Developing novel technologies and diagnostics

CRISPR SeroSeq

CRISPR-Cas systems can be found in multiple bacterial genomes including Salmonella. Historically bacteria have used these systems as an adaptive immune system through the acquisition of foreign DNA. Salmonella has two CRISPR arrays, CRISPR1 and CRISPR2. These arrays are no longer acquiring new DNA (spacers) and their high conservation within a Salmonella serovar lends them to be excellent molecular serotyping tools.

Because different Salmonella serovars have varying association with human and animal illness incidence and severity (i.e. different serovar pose different risks), being able to detect and distinguish different serovars is important.

It is becoming increasingly evident that Salmonella populations in animals and the environment. These complex populations can be resolved using high resolution deep-serotyping approaches. CRISPR-SeroSeq (Serotyping by sequencing CRISPR) was the first amplicon-based next-generation sequencing tool to detect multiple Salmonella serovars in a a single sample. Conceptually, this is similar to 16S microbiome sequencing, with CRISPR spacers as the target amplicon.

Schematic of CRISPR-SeroSeq deep serotyping approach to profile the relative abundance of multiple serovars in a single sample.

Salmonella qPCR

For targeted diagnostics, qPCR assays can be designed against serovar-specific CRISPR spacers to enable rapid serotyping. We have shown that these work on pure isolates or in mixed cultures containing multiple serovars.

Salmonella PCR

Live attenuated Salmonella serovar Typhimurium vaccines are widely used in industry and are a critical pre-harvest tool for Salmonella control in poultry. We have developed a PCR assay to distinguish between field/native serovar Typhimurium isolates and one commercially available vaccine strain. Where WGS data is available for a serovar Typhimurium isolate, BLAST can be used to determine whether it is the vaccine strain, as shown in the video below:

CRISPR subtyping

Macro-evolution of CRISPR arrays leads to small strain-to-strain differences in CRISPR arrays, typically by absence or duplication of a spacer. These differences can be resolved from CRISPR-SeroSeq reads and also derived from amplification and sequencing of the each array, making them a useful subtyping tool to distinguish between different strains.

Relevant papers:

Raccoursier et al. (2024) In silico and PCR screening for a live attenuated Salmonella Typhimurium vaccine strain. Avian diseases. 68(1), 18–24.

Thompson et al. (2018) High resolution identification of multiple Salmonella serovars in a single sample by using CRISPR SeroSeq. Applied and Environmental Microbiology. 84(21):e01859-18.

Richards et al. (2020) Conserved CRISPR arrays in Salmonella enterica serovar Infantis can serve as qPCR targets to detect Infantis in mixed serovar populations. Letters in Applied Microbiology. 71(2):138-145.

Siceloff et al. (2021) Antimicrobial resistance hidden within multiserovar Salmonella populations. Antimicrobial Agents and Chemotherapy. 65(6):e00048-21.

Shariat et al. (2013) The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis. Food Microbiology. 34(1):164-173.

Vosik et al. (2018) CRISPR typing and antibiotic resistance correlates with polyphyletic distribution in human isolates of Salmonella Kentucky. Foodborne Pathogens and Disease. 15(2):101-108.

Shariat et al. (2015) Characterization and evolution of Salmonella CRISPR-Cas systems. Microbiology. 161(2):374-386.