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

Abstract

d Salmonella enterica subsp. enterica serovar Newport (S. Newport) is the third most prevalent cause of food-borne salmonellosis. Rapid, efficient, and accurate methods for identification are required to track specific strains of S. Newport during outbreaks. By exploiting the hypervariable nature of virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs), we previously developed a sequence-based subtyping approach, designated CRISPR-multi-virulence-locus sequence typing (CRISPR-MVLST). To demonstrate the applicability of this approach, we analyzed a broad set of S. Newport isolates collected over a 5-year period by using CRISPR-MVLST and pulsed-field gel electrophoresis (PFGE). Among 84 isolates, we defined 38 S. Newport sequence types (NSTs), all of which were novel compared to our previous analyses, and 62 different PFGE patterns. Our data suggest that both subtyping approaches have high discriminatory abilities (>0.95) with a potential for clustering cases with common exposures. Importantly, we found that isolates from closely related NSTs were often similar by PFGE profile as well, further corroborating the applicability of CRISPR-MVLST. In the first full application of CRISPR-MVLST, we analyzed isolates from a recent S. Newport outbreak. In this blinded study, we confirmed the utility of CRISPR-MVLST and were able to distinguish the 10 outbreak isolates, as defined by PFGE and epidemiological data, from a collection of 20 S. Newport isolates. Together, our data show that CRISPR-MVLST could be a complementary approach to PFGE subtyping for S. Newport.

Comparative Analysis of Subtyping Methods against a Whole-Genome-Sequencing Standard for Salmonella enterica Serotype Enteritidis

“…The majority of CRISPR alleles were determined using the WGS data. For the few CRISPR sequences where we were unable to extract the CRISPR sequences, we PCR amplified and sequenced the CRISPR array as previously described (12). To depict the clustering of subtypes determined by CRISPR-MVLST, the binary distribution (presence or absence) of every spacer in CRISPR1 and CRISPR2 and every SNP in fimH and sseL was profiled for each isolate.…”

“…Both MLVA and CRISPR-MVLST have been assessed in Salmonella based on these criteria (7,8,(10)(11)(12)(13). Evaluation of subtyping methods is often conducted through comparisons with PFGE; however, PFGE is not sufficiently discriminatory against clonal organisms such as S. enterica serotype Enteritidis and its utility as a benchmark for other subtyping techniques can be compromised.…”

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

“…Thus, elevated acquisition and turnover of CRISPR spacers of type I-F and I-E CRISPR-Cas systems in Y. pestis and Salmonella spp., respectively, do allow for high-resolution typing. Indeed, a recent study of Salmonella enterica established that CRISPR typing in combination with a method exploiting the hypervariability of the virulence genes, CRISPR-MVLST, was useful for subtyping purposes (41).…”

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].…”