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 Mycobacterium tuberculosis Beijing B0/W148 cluster isolates.

The Mycobacterium tuberculosis (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.

Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays.

BACKGROUND: The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays. METHODS: The method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested. RESULTS: The sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens. CONCLUSIONS: The combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.

Analysis of mutations in the gyrA and gyrB genes and their association with the resistance of Mycobacterium tuberculosis to levofloxacin, moxifloxacin and gatifloxacin.

The purpose of the present study was to analyse mutations in the gyrA and gyrB genes of Mycobacterium tuberculosis and define the possible correlation between these mutations and resistance to levofloxacin (LVX), moxifloxacin (MFX) and gatifloxacin (GAT), based on their MICs. One hundred and forty-two M. tuberculosis clinical isolates were collected from pulmonary tuberculosis patients in the Moscow region. All M. tuberculosis strains were tested for drug susceptibility to rifampicin and isoniazid using the BACTEC MGIT 960 System and to ofloxacin (OFX) using the absolute concentration method on solid Lowenstein-Jensen slants. All in all, 68 strains were selected at random (38 strains were resistant and 30 were susceptible to OFX) for further analysis using the TB-BIOCHIP-2 test system and DNA sequence analysis. The MICs of LVX, MFX and GAT for selected strains were determined using the BACTEC MGIT 960 System. Mutations in the gyrA gene were observed in 36 out of 38 (94.7 %) OFX-resistant M. tuberculosis strains. Asn538Asp and Asp500His substitutions in the gyrB gene only were found in two (5.3 %) strains. Twenty-nine out of 30 OFX-sensitive M. tuberculosis strains had no mutations in either gene. One (3.3 %) OFX-sensitive M. tuberculosis strain carried an Arg485His substitution in gyrB. The results of our investigation showed that there is no clear correlation between the type of mutation in the genes gyrA and gyrB, and the MIC levels of LVX, MFX and GAT for resistant strains. Mutations in gyrA and Asn538Asp, and Asp500His substitutions in gyrB were associated with cross-resistance of M. tuberculosis to fluoroquinolones. The substitution Arg485His in gyrB does not confer resistance to LVX, MFX and GAT in M. tuberculosis.