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.

Figure 1. CRISPR/Cas system structures from 39 available genome sequences for S. enterica and S. bongori.

Two CRISPR loci (CRISPR1 and CRISPR2) are present in all genomes. The CRISPR-associated (cas) genes cas2, cas1, cse3, cas5e, cse4, cse2, and cas3 genes of the “Ecoli” subtype are located between the CRISPR loci. The most frequent structure, A, is represented by S. enterica serotype Typhimurium strain LT2. Structures B to E are represented by S. enterica serotypes Choleraesuis strain SC-B67, Javiana strain GA_MM04042433, Paratyphi B strain SPB7, and S. enterica subsp. arizonae serotype 62:z4,z23:- strain CDC346-86, respectively. Structure F is represented by S. enterica serotype Typhi strain Ty2. Black diamonds represent direct repeats, with colored diamonds indicating spacers. The CRISPR1 locus of serotype Typhi strain Ty2 is enlarged. The primers used to amplify and sequence the CRISPR loci for the spacer inventory are indicated by horizontal arrows.

Figure 2. Multilocus sequence typing and CRISPR spacer content of 34 S. enterica strains and isolates with the antigenic formula 6,7:c:1,5.

Based on additional biochemical characters, we can identify five subserotypes: Choleraesuis sensu stricto, Choleraesuis variant Kunzendorf, Paratyphi C (human-restricted), Typhisuis (pig-restricted) and Decatur. MLST (a) and CRISPR data (b) show that Choleraesuis, Paratyphi C and Typhisuis share a common ancestor, whereas Decatur is made up of at least five unrelated populations. The numbers in panel “a” correspond to the allelic difference between STs. The size of the circle is not correlated with the number of strains with the corresponding ST. The exact name of the spacers and the spacer content of Decatur strains from panel b can be found in Table S2.

Figure 3. CRISPR sizing by PCR for the rapid comparison of Salmonella spp isolates.

Results of PCR amplification for 8 S. enterica serotype Typhimurium isolates collected from the same city during a single week (cluster E in Table S7). Three cases were from the same food poisoning cluster (the food isolate was also tested), whereas the other cases were unrelated.

Figure 4. Overview of the bead-based CRISPOL assay for S. enterica serotype Typhimurium developed here.

The estimated time for each step is based on the testing of 65 isolates in a 96-well plate. The amplification step spans from the preparation of thermolysates to the gel electrophoresis of PCR products.

Figure 5. Dendrogram presentation of the 245 distinct CRISPOL types detected among 2,200 isolates of S. enterica serotype Typhimurium or its monophasic variant of antigenic formula 1,4,,12:i:-.

Black squares indicate presence of the spacer, as detected by the corresponding probe, whereas white indicates an absence of the spacer. For the determination of CRISPOL types (CTs), each of the 68 spacers was treated as a numerical character indicating absence (0) or presence (1 for all spacers except BraB14, for which an arbitrary value of 10 was assigned) in BioNumerics 6.5 software (Applied Maths, Sint-Martens-Latem, Belgium). Similarities between CTs were assessed by calculating the Pearson product-moment, and a dendrogram was constructed by the unweighted pair group method with arithmetic mean (UPGMA). The four SNP-variant spacers targeted by probes 69 to 72 are shown but were excluded from the phylogenetic analysis, as they were not independent. A indicates a group of profiles derived from CT1, the main type of emerging monophasic isolates. B indicates a group of profiles derived from CT21, which is associated with multidrug-resistant DT104 serotype Typhimurium isolates. C indicates a group of serotype Typhimurium isolates of ST36 that may have one or two specific spacers on the leader side of CRISPR1 (BraB14) and CRISPR2 (STMB35).

Figure 6. Distribution of selected CRISPOL types of S. enterica serotype Typhimurium and S. enterica with antigenic formula 1,4,,12:i :- isolated from humans in France between January 1 and July 4 2010.

Over this period, all 1,084 isolates (one per patient) were CRISPOL-typed and two outbreaks were investigated. Outbreak A (≈40 cases) was due to the consumption of a raw milk cheese contaminated with a CT62 highly multidrug-resistant S. enterica serotype Typhimurium strain, whereas outbreak B (≈50 cases) was caused by the consumption of a dried pork sausage contaminated with a CT136 S. enterica 4,12:i:- strain. The third peak, corresponding to CT21, in May was neither detected nor investigated, as CRISPOL typing was carried out retrospectively.