Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are found in ~50% of all bacterial species, including Salmonella. CRISPR elements comprise several unique short sequences, called spacers, which are interspaced
PND-1186 cost by conserved direct repeats. In some bacteria, homology between a spacer and a complementary target nucleic acid results in degradation of the target by sequence-specific endonucleases, providing protection from exogenous bacteriophage or plasmid DNA [reviewed in . Due to both acquisition and loss of these spacer elements, CRISPRs represent arguably the most rapidly evolving prokaryotic loci [29–31]. Sequence analysis of CRISPR loci has been used to subtype clinical isolates of Salmonella[32–34], Escherichia coli[35, 36], group A Streptococcus and Campylobacter species . Salmonella contains two of these non-coding loci, which are comprised of direct selleck repeats of 29 nucleotides separated by spacers of 32 nucleotides (Figure 1). Generally, CRISPR polymorphisms between Salmonella strains are due to deletion or repetition of one or more spacers, termed ‘spacer microevolution’ [32–34, 39, 40]. An extensive investigation of 738 isolates, representing several different serovars, showed that polymorphisms within
the CRISPR loci correlate highly with serovar, with isolates from individual serovars bearing distinct CRISPR patterns . Figure 1 Salmonella CRISPR loci. Salmonella have two CRISPR loci, selleck inhibitor CRISPR1 and CRISPR2 comprised of direct repeats of 29 nucleotides (black diamonds) separated by spacers (empty rectangles). There is an A-T rich leader sequence why upstream of each locus (shaded rectangle) and the CRISPR-associated genes (cas) are upstream of the CRISPR1
locus (grey boxed arrow). Primers used for amplification are shown in blue and red for CRISPR1 and CRISPR2, respectively. We recently developed a sequence-based subtyping assay (multi-virulence locus sequence typing; MVLST) for Salmonella that involves the sequencing of two virulence genes, fimH1 (fimH) and sseL, in addition to CRISPR sequencing . Preliminary studies showed that this approach, termed CRISPR-MVLST, provided better discrimination than either CRISPR or MVLST alone and, importantly, exhibited strong epidemiologic concordance among eight out of nine of the most common illness-causing Salmonella enterica serovars , including both S. Heidelberg and S. Typhimurium outbreak strains. Subsequently, among a large number of clinical isolates of the highly clonal S. Enteritidis, a combination of CRISPR-MVLST and PFGE was required to provide a sufficient discriminatory power . Among a large set of S. Newport clinical isolates, CRISPR-MVLST provides similar discrimination to PFGE . To further determine the functionality of this new subtyping approach, we investigated the discriminatory power of both CRISPR-MVLST and PFGE among a larger and unbiased collection of clinical S. Typhimurium and S.