Molecular epidemiological monitoring of the tuberculosis pathogen in the Arkhangelsk region

Introduction. Against the background of improvement of the main epidemiological indicators (morbidity and mortality) for tuberculosis in the Arkhangelsk region, the proportion of newly diagnosed tuberculosis patients with multidrug-resistant pathogen (MDR-TB) increased from 18.7% in 2002 to 33.8% in 2018. The purpose of this study was the genotypic characterization of Mycobacterium tuberculosis strains obtained from newly diagnosed tuberculosis patients in the Arkhangelsk region in 2018. Materials and methods. 89 M. tuberculosis strains isolated in 2018 from newly diagnosed tuberculosis patients were studied. Beijing genotype, its clusters B0/W148 and Central Asian/Russian were determined by PCR detection of the specific markers: IS6110 insertions in the dnaA-dnaN region, mutations in codons 48 of the mutT4 gene (CGG GGG) and 58 of the mutT2 gene (GGA CGA), IS6110 insertions in the Rv2664 region-Rv2665 and Rv1359-Rv1360, substitutions G A in the sigE gene. Non-Beijing strains were spoligotyped. Results. Drug resistance was detected in 41.6% (37/89), MDR — in 33.7% of strains. In 90% (27/30) of MDR strains, resistance to rifampicin and isoniazid was due to rpoB Ser531Leu and katG Ser315Thr mutations. Following M. tuberculosis genotypes were identified: Beijing (67.4%), T (14.6%), Ural (4.5%), Haarlem (4.5%), LAM (2.3%) and CAS1-Delhi (1.1%). Among the Beijing strains, clusters Central-Asian/Russian (60%; 36/60) and B0/W148 (30%; 18/60) prevailed. The majority of MDR strains belonged to the Beijing family (93.3%; 28/30), of which 64.3% (18/28) and 21.4% (6/28) belonged to clusters B0/W148 and Central-Asian/Russian, respectively. Conclusion. In heterogeneous population of the causative agent of tuberculosis in the Arkhangelsk region, the most common strains were those of the Beijing genotype; in 2018 its share increased to 67.4% (40.4% in 1998–1999). Among MDR strains, the proportion of Beijing reached 93.3%, of which more than half (64.3%) belonged to the epidemiologically and clinically significant in Russia cluster B0/W148

Genome-Wide Mycobacterium tuberculosis Variation (GMTV) Database: A New Tool for Integrating Sequence Variations and Epidemiology

Background Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome. Description Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes. Conclusions Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains

Unusual Large-Scale Chromosomal Rearrangements in <i>Mycobacterium tuberculosis</i> Beijing B0/W148 Cluster Isolates

<div><p>The <i>Mycobacterium tuberculosis</i> (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.</p></div

Genotyping and drug resistance data of the B0/W148 strains sequenced in this study.

<p>¹ RIF - rifampicin, INH - isoniazid, EMB - ethambutol, STR - streptomycin, PZA - pyrazinamide, ETH - ethionamide, AMI- amikacin, CAPR - capreomycin, OFL – ofloxacin.</p><p>² B0 designation according to Narvskaya <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Narvskaya1" target="_blank">[6]</a>, W148 according to Bifani <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Bifani1" target="_blank">[14]</a>.</p><p>³ SITVITWEB was used for identification of data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Demay1" target="_blank">[15]</a>.</p><p>⁴ 24 – VNTR: s154, s580, s960, s1644, s2059, s2531, s2687, s2996, s3007, s3192, s4348, s802, s2165, s2461, s577, s2163, s4052, s4156, s424, s1955, s2347, s2401, s3171, s3690 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Supply1" target="_blank">[16]</a>.</p

Southern blot analysis of H37Rv and SP21 MTB strains.

<p>Genomic DNA was digested with <i>Mlu</i>I and hybridized with the fluorescent labeled probes obtained by amplification. The probes are listed at the top of the lanes (from A to H). (A) Hybridization patterns of H37Rv strain. The order of probes corresponds to the order of complementary sequence sites in the genome of H37Rv. (B) Hybridization pattern of SP21 strains. The order of probes corresponds to the order of complementary sequence sites in the genome of H37Rv. (C) Hybridization patterns of SP21 strain. The order of probes is rearranged in accordance with the expected order of complementary sequence sites in the inverted genome (Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971.s005" target="_blank">Text S1</a>). The merged bands from probes complementary to the boundaries of recombination junctions are boxed. M, marker strain Mt14323 (Mycobacterial Reference Laboratory, National Public Health Institute (Turku, Finland)).</p

Results of PCR verification of inversions.

<p>Electrophoregram of PCR products obtained for MTB strains during the amplification with primer sets 1–8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone-0084971-t003" target="_blank">Table 3</a>).(A) SP 21 B0/W148 Beijing strain and (B) SP 5 non-B0/W148 Beijing strain. Lanes 1–8 correspond to primer sets 1–8; M is a marker GeneRuler 100 bp Plus DNA Ladder (Fermentas, SM0324); K- is a negative control.</p

Schematic presentation of duplicated bases flanking the IS<i>6110</i> insertion sites between LCBs II&III and III&IV.

<p>Schematic presentation of duplicated bases flanking the IS<i>6110</i> insertion sites between LCBs II&III and III&IV.</p

Schematic representation of genome rearrangements' and primer design strategy.

<p>Each local collinear block (LCB) corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone-0084971-t002" target="_blank">Table 2</a> and is represented by a different color.</p