WHOLE GENOME SEQUENCING OF MYCOBACTERIUM TUBERCULOSIS ISOLATE: CLINICAL AND EPIDEMIOLOGICAL SIGNIFICANCE
Objective. To perform the first whole genome sequencing (WGS) of Mycobacterium tuberculosis in Belarus and bio-informational assessment of the genome structure and to evaluate the importance for the practical physiology. Materials and methods. The strain of М. tuberculosis 5005 was the studied and it's phenotypic properties were downloaded in GenBank, NCBI (accession number SAMN14150054). The WGS was performed by both MiSeq, Illumina and MiniOn, Nanopore. Genome assembly dated Pebruary, 14, 2020 was carried out by software: SPAdes v. 3.14; A5-miseq v. 20160825; Plye v. 2.7b; Canu v. 1.8. The genome coverage was 200.0x. Results. The sequenced genome of M. tuberculosis 5005 was downloaded in GenBank with the accession number CP049108.1. It was highly identical to the reference strain H37Rv (average nucleotide identity 99.92%) and comprised 4121 genes, 4070 CD's 3786 of which were protein coding. In the genome of M. tuberculosis 5005 deletions (n=29), insertions (n=23), nucleotide replacements - 775, others - 22 were identified - totally 849 mutations. The frequency of single nucleotide polymorphism (SNP) was 17.5 per 100 kbp, of indels - 1.15 per 100 kbp. Basing on the MIRU-VNTR profiles evaluation the strain was typed as Beijing genotype. It was assigned to group one basing on the structure of codon 463 of katG gene and of codon 95 of gyrA gene. Mutations with bases replacements А/T^G/С were more frequent than G/С^-А/Т: 2.15 mutations per 100 kbp of all А+T nucleotides compared to 0.896 per 100 kbp of all G+C bases. The difference in the А/T^^G/С replacements versus G/С^А/Т was based on a higher rate of the Т^С mutations (126 replacements versus 57 events of the С^Т mutations). The shift to a higher frequency of A/T^G/С mutations resulting in high contents of G and С in DNA might have impact on the M. tuberculosis adaptation and survival in UV-light rich environment. The whole genome sequencing of M. tuberculosis 5005 provided accurate data regarding mutations in the gens responsible for resistance to rifampicin, isoniazid, ethambutol, aminoglycosides, ethionamide/protionamide (phenotypic and genetic data of resistance were relevant). Concordance of genetic and phenotypic data regarding susceptibility to linezolid and paraaminosalicylic acid was registered. In spite of mutation presence in genes associated with the resistance, M. tuberculosis 5005 demonstrated susceptibility to the following anti-TB drugs: to the fluoroquinolones (mutations gag61cag, agc284acc, ggc2003gac in gene gyrA were recorded), clofazimine and bedaquiline (G deletion between codons 272-271 in gene pepQ), cycloserine ( mutation cgg278ctg in gene cycA), delamanide (mutation ttt960ttc in gene fgd1). All the mutations recorded in gyrA, pepQ, cycA, fgd1genes may have compensatory effect and be not responsible for resistance formation. Conclusion. The results obtained regarding the M. tuberculosis 5005 genome structure broaden the insight on the genetic structure of M. tuberculosis circulating in Belarus and allow predict the disease course and the therapy outcome.