New clustered regularly interspaced short palindromic repeat locus spacer pair typing method based on the newly incorporated spacer for Salmonella enterica

Abstract

A clustered regularly interspaced short palindromic repeat (CRISPR) typing method has recently been developed and used for typing and subtyping of Salmonella spp., but it is complicated and labor intensive because it has to analyze all spacers in two CRISPR loci. Here, we developed a more convenient and efficient method, namely, CRISPR locus spacer pair typing (CLSPT), which only needs to analyze the two newly incorporated spacers adjoining the leader array in the two CRISPR loci. We analyzed a CRISPR array of 82 strains belonging to 21 Salmonella serovars isolated from humans in different areas of China by using this new method. We also retrieved the newly incorporated spacers in each CRISPR locus of 537 Salmonella isolates which have definite serotypes in the Pasteur Institute's CRISPR Database to evaluate this method. Our findings showed that this new CLSPT method presents a high level of consistency (kappa = 0.9872, Matthew's correlation coefficient = 0.9712) with the results of traditional serotyping, and thus, it can also be used to predict serotypes of Salmonella spp. Moreover, this new method has a considerable discriminatory power (discriminatory index [DI] = 0.8145), comparable to those of multilocus sequence typing (DI = 0.8088) and conventional CRISPR typing (DI = 0.8684). Because CLSPT only costs about $5 to $10 per isolate, it is a much cheaper and more attractive method for subtyping of Salmonella isolates. In conclusion, this new method will provide considerable advantages over other molecular subtyping methods, and it may become a valuable epidemiologic tool for the surveillance of Salmonella infections.

FIG 1

The new method is based only on the newly incorporated spacers adjoining the leader array in both CRISPR loci. We used the spacer adjoining the leader array in each CRISPR locus to form a spacer pair to represent each isolate. Spacers and direct repeats were visualized as described by L. Fabre (14) et al.

FIG 2

A minimum spanning tree has been constructed based on CLSPTs using the strains listed in Table 2. In the tree, the corresponding serotypes are circled. CLSPT types are represented by circles, and the size of a circle indicates the number of strains with this particular type. There is no ambiguous result such as one CLSPT type corresponding to two or more serotypes. The halos surrounding the various types denote the groupings obtained by Bionumerics analysis, which indicate that they may be separated from the related phage/plasmid pool. A minimum neighbor difference of 1 was used for the creation of groups.

FIG 3

PFGE dendrogram of 82 Salmonella strains, with strain number, serotype, ST, and PT for each strain.