Efficient Discrimination within aCorynebacterium diphtheriaeEpidemic Clonal Group by a Novel Macroarray-Based Method

Abstract

The diphtheria epidemic in Russia and neighboring countries in the 1990s (140,000 cases, 4,000 deaths in 1991 to 1996 [39]) stimulated research activities on Corynebacterium diphtheriae, a causative agent of the disease. A number of the typing methods available at that time (multilocus enzyme electrophoresis [MLE], pulsed-field gel electrophoresis [PFGE], ribotyping, and randomly amplified polymorphic DNA [RAPD] analysis) and newer methods (amplified fragment length polymorphism analysis) were applied for interstrain differentiation of the pathogen (6,7,8,20,22,23,28,29,33,36,40). These methods allowed the identification of a clonal group of closely related strains responsible for the epidemic in Russia and all other countries of the former Soviet Union and to trace strains exported into other countries (6,22,23,32,33). These strains were indistinguishable by PFGE, RAPD analysis, and amplified fragment length polymorphism analysis and very similar by ribotyping (there were two principal profiles, "Rossija" and "Sankt-Peterburg," which differed by one band [6, 14, 33]). Minor rare variants were identified by RAPD and ribotyping techniques (22), and a total of 27 types similar by Ͼ80% were identified by MLE typing of all strains of this clonal group studied to date (32, 33). However, MLE, PFGE, and ribotyping are time-consuming and rather cumbersome methods, while RAPD analysis lacks interlaboratory reproducibility and hence exchangeability of results. To identify and rapidly monitor subtle changes in the genome structure at an infraclonal level during and between epidemics, fast, simple, portable, and discriminatory molecular typing methods of C. diphtheriae are still needed.Repetitive genome sequences present important sources of intraspecies variation. A new family of such loci (clustered, regularly interspaced, short palindromic repeats [CRISPR]) has recently been identified by in silico analysis of many bacterial species (19). This family is characterized by direct repeats (DR) varying in size from 21 to 37 bp, interspaced by similarly sized nonrepetitive sequences (variable spacers). DR and adjacent variable spacers form direct variant repeats (DVR) (20). The DNA reverse-hybridization method was developed to study variation in the Mycobacterium tuberculosis DR locus (the presence or absence of 43 different spacers) by using the macroarray format; this method was named "spoligotyping" (spacer oligonucleotide typing [20]) and has been widely used for epidemiological and phylogenetic purposes (12,20,35).In 2003, a complete genome sequence of the C. diphtheriae epidemic strain of biotype gravis ribotype Sankt-Peterburg was published (4). This publication made possible a more thorough, precise, and comprehensive search of candidate polymorphic loci for the development of new typing methods for this pathogen. In the present study, we identified in silico a large DR region in the genome of C. diphtheriae and developed a reverse-hybridization macroarray-based method to study its polymorphism. Using this method, we e...

Multilocus Sequence Typing Identifies Evidence for Recombination and Two Distinct Lineages of Corynebacterium diphtheriae

“…As with AFLP, PFGE, and RAPD studies (3,4,5), MLST was unable to differentiate between the two predominant ribotypes associated with the FSU outbreak: Sankt-Peterburg and Rossija. Spoligotyping studies of this epidemic clonal group showed a clear divergence between these two ribotypes and suggested that the Rossija ribotype may have originated from one particular subpopulation of ribotype Sankt-Peterburg (21,22). Likewise, it is clear by MLST that the Vladimir and Lyon ribotypes are clonally derived (eBURST group 5), as are the Cluj and Gatchina ribotypes (eBURST group 11).…”

“…Traditionally these techniques were based upon serologic, phage, and biotyping methods. However, since the methods provide limited resolution, molecular typing techniques, including amplified fragment length polymorphisms (AFLP) (4), random amplified polymorphic DNA (RAPD) (3,24), multilocus enzyme electrophoresis (MEE) (28), spoligotyping (21), and pulsed-field gel electrophoresis (PFGE) (5), have been developed and show significant intraspecies genetic diversity. Recently a comparison of the different typing techniques was performed (6).…”

CRISPRs: Molecular Signatures Used for Pathogen Subtyping

“…Traditional spoligotyping has also been applied to Corynebacterium diptheriae (61)(62)(63) and Legionella pneumophila (64). Since not all Legionella spp.…”

The Family Corynebacteriaceae

“…The method was evaluated with 154 clinical strains of Corynebacterium diphtheriae previously identified as belonging to the ribotypes Sankt-Peterburg and Rossija. The 154 strains were subdivided into 34 spoligotypes, comprising 14 unique strains and 20 types shared by 2-46 strains (Mokrousov et al 2005). Therefore, spoligotyping allows efficient discrimination within an epidemic clonal group of Corynebacterium diphtheriae and is applicable to phylogenetic reconstruction and epidemiological investigations (Mokrousov et al 2005(Mokrousov et al , 2009Trost et al 2012).…”

“…Therefore, the MLST scheme provides a valuable tool for monitoring and characterizing Corynebacterium diphtheriae strains . Moreover, a macroarray-based spoligotyping method was evaluated for Corynebacterium diphtheriae strain typing (Mokrousov et al 2005(Mokrousov et al , 2007(Mokrousov et al , 2009. The spoligotyping method is based on simultaneous reverse-hybridization analysis of two clustered regularly interspaced short palindromic repeats (CRISPR) loci present in the genome of Corynebacterium diphtheriae.…”