org/10.5588/ijtld.15.0389

4. Extensively drug-resistant tuberculosis: epidemiology and management challenges / K. Dheda [et al.] // Infect. Dis. Clin. – 2010. – Vol. 24, N 3. – P. 705–725. https://doi.org/10.1016/j.idc.2010.05.001

5. Extensively drug-resistant tuberculosis, Italy and Germany / G. B. Migliori [et al.] // Emerg. Infect. Dis. – 2007. – Vol. 13, N 5. – P. 780–782. https://doi.org/10.3201/eid1305.060200

6. Extensively drug–resistant, tuberculosis in South Korea: risk factors and treatment outcomes among patients at a tertiary referral hospital / C. Y. Jeon [et al.] // Clin. Infect. Dis. – 2008. – Vol. 46, N 1. – P. 42–49. https://doi.org/10.1086/524017

7. Collins, C. H. In–vitro susceptibility of mycobacteria to ciprofloxacin / C. H. Collins, A. H. Uttley // J. Antimicrob. Chemother. – 1985. – Vol. 16, N 5. – P. 575–580. https://doi.org/10.1093/jac/16.5.575

8. Drlica, K. Fluoroquinolones: action and resistance / K. Drlica, M. Malik // Curr. Top Med. Chem. – 2003. – Vol. 3, N 3. – P. 249–282. https://doi.org/10.2174/1568026033452537

9. Epidemiology and clinical management of XDR–TB: a systematic review by TBNET / G. Sotgiu [et al.] // Eur. Resp. J. – 2009. – Vol. 33, N 4. – P. 871–881. https://doi.org/10.1183/09031936.00168008

10. Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success / L. S. Redgrave [et al.] Trends Microbiol. – 2014. – Vol. 22, N 8. – P. 438–445. https://doi.org/10.1016/j.tim.2014.04.007

11. Multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis: epidemiology and control / A. Matteelli [et al.] // Expert Rev. Anti-infect. Ther. – 2007. – Vol. 5, N 5. – P. 857–871. https://doi.org/10.1586/14787210.5.5.857

12. Alangaden, G. J. The clinical use of fluoroquinolones for the treatment of mycobacterial diseases / G. J. Alangaden, S. A. Lerner // Clin. Infect. Dis. – 1997. – Vol. 25, N 5. – P. 1213–1221. https://doi.org/10.1086/516116

13. Cambau, E. Amplification and nucleotide sequence of the quinolone resistance-determining region in the gyrA gene of mycobacteria / E. Cambau, W. Sougakoff, V. Jarlier // FEMS Microbiol. Lett. – 1994. – Vol. 116, N 1. – P. 49–54. https://doi.org/10.1111/j.1574-6968.1994.tb06674.x

14. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations / H. E. Takiff [et al.] // Antimicrob. Agents Chemother. – 1994. – Vol. 38, N 4. – P. 773–780. https://doi.org/10.1128/AAC.38.4.773

15. Comprehensive treatment of extensively drug-resistant tuberculosis / C. D. Mitnick [et al.] // New Engl. J. Med. – 2008. – Vol. 359, N 6. – P. 563–574. https://doi.org/10.1056/NEJMoa0800106

16. Piddock, L. J. V. Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992–1994 / L. J. V. Piddock // Drugs. – 1995. – Vol. 49, suppl. 2. – P. 29–35. https://doi.org/10.2165/00003495-199500492-00006

17. Whole-genome sequencing has the potential to improve treatment for rifampicin-resistant tuberculosis in high-burden settings: a retrospective cohort study / H. Cox [et al.] // J. Clin. Microbiol. – 2022. – Vol. 60, N 3. – P. e02362–21. https://doi.org/10.1128/jcm.02362-21

18. Multidrug-resistant tuberculosis in Europe, 2010–2011 / G. Günther [et al.] // Emerg. Infect. Dis. – 2015. – Vol. 21, N 3. – P. 409. https://doi.org/10.3201/eid2103.141343

19. Yin, X. Mutation characterization of gyrA and gyrB genes in levofloxacin-resistant Mycobacterium tuberculosis clinical isolates from Guangdong Province in China / X. Yin, Z. Yu // J. Infect. – 2010. – Vol. 61, N 2. – P. 150–154. https://doi.org/10.1016/j.jinf.2010.05.001

20. Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia / I. Mokrousov [et al.] // Antimicrob. Agents Chemother. – 2008. – Vol. 52, N 8. – P. 2937–2939. https://doi.org/10.1128/AAC.00036-08

21. Fluoroquinolone susceptibility among Mycobacterium tuberculosis isolates from the United States and Canada / L. Bozeman [et al.] // Clin. Infect. Dis. – 2005. – Vol. 40, N 3. – P. 386–391. https://doi.org/10.1086/427292