CRISPR 2 spacer architecture analysis and virulotyping for epidemiological study of Salmonella enterica subsp. Enterica circulating in northern Thailand (2015 -2017)

Abstract

Background: Salmonella enterica subsp. enterica, particularly serotype S. 4[5],12:i:-, S. Typhimurium, and S. Enteritidis, represents a significant causative agent of diarrhea, particularly impacting children and immunocompromised individuals on a global scale. Molecular typing of Salmonella spp. has a vital role in understand Salmonella epidemiology. Objective: The objective of this study is to utilize CRISPR 2 spacer analysis coupled with multiple-locus variable number tandem-repeat (VNTR) analysis and virulotyping to perform molecular typing and potential subtyping of Salmonella spp. Materials and methods: CRISPR 2 - multiple-locus variable number tandem-repeat (VNTR) analysis, complemented by additional virulotyping, were performed to rapidly characterize those Salmonella isolates including eight unidentified strains. Serotype-specific CRISPR 2 amplicons were subjected to sequencing and the obtained sequences were blasted with corresponding whole-genome sequencing (WGS) data in order to extract CRISPR 2 information, especially the number and sequence of spacers which were then utilized to predict Salmonella serotypes. Moreover, the similar CRISPR 2 spacer architectures to the corresponding WGS offered the prediction of multilocus sequence types (MLST). Results: S. 4,[5],12:i:-, S. Typhimurium, S. Enteritidis, S. Weltevraden, and S. Derby exhibited distinct clustering, while eight unidentified Salmonella serotypes displayed unique CRISPR 2-MLVA profiles. Through subsequent sequence analysis and comparison with publicly available whole-genome sequencing data, serotype-specific CRISPR 2 amplicon lengths and spacer architectures were unveiled, enabling precise prediction of MLST types. Intriguingly, a linear correlation emerged between CRISPR 2 amplicon length (500-2000 bps) and the number of spacers (6-32) across diverse Salmonella serotypes. Critically, the molecular signatures of CRISPR 2 amplicons accurately predicted the identity of eight unknown Salmonella isolates, aligning with conventional serotyping standards. Furthermore, MLST sequences for prevalent S. 4,[5],12:i:-, S. Typhimurium, and S. Enteritidis were unveiled as ST 34, ST 19, and ST 10, respectively. Subtyping of S. 4,[5],12:i:- using the sopE1 procession (a bacteriophage gene) revealed two major subtypes within ST 34. These subtypes encompassed all six virulent genes, including InvA, bcfC, csgA, agfA, sodC1, and gipA, either with sopE1 (N=8) or without sopE1 (N=10). These findings contribute preliminary insights into the genetic diversity and subtyping of S. 4,[5],12:i:-. Conclusion: The combination of CRISPR 2 sequence analysis and virulotyping emerged as a potent epidemiological tool, facilitating the identification of Salmonella serotypes and potentially informative subtypes, thereby aiding in the surveillance, and tracking of Salmonella transmission in northern Thailand.