WHOLE GENOME SEQUENCING OF Mycobacterium tuberculosis LAM GENOTYPE STRAIN WITH MULTIPLE DRUG RESISTANCE

Objective: to perform whole-genome sequencing (WGS) of Mycobacterium tuberculosis and evaluate its importance for practical phthisiology. Materials and methods. We studied M. tuberculosis strain 4860, which phenotypic properties were in GenBank, NCBI (accession number SAMN14598146). High-throughput genome sequencing was carried out using MiSeq (Illumina) and MinION (Nanopore) platforms. A genome assembly using the software Flye v. 2.7b; Unicycler v. 0.4.9b was performed on April 10, 2020. A genome coverage was 200x. Results. The sequenced genome of M. tuberculosis 4860 was downloaded to GenBank with accession number CP053092.1. It was highly identical to reference strain H37Rv (the average nucleotide identity was 99.96%) and comprised 4121 genes, 4121 reading frames, including 3972 protein-coding genes. GC-content was 65.62%. The frequency of mutations was 35.89 per 100 kbp, indels – 6.99, while the previously sequenced M. tuberculosis strain 5005 (NCBI, accession number CP049108) referred to Beijing genotype B0/W148 had a higher frequency of mutations and indels, 45.97 and 12.70 per 100 kbp respectively. The most frequent dinucleotides were CG, GC, СС, GG, which accounted for 12.73; 11.49; 9.41; 9.22% respectively; the share of others ranged from 1.69% (ТА) to 6.29% (TG). Based on the MIRU-VNTR profile, the strain was typed as LAM genome. It was established that in M. tuberculosis 4860 А/Т → G/С mutations were more frequent than G/С → А/Т substitutions. The resistome analysis revealed that M. tuberculosis 4860 did not have mutations associated with resistance to amikacin (rrs, eis), ethionamide (ethA, ethR, inhA), PAS (folC, dfrA,thyX, ribD), linezolid (rrl, rplC), bedaquiline (atpE, pepQ, Rv0678), clofazimine (pepQ), or fluoroquinolones (gyrA, gyrB). M. tuberculosis 4860 had mutations associated with resistance to rifampicin, isoniazid, ethambutol, cycloserine, and pyrazinamide. Conclusion. The data on the whole genome structure of M. tuberculosis 4860 have demonstrated that high-throughput next generation sequencing techniques enable to evaluate M. tuberculosis resistance to TB drugs and assess similarities with strains from other geographical regions by genotyping.