Corynebacterium diphtheriae spoligotyping based on combined use of two CRISPR loci.

A large diphtheria epidemic in the 1990s in Russia and neighboring countries underlined the importance of permanent surveillance of the circulating and emerging clones of Corynebacterium diphtheriae, and hence there is a need for highly discriminatory, simple and portable typing methods. In the complete genome sequence of C. diphtheriae strain NCTC13129, we previously identified in silico two clustered, regularly interspaced short palindromic repeat (CRISPR) loci, and developed a macroarray-based method to study polymorphism in the larger DRB locus.

Rapid detection of the Mycobacterium tuberculosis Beijing genotype and its ancient and modern sublineages by IS6110-based inverse PCR.

The Mycobacterium tuberculosis Beijing genotype strains appear to be hypervirulent and associated with multidrug-resistant tuberculosis. Therefore, the development of a both rapid and simple method to detect the M. tuberculosis Beijing genotype is of clinical interest per se.

Efficient discrimination within a Corynebacterium diphtheriae epidemic clonal group by a novel macroarray-based method.

A large diphtheria epidemic in the 1990s in Russia and neighboring countries was caused by a clonal group of closely related Corynebacterium diphtheriae strains (ribotypes Sankt-Peterburg and Rossija). In the recently published complete genome sequence of C. diphtheriae strain NCTC13129, representative of the epidemic clone (A.

Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations.

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al.

Phylogenetic reconstruction within Mycobacterium tuberculosis Beijing genotype in northwestern Russia.

A selection of genetic markers was used to study the evolution of Mycobacterium tuberculosis Beijing family strains in northwestern Russia. A total of 221 of 434 epidemiologically unlinked isolates studied in 1996-2001 belonged to the Beijing family as determined by standard spoligotyping (signals 35-43). Ninety-six percent of these Beijing isolates ("typical") were closely related in IS6110-RFLP (D > 0.

Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation.

We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC-->ACC and ACA (Ser-->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inh(r) strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States.

Molecular characterization of multiple-drug-resistant Mycobacterium tuberculosis isolates from northwestern Russia and analysis of rifampin resistance using RNA/RNA mismatch analysis as compared to the line probe assay and sequencing of the rpoB gene.

This investigation evaluated the potential of RNA/RNA mismatch analysis for the detection of rifampin resistance among 38 multiple-drug-resistant (MDR) isolates of Mycobacterium tuberculosis from northwestern Russia. The results obtained were compared with a commercialized line probe assay and rpoB sequencing, and the genetic diversity of the isolates was also investigated in parallel using spoligotyping and variable number of tandem DNA repeats (VNTR). The mismatch analysis revealed 3 distinct RNA cleavage profiles permitting the subdivision of the strains into mutation groups 1 to 3, the most common being group 1 (28 of 38 isolates) that contained a majority of strains with a TCG531>TTG (Ser->Leu) mutation, followed by group 2 (6 of 38 isolates) characterized by different mutations in the codon CAC526 (His), and group 3 (4 of 38 isolates), all characterized by a GAC516(Asp) mutation.

Detection of ethambutol-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting embB306 mutations.

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele.

High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001.

A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC-->ACC (Ser-->Thr), which is associated with INH resistance. This analysis revealed a 93.

Novel IS6110 insertion sites in the direct repeat locus of Mycobacterium tuberculosis clinical strains from the St. Petersburg area of Russia and evolutionary and epidemiological considerations.

A modification of spoligotyping with primers derived from the direct repeat (DR) and IS6110 sequences was used to identify IS6110 insertions in the DR locus of Mycobacterium tuberculosis clinical strains from the St. Petersburg area of Russia. Novel IS6110 insertions were identified: (i) in two epidemiologically unlinked Beijing family strains, an asymmetrical direct insertion in DR37; (ii) in a non-Beijing strain, an asymmetrical insertion in the opposite orientation in DR38; (iii) in another non-Beijing strain, a direct insertion in DR38 and one in the opposite orientation in DR14 (DR numbering is according to standard spoligotyping).