A whole genome sequencing was performed of strain M. tuberculosis 11502 (NCBI biosamples database, access code SAMN17832565) that was assigned to the Beijing genotype subtype B0/W148 of cluster 100-32, based on the MIRU- VNTR loci (n = 24) structure, a nd t hat exhibited pre-extended d rug resistance. M. tuberculosis 11502 was resistant to isoniazid, rifampicin, ethambutol, levofloxacin, and ethionamide, which correlated with the presence of mutations in the genes: resistance to isoniazid – the mutations in the fabG1 promoter (p.-8T>C), the katG promoter (p.S315T), to ethionamide – the mutations in ethA (deletion of T at position 4 335 027 (gatgc-gagc)); to fluoroquinolones – in the gyrA gene (p.D94G); to ethambutol – in the embB gene (p.M306I); to streptomycin – in the rpsL gene (p.K43R). M. tuberculosis 11502 genome (Gen- Bank NCBI access code – CP070338) contained 4 420 561 base pairs, 4 104 genes, 4 053 CDSs (coding proteins – 3 874) and differed from reference strain M. tuberculosis H37Rv by the presence of 2 055 mutations. A slight drift of mutations towards the G+C accumulation was revealed, which indicates the importance of maintaining a high G+C content in the Mycobacterium spp.genome Strain M. tuberculosis 11502 has a higher number of mutations in comparison to previously sequenced M. tuberculosis 4860 (GenBank Access Code, NCBI: CP053092) belonging to the LAM genotype (2055 vs. 1577 mutations), which may be a consequence of a longer circulation of M. tuberculosis 11502, or some biological features providing the promutagenic effect.
The variability assessment of PE/PPE genes, as well as of DNA repair, replication, and recombination system genes may drive the concept of mechanisms of Mycobacterium tuberculosis evolution and adaptation.The aim is to study the variability of PE_PGRS genes, 3R-system genes (DNA repair, recombination, and replication) to assess the mechanisms of evolutionary changes in M. tuberculosis.Whole genome sequencing of M. tuberculosis 11502 (the Beijing genotype subtype B0/W148 cluster 100-32), M. tuberculosis 5005 (the Beijing genotype subtype B0/W148), M. tuberculosis 4860 (the LAM genotype) strains was performed. They were isolated from patients with newly diagnosed pulmonary tuberculosis. Genomes were uploaded to the GanBank, NCBI: M. tuberculosis 11502 – access code: CP070338.1, M. tuberculosis 5005 – access code: CP053092.1, M. tuberculosis 4860 – access code: CP049108.1. A reference genome (M. tuberculosis H37Rv; NC_000962.3) was used for genetic analysis. In the M. tuberculosis 11502 genome, 44.4 ± 6.8 % of genes (24 genes out of 54) were revealed in the mutations related to the 3R system, while in M. tuberculosis 4860– 29.6 ± 6.2 % (16 genes out of 54). In the 3R system genes, a slight shift of mutations towards replacement by adenine and thymine was revealed, while the entire genome of M. tuberculosis 11502 (compared to M. tuberculosis H37Rv) demonstrated mutations, resulting in a slight accumulation of G + C. Mutations in the 3R system genes may lead to the suboptimal activity of proteins responsible for the DNA-repair, resulting in the upsurge of mutation frequency and promoting adaptive evolution. PE_PGRS genes in the genome of M. tuberculosis 11502, 4860, and 5005 exhibited a high variability and their variability diverged among different members of this gene family. A high level of tetranucleotides CGGC was found in the majority of PE_PGRS family genes, where their proportion varied from 2.11 to 8.42 %, while an average proportion of CGGC in the M. tuberculosis genome was 1.62 %. Some genes in the M. tuberculosis genome were detected to carry no tetranucleotides CGGC (Rv0011, Rv0100, Rv0460, Rv0616A, Rv0691A, Rv0722, Rv0863, Rv0909, Rv1038c, Rv1197, Rv2347c, Rv2452c, and Rv3330c). The DNA conformation analysis at the mutation sites in the genes, associated with resistance to anti-tuberculosis drugs, showed that the secondary DNA structures were mainly formed by nucleotides CGGC, GCGC, GGG, GGGG, CTGC, and mutations occurred, predominantly, at the sites of forming secondary DNA structures (hairpins) where the redistribution of energy and charges can influence the accuracy of replication and result in replication errors and a mutation event. A number of additional factors can influence the probability of a mutation event. These are the factors that can neutralize the energy changes in the DNA secondary structures, and can affect the accuracy of DNA-repair and replication (mutations in the gyrA gene, in the 3R-system genes).
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.
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