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

摘要

Salmonella enterica is represented by 2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. S. enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology. Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected. Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample. Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 0.01 of the sample. Poultry is a major reservoir of Salmonella spp., 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 Salmonella spp. 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. Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected. In another sample, reads for the minority serovar comprised 0.003 of the total number of Salmonella spacer reads. The most abundant serovars identified were Salmonella enterica serovars 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 the surveillance of Salmonella species.