Whole genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates collected in the Czech Republic, 2005-2020

Abstract

The emergence and spread of resistant tuberculosis (TB) pose a threat to public health, so it is necessary to diagnose the drug-resistant forms in a clinically short time frame and closely monitor their transmission. In this study, we carried out a first whole genome sequencing (WGS)-based analysis of multidrug resistant (MDR) M. tuberculosis strains to explore the phylogenetic lineages diversity, drug resistance mechanisms, and ongoing transmission chains within the country. In total, 65 isolates phenotypically resistant to at least rifampicin and isoniazid collected in the Czech Republic in 2005-2020 were enrolled for further analysis. The agreement of the results obtained by WGS with phenotypic drug susceptibility testing (pDST) in the determination of resistance to isoniazid, rifampicin, pyrazinamide, streptomycin, second-line injectables and fluoroquinolones was more than 80%. Phylogenetic analysis of WGS data revealed that the majority of MDR M. tuberculosis isolates were the Beijing lineage 2.2.1 (n = 46/65; 70.8%), while the remaining strains belonged to Euro-American lineage. Cluster analysis with a predefined cut-off distance of less than 12 single nucleotide polymorphisms between isolates showed 19 isolates in 6 clusters (clustering rate 29.2%), located mainly in the region of the capital city of Prague. This study highlights the utility of WGS as a high-resolution approach in the diagnosis, characterization of resistance patterns, and molecular-epidemiological analysis of resistant TB in the country.

Figure 1

Incidence of tuberculosis and number of deaths during 2005–2020 in the Czech Republic.

Figure 2

Maximum likelihood phylogenetic tree generated from 64 MDR isolates collected in the Czech Republic during the years 2005–2020 together with their lineage, cluster assignment, and genotypic resistance to first- and second-line antituberculosis drugs. The tree was annotated using iTOL v6 (https://itol.embl.de/). The first column denotes the lineages. The next 5 columns show genomic relatedness within clusters (CL12, CL5, CL2 and CL0—sets the maximum number of SNPs that differ from the genetically closest isolate). Mutations encoded resistance are represented by filled circles (presence of mutation) or empty circles (absence of mutation) icons. MDR multi-drug resistant, XDR extensively-drug resistant, drug-resistant (including mono- and poly-resistant).

Figure 3

Minimum spanning tree based on SNP differences between the strains, including the XDR-TB and MDR-TB strains collected in Czech Republic during 2005 and 2020. Maximum distance set to 12 SNPs for linked transmission. Distant matrix generated from MTBseq (version 1.0.2, https://github.com/ngs-fzb/MTBseq_source) and a minimum spanning tree was constructed using GrapeTree software (https://achtman-lab.github.io/GrapeTree/MSTree_holder.html).