Novel macroarray-based method of Corynebacterium diphtheriae genotyping: evaluation in a field study in Belarus

Abstract

In this study, we evaluated a novel macroarray-based spoligotyping method for Corynebacterium diphtheriae strain typing. A total of 20 C. diphtheriae biotype gravis toxigenic isolates collected in Belarus from suspected foci of diphtheria infection (diphtheria cases, carriers, or contacts) were subjected to DNA fingerprinting. All strains had an identical ribotyping profile that was identified as ribotype 'Rossija' by comparison with the international ribotype database at the Institut Pasteur of Paris. A spoligotyping method based on simultaneous reverse-hybridization analysis of two CRISPR (clustered, regularly interspaced short palindromic repeats) loci differentiated these strains into three spoligotypes. Comparison of the spoligotyping results with the epidemiological linkage network helped us to resolve suspected links in the chains of transmission. To conclude, the C. diphtheriae spoligotyping method demonstrated its utility in the field study, in particular, underlining the importance of the use of both CRISPR loci. The generated discrete data can be presented in digital binary format and be easily exchanged between laboratories and stored in local and global databases.

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

“…Louwen et al thus established that not only the spacer variation of the CRISPR array but also single nucleotide polymorphisms (SNPs) of the cas genes in C. jejuni are useful for typing purposes (30). Likewise, in Corynebacterium diphtheriae, the CRISPR-Cas systems (type I-E and type II-C) were found to be useful for typing purposes (32,33). Remarkably, spoligotyping, a technique making use of sequence information contained within the CRISPR spacers, was found to provide enhanced discriminatory power in C. diphtheriae compared to pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and AFLP analyses (33).…”

“…Likewise, in Corynebacterium diphtheriae, the CRISPR-Cas systems (type I-E and type II-C) were found to be useful for typing purposes (32,33). Remarkably, spoligotyping, a technique making use of sequence information contained within the CRISPR spacers, was found to provide enhanced discriminatory power in C. diphtheriae compared to pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and AFLP analyses (33). Furthermore, spoligotyping had discriminatory power for subtyping of Legionella pneumophila strains and enabled the identification of environmental sources that caused clinical outbreaks (34).…”

Legionella pneumophila Sequence Type 1/Paris Pulsotype Subtyping by Spoligotyping

“…The diversity of CRISPR spacer sequences has been used as the basis for genotyping several pathogens, and these spoligotyping methods have demonstrated comparable in index of discrimination values to those of the gold standard genotyping methods, such as PFGE, multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP), or multiple-locus variable-number tandem-repeat analysis (MLVA), demonstrating the usefulness of this method for typing purposes (14,16,17,20). In this study, using the genome of the Paris CIP107629 reference strain, we explored the diversity of CRISPR spacers and developed a spoligotyping tool that was able to discriminate L. pneumophila ST1/Paris pulsotype isolates that were formerly undistinguishable with PFGE and SBT (5).…”

“…ST1/Paris pulsotype isolates have also been detected in clinical and environmental samples from several other countries around the world, including Switzerland, Italy, Spain, Sweden, the United States, Japan, Senegal, and Canada (1, 4). The high isolation rate of this strain in clinical and environmental samples makes it difficult, and frequently impossible, to identify the environmental source of an infection during epidemiological investigations.Recent studies have demonstrated the value of using the diversity of CRISPR spacers as genotyping markers for several pathogenic agents, and spoligotyping tools have been successfully developed for this purpose (14,17,19,20). The aim of this study was to design the first spoligotyping tool for subtyping L. pneumophila ST1/Paris pulsotype isolates and to evaluate its performance and efficiency on a collection of clinical and environmental isolates from France.…”

“…Recent studies have demonstrated the value of using the diversity of CRISPR spacers as genotyping markers for several pathogenic agents, and spoligotyping tools have been successfully developed for this purpose (14,17,19,20). The aim of this study was to design the first spoligotyping tool for subtyping L. pneumophila ST1/Paris pulsotype isolates and to evaluate its performance and efficiency on a collection of clinical and environmental isolates from France.…”