CRISPR Typing and Subtyping for Improved Laboratory Surveillance of Salmonella Infections

Abstract

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.

Subtyping of Salmonella enterica Serovar Newport Outbreak Isolates by CRISPR-MVLST and Determination of the Relationship between CRISPR-MVLST and PFGE Results

“…Variations in the numbers and compositions of spacers within different CRISPR alleles result in different-sized CRISPR PCR products. By virtue of this observation, it has been suggested and shown by Weill and colleagues that size determination by simple gel electrophoresis of CRISPR amplicons can provide rapid initial typing of S. Typhimurium isolates (27). This screening approach would be particularly valuable during the midst of an outbreak and in public health laboratories in developing countries where the resources for PFGE or other in-depth subtyping techniques do not exist.…”

“…Due to both acquisition and loss of these spacer elements, CRISPRs arguably represent the most rapidly evolving prokaryotic loci (19)(20)(21). Although originally used for spacer oligonucleotide typing, or spoligotyping, for Mycobacterium tuberculosis (22,23), we and others have successfully exploited CRISPR spacer sequence differences for subtyping several different pathogens, including group A Streptococcus (24), Campylobacter species (25), Salmonella (15,(26)(27)(28), and Shiga toxin-producing Escherichia coli (29,30). In Salmonella, CRISPR spacer compositions are highly conserved at the serovar level, suggesting that CRISPR sequence typing alone may be sufficient to detect, identify, and distinguish serotypes (27).…”

“…Although originally used for spacer oligonucleotide typing, or spoligotyping, for Mycobacterium tuberculosis (22,23), we and others have successfully exploited CRISPR spacer sequence differences for subtyping several different pathogens, including group A Streptococcus (24), Campylobacter species (25), Salmonella (15,(26)(27)(28), and Shiga toxin-producing Escherichia coli (29,30). In Salmonella, CRISPR spacer compositions are highly conserved at the serovar level, suggesting that CRISPR sequence typing alone may be sufficient to detect, identify, and distinguish serotypes (27). CRISPR analysis of a limited set of S. Typhimurium isolates has shown that this approach is discriminatory enough for laboratory surveillance of Salmonella infections (27).…”

“…In Salmonella, CRISPR spacer compositions are highly conserved at the serovar level, suggesting that CRISPR sequence typing alone may be sufficient to detect, identify, and distinguish serotypes (27). CRISPR analysis of a limited set of S. Typhimurium isolates has shown that this approach is discriminatory enough for laboratory surveillance of Salmonella infections (27). MVLST is an adaptation of MLST schemes and involves sequence analysis of virulence genes instead of housekeeping genes (31)(32)(33).…”

CRISPR– cas Loci Profiling of Cronobacter Sakazakii Pathovars

“…Consequently, spacer arrays can be used as alternative targets for molecular subtyping and may offer higher strain resolution than MLST and PFGE. Subtyping protocols based on CRISPR-cas systems have been proposed for Salmonella and these have included combined analysis with multi-virulence-locus sequence typing and PFGE [28][29][30]. In contrast, CRISPR typing cannot be used for all E. coli strains as it is absent from the extra intestinal phylogenetic group B2 but can be used for specific identification of enterohemorrhagic and Shiga toxin producing E. coli serotypes [27,31,32].…”