High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq

Abstract

is represented by >2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. is the leading bacterial cause of foodborne illness in the United States with well-established detection methodology. Current surveillance protocols rely on characterization of a few colonies to represent an entire sample, thus minority serovars remain undetected. contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer content of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq, to identify multiple serovars present in a single sample. Using mixed genomic DNA from two serovars, we were able to confidently detect a serovar that constituted 0.01% of the sample.Poultry is a major reservoir for, including serovars that are frequently associated with human illness, as well as those that are not. Numerous studies have examined the prevalence and diversity of in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars. CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility. 91% of samples harbored multiple serovars, including one where four different serovars were detected.In another sample, reads for the minority serovar comprised 0.003% of the total number of spacer reads. The most abundant serovars identified were Montevideo, Kentucky, Enteritidis, and Typhimurium. CRISPR-SeroSeq also differentiated between multiple strains of some serovars. This high resolution of serovar populations has the potential to be utilized as a powerful tool in surveillance of is the leading bacterial cause of foodborne illness in the United States and is represented by over 2600 distinct serovars. Some of these serovars are pathogenic in humans, while others are not. Current surveillance for this pathogen is limited by detection of only the most abundant serovars, due to the culture-based approaches that are used. Thus pathogenic serovars that are present in a minority remain undetected. By exploiting serovar-specific differences in the CRISPR arrays of , we have developed a highthroughput sequencing tool to be able to identify multiple serovars in a single sample and tested this in multiple poultry samples. This novel approach allows differences in dynamics of individual serovars to be measured and can have a significant impact on understanding the ecology of this pathogen with respect to zoonotic risk and public health.

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

“…Importantly, these conserved sequences comprise the binding sites for the Cas1-Cas2e complex and the integration host factor (IHF), both of which are essential for uptake of new spacers into leader-repeat junctions of type I-E arrays 28,27 . Next, we searched for evidence of preferential acquisition of spacers in the leader-end of IV-A3 CRISPR arrays, a phenomenon described for some CRISPR-Cas systems 29,30,31,32 . However, clustering of related IV-A3 CRISPR loci and a comparison of their spacer contents did not reveal any clear support for this ( Supplementary Fig.…”

CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems

“…The system comprises two major molecular constituents: A set of cas genes and CRISPR arrays ( Figure 1). CRISPR arrays have been widely used in the serotyping of Salmonella according to the variety in the spacers [6,7], and it was shown that the results from CRISPR typing had a good correspondence with whole genome sequence typing [8,9]. Furthermore, CRISPR-multi-virulence-locus sequence typing (MVLST) has been frequently used to subtype Salmonella serovars [10,11].…”

Pig as a reservoir of CRISPR type TST4 Salmonella enterica serovar Typhimurium monophasic variant during 2009–2017 in China

“…The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) typing has been used as a high-resolution typing method of a broad range of bacteria. Thus far, CRISPR typing has been widely used to subtype Salmonella isolates belonging to identical serotypes including Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Pullorum [10][11][12][13][14]. Such studies have demonstrated that CRISPR typing is efficient in discriminating isolates from different sources and time periods.…”