The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.
В связи с важностью детекции генотипов и генетических подтипов для изучения особенностей циркулирующей популяции M. tuberculosis в условиях демографических изменений и использования новых противотуберкулезных лекарственных средств необходимо разработать методы ускоренной идентификации актуальных для Беларуси генетических типов: Beijing, Beijing B0/W148, а также T (Т1), LAM (LAM 9), U, X, Manu 2, H4.С помощью ПЦР в реальном времени были исследованы изоляты M. tuberculosis (n=250) и распределены на принадлежащие к генотипу Beijing и не относящиеся к нему, последние были изучены с помощью разработанных методов ПЦР в реальном времени на наличие мутаций, специфичных для генотипов Haarlem, TUR, URAL. Проведена оценка возможности использования мутаций Gly594Glu в гене rpoC, Ala1075Thr в гене Rv1009, TCA940TGA в гене Rv1967 для идентификации генотипов Haarlem, TUR, Ural соответственно.Показана способность дифференцировать подтип Haarlem от других генетических вариантов по мутации Gly594Glu (GGG594GAG) в гене rpoC с использованием разработанных праймеров и парных зондов. Показана способность разработанных методов ПЦР в реальном времени дифференцировать мутантные и дикие аллели Rv1009 в 1075 нуклеотиде (GCG1075ACG) и в гене mce3B в 940 нуклеотиде TCA940TGA и на основании наличия мутаций идентифицироватьM. tuberculosis генетических подтипов TUR и Ural.Метод ПЦР в реальном времени может быть использован для быстрой идентификации генетических подтипов M. tuberculosis – Haarlem, TUR, Ural – по мутациям Gly594Glu в гене rpoC, Ala1075Thr в гене Rv1009, TCA940TGA в гене Rv1967 соответственно. Due to the importance of studies on genotyping of Mycobacterium tuberculosis to characterise circulating population of them under demographic changes and the use of new anti-TB drugs, itis actual to develop methods for rapid genotyping of M. tuberculosis than spoligotyping, RFLP- IS6110, MIRU-VNTR. The surveillance of such genotypes and linages as Beijing, Beijing B0 / W148 genotypes, T (T1), LAM (LAM 9), U, X, Manu 2, H4 are important for Belarus.M. tuberculosis isolates (n=250) were studied by real-time PCR and distributed into Beijing and non- Beijing groups; the latter was studied by real-time PCR for the presence of determinants specific for Haarlem, TUR, Ural genotypes such as mutations Gly594Glu in the rpoC gene, Ala1075Thr in the Rv1009 gene, TCA940TGA in the Rv1967 gene respectively. Validity of these mutations for Haarlem, TUR, Ural genotypes detection has been evaluated.The Haarlem subtype can be distinguished from other genetic lineages by the presence of Gly594Glu mutation (GGG594GAG) in the rpoC gene; it was confirmed by real time PCR with developed primers and linear hydrolysis probes. It was shown the ability to differentiate genetic linages TUR and Ural by mutation in the gene Rv1009 in position 1075 nt (GCG1075ACG) and in the mce3B gene in position 940 nt (TCA940TGA).Real-time PCR can be used for the rapid identification of M. tuberculosis genetic linages – Haarlem, TUR, Ural basing on the presence following mutations – Gly594Glu in the rpoC gene, Ala1075Thr in the Rv1009 gene, TCA940TGA in the Rv1967 gene, respectively.
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).
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