Background Clustered regular interspaced short palindromic repeats (CRISPRs) appear in nearly half of all bacterial genomes. Spacer oligonucleotide typing (spoligotyping) has detected the unique sequences (spacers) found between CRISPR repeats. Polymorphism in presence or absence of spacers provides the basis for spoligotyping Mycobacterium tuberculosis strains. As was recently reported, CRISPRs have an important role in limiting horizontal gene transfer (HGT). Mycobacterium tuberculosis strains that have deleted chromosomal DR regions (CRISPR genetic family members) may potentially be more susceptible to HGT, and eventually acquire new types of antibiotic resistance and new mechanisms for evasion of immune surveillance.
Materials and Methods NMRL has genotyped all new Mycobacterium tuberculosis isolates by spoligotyping and MIRU-VNTR typing (mycobacterial interspersed repeat units – variable number of tandem repeats). Three “missing DR region” tuberculosis cases were found from 1516 genotyped strains during years 2008-2013. Two clinical isolates from each of the 3 “missing DR region” tuberculosis cases, 3 additional isolates with intact DR regions and one control strain were sequenced by NGS and analyzed (bioinformatics) in TSRILJC.
Results and Conclusions NGS showed that the six isolates, from the 3 tuberculosis cases that had not yielded amplicons on spoligotyping, deleted all or most of their DR regions, including repeats and spacers. Some of the genes for CRISPR-associated proteins were also deleted. Surprisingly, each deleted DR region had its own characteristic deletion boundaries. This is the first published evidence of Mycobacterium tuberculosis strains from different MIRU-VNTR lineages showing deletions of their DR regions and ORFs surrounding the DR regions.
