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

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

Comparative phylogenetic analysis of strains under study and 12 whole genomes from the NCBI database.

<p>Phylogenetic tree based on all SNPs of genomes was constructed using the Neighbor-Joining algorithm. Evolutionary distances were calculated using p-distance method.</p

Genome rearrangements' representation for W-148 Progenitor I like H37Rv, W-148 Progenitor II and W-148 genomes.

<p>Each local collinear block (LCB) I–V is represented by a different color. Upside-down blocks (LCBs II and IV) represent the location of the reverse strand, which means an inversion has occurred. Asterisk indicates a terminus of a replication site. Terminus of a replication site was calculated based on GraphDNA (GC-skew mode) software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Thomas1" target="_blank">[19]</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

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

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

Description of five local collinear blocks (LCBs) between H37Rv and W-148.

<p>- LCB III conserved according to initial mechanistic analysis, but re-arranged according to our hypothesis</p