OBJECTIVE: The resistance of Mycobacterium (M.) tuberculosis to antituberculosis drugs poses a major threat to global public health. Whole genome sequencing (WGS) is an increasingly preferred method in the diagnostics and monitoring of the transmission dynamics of resistant forms of tuberculosis (TB). The aim of the study was to, for the first time, use the sequencing-based analysis to study the transmission and resistance patterns of a systematic and recent collection of extensively drug resistant (XDR) and multidrug resistant tuberculosis (MDR-TB) isolates and to expand our knowledge about drug resistant (DR) TB epidemiological dynamics in Slovakia. DESIGN: A total of 495 patients with pulmonary TB, who were referred to National Reference Laboratory for Mycobacteriology (Vyšné Hágy, Slovakia) in the years 2018-2019, were studied. Out of the total of 495 patients, 4 XDR-TB (0.8%) and 8 (1.6%) MDR-TB isolates were identified by conventional drug susceptibility testing on Löwenstein-Jensen solid medium and subjected to whole genome sequencing. Sequencing data were evaluated for molecular-epidemiological analysis and identification of resistance patterns. RESULTS: Phylogenetic and cluster analysis showed extensive recent transmission events and the predominance of Euro-American lineage 4.7 in Slovakia. However, phylogenetic analysis revealed the circulation of several lineages that originally occurred in Eastern European countries. Resistance patterns for first- and second-line antituberculosis drugs characterized by whole genome sequencing were in high concordance with the results of phenotypic drug susceptibility testing. CONCLUSION: Forty percent of at least MDR-TB isolates were not genetically linked, indicating that appropriate measures should be taken to monitor and prevent the spread of drug-resistant tuberculosis within the country as well as in other regions.

Biomedical Center Martin Department of Molecular Medicine Jessenius Faculty of Medicine in Martin Comenius University in Bratislava Slovakia Department of Clinical Biochemistry Jessenius Faculty of Medicine in Martin Comenius University in Bratislava Slovakia

Department of Pharmacology and Biomedical Center Martin Jessenius Faculty of Medicine Comenius University Slovakia

Emerging Bacterial Pathogens Unit IRCCS San Raffaele Scientific Institute Milan Italy

Faculty of Health Catholic University Ružomberok Slovakia

International Reference Laboratory of Mycobacteriology Statens Serum Institut Copenhagen Denmark

National Institute of Tuberculosis Lung Diseases and Thoracic Surgery Vyšné Hágy Slovakia Faculty of Health Catholic University Ružomberok Slovakia

National Reference Laboratory for Mycobacteria National Institute of Public Health Praha Czech Republic

Zobrazit více v PubMed

Analýza situácie TBC na Slovensku – NÚTPCHaHCH Vyšné Hágy n.d. https://www.hagy.sk/narodny-register-tbc/analyza-situacie-tbc-na-slovensku/ (accessed September 8, 2021).

Cirillo D.M., Miotto P., Tortoli E. Evolution of Phenotypic and Molecular Drug Susceptibility Testing. Adv Exp Med Biol. 2017;1019:221–246. doi: 10.1007/978-3-319-64371-7_12. PubMed DOI

Dohál M., Porvazník I., Pršo K., Rasmussen E.M., Solovič I., Mokrý J. Whole-genome sequencing and Mycobacterium tuberculosis: Challenges in sample preparation and sequencing data analysis. Tuberculosis. 2020;123:101946. doi: 10.1016/j.tube.2020.101946. PubMed DOI

Nikolayevskyy V., Niemann S., Anthony R., van Soolingen D., Tagliani E., Ködmön C., et al. Role and value of whole genome sequencing in studying tuberculosis transmission. Clin Microbiol Infect. 2019;25(11):1377–1382. PubMed

Jagielski T., van Ingen J., Rastogi N., Dziadek J., Mazur P.K., Bielecki J. Current methods in the molecular typing of mycobacterium tuberculosis and other Mycobacteria. Biomed Res Int. 2014;2014:1–21. doi: 10.1155/2014/645802. PubMed DOI PMC

Jamieson F.B., Teatero S., Guthrie J.L., Neemuchwala A., Fittipaldi N., Mehaffy C. Whole-genome sequencing of the Mycobacterium tuberculosis manila Sublineage results in less clustering and better resolution than Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and Spoligotyping. J Clin Microbiol. 2014;52(10):3795–3798. doi: 10.1128/JCM.01726-14. PubMed DOI PMC

Ford C.B., Shah R.R., Maeda M.K., Gagneux S., Murray M.B., Cohen T., et al. Mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial differences in the emergence of drug-resistant tuberculosis. Nat Genet. 2013;45(7):784–790. doi: 10.1038/ng.2656. PubMed DOI PMC

Reiling N., Homolka S., Walter K., Brandenburg J., Niwinski L., Ernst M., et al. Clade-Specific Virulence Patterns of Mycobacterium tuberculosis Complex Strains in Human Primary Macrophages and Aerogenically Infected Mice. mBio. 2013;4(4) doi: 10.1128/mBio.00250-13. PubMed DOI PMC

Technical Report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis n.d. https://www.who.int/publications/i/item/WHO-CDS-TB-2018.5 (accessed September 8, 2021).

Kohl T.A., Utpatel C., Schleusener V., De Filippo M.R., Beckert P., Cirillo D.M., et al. MTBseq: a comprehensive pipeline for whole genome sequence analysis of Mycobacterium tuberculosis complex isolates. PeerJ. 2018;6:e5895. doi: 10.7717/peerj.589510.7717/peerj.5895/fig-110.7717/peerj.5895/fig-210.7717/peerj.5895/fig-310.7717/peerj.5895/table-110.7717/peerj.5895/supp-110.7717/peerj.5895/supp-2. PubMed DOI PMC

I. Cancino-Muñoz M. Moreno-Molina V. Furió G.A. Goig M. Torres-Puente Á. Chiner-Oms et al. Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis J Infect Dis 220 2 2019 2019 316 320. PubMed PMC

Feuerriegel S., Schleusener V., Beckert P., Kohl T.A., Miotto P., Cirillo D.M., et al. PhyResSE: A web tool delineating Mycobacterium tuberculosis antibiotic resistance and lineage from whole-genome sequencing data. J Clin Microbiol. 2015;53(6):1908–1914. doi: 10.1128/JCM.00025-15. PubMed DOI PMC

Coll F., McNerney R., Preston M.D., Guerra-Assunção J.A., Warry A., Hill-Cawthorne G., et al. Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences. Genome Med. 2015;7(1) doi: 10.1186/s13073-015-0164-0. PubMed DOI PMC

Zhou Z., Alikhan N.-F., Sergeant M.J., Luhmann N., Vaz C., Francisco A.P., et al. GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Res. 2018;28(9):1395–1404. PubMed PMC

Walker T.M., Ip C.LC., Harrell R.H., Evans J.T., Kapatai G., Dedicoat M.J., et al. Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study. Lancet Infect Dis. 2013;13(2):137–146. doi: 10.1016/S1473-3099(12)70277-3. PubMed DOI PMC

Walker T.M., Merker M., Knoblauch A.M., Helbling P., Schoch O.D., van der Werf M.J., et al. A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study. Lancet Infect Dis. 2018;18(4):431–440. doi: 10.1016/S1473-3099(18)30004-5. PubMed DOI PMC

Fonseca J.D., Knight G.M., McHugh T.D. The complex evolution of antibiotic resistance in Mycobacterium tuberculosis. Int J Infect Dis. 2015;32:94–100. doi: 10.1016/J.IJID.2015.01.014. PubMed DOI

Merker M., Nikolaevskaya E., Kohl T.A., Molina-Moya B., Pavlovska O., Brännberg P., et al. Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Beijing Clades, Ukraine, 2015 - Volume 26, Number 3—March 2020 - Emerging Infectious Diseases journal - CDC. Emerg Infect Dis. 2020;26(3):481–490. PubMed PMC

Kovalev SY, Kamaev EY, Kravchenko MA, Kurepina NE, Skorniakov SN. Genetic analysis of Mycobacterium tuberculosis strains isolated in Ural region, Russian Federation, by MIRU-VNTR genotyping n.d. PubMed

E. Tagliani R. Anthony T.A. Kohl A. de Neeling V. Nikolayevskyy C. Ködmön et al. Use of a whole genome sequencing-based approach for Mycobacterium tuberculosis surveillance in Europe in 2017–2019: an ECDC pilot study 2002272 10.1183/13993003.02272-2020 10.1183/13993003.02272-2020.Supp1 10.1183/13993003.02272-2020.Shareable1. PubMed

Siu G.K.H., Yam W.C., Zhang Y., Kao R.Y.T. An upstream truncation of the furA-katG operon confers high-level isoniazid resistance in a Mycobacterium tuberculosis clinical isolate with no known resistance-associated mutations. Antimicrob Agents Chemother. 2014;58(10):6093–6100. doi: 10.1128/AAC.03277-14. PubMed DOI PMC

Makafe G.G., Cao Y., Tan Y., Julius M., Liu Z., Wang C., et al. Role of the Cys154Arg substitution in ribosomal protein L3 in oxazolidinone resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2016;60(5):3202–3206. doi: 10.1128/AAC.00152-16. PubMed DOI PMC

Sun Q., Xiao T.-Y., Liu H.-C., Zhao X.-Q., Liu Z.-G., Li Y.-N., et al. Mutations within embCAB are associated with variable level of ethambutol resistance in Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother. 2018;62(1) doi: 10.1128/AAC.01279-17. PubMed DOI PMC

khan M.T., Malik S.I., Ali S., Masood N., Nadeem T., Khan A.S., et al. Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa. Pakistan. BMC Infect Dis. 2019;19(1) doi: 10.1186/s12879-019-3764-2. PubMed DOI PMC

J M, I P, P K, M D, I S. Detection of resistance to anti-tuberculosis drugs in the clinical isolates of Mycobacterium tuberculosis from Slovakia through comparison between phenotypic and genetic methods and evaluation of resistance levels with clinical parameter. J Physiol Pharmacol 2019;70:105–14. https://doi.org/10.26402/JPP.2019.1.10. PubMed

Coll F., Phelan J., Hill-Cawthorne G.A., Nair M.B., Mallard K., Ali S., et al. Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis. Nat Genet. 2018;50(2):307–316. doi: 10.1038/s41588-017-0029-0. PubMed DOI

Shitikov E.A., Bespyatykh J.A., Ischenko D.S., Alexeev D.G., Karpova I.Y., Kostryukova E.S., et al. Unusual Large-Scale Chromosomal Rearrangements in Mycobacterium tuberculosis Beijing B0/W148 Cluster Isolates. PLoS ONE. 2014;9(1):e84971. doi: 10.1371/journal.pone.0084971. PubMed DOI PMC

Tuberculosis in Ukrainian War Refugees and Migrants in the Czech Republic and Slovakia: A Molecular Epidemiological Study