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Using Crispr-Seroseq to Investigate Salmonella Ecology and the Prevalence of Antibiotic Resistance During Poultry Processing

Shariat, N.
Gettysburg College
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We plan to use CRISPR-SeroSeq, a novel and high-resolution molecular tool, to detect and identify all Salmonella serovars present in individual poultry samples without the need for isolation of individual colonies, and to investigate the presence of antibiotic resistance in these serovars. If successful, our molecular approach to identify all Salmonella serovars could be implemented into routine Salmonella surveillance protocols. Moreover, with regards to antibiotic resistance, this work will provide a population-wide understanding of antibiotic resistance in Salmonella, which will aid future mitigation strategies. The goals of this proposal are two-fold:1) To provide more accurate estimates as to the presence of background Salmonella serovars in chickens, from harvest through processing.2) To determine the prevalence of different antibiotic resistance patterns in background serovars.

We have two specific aims to accomplish these goals, the objectives and proposed time required are listed below each specific aim. Specific Aim 1: Use CRISPR-SeroSeq to detect lesser prevalent Salmonella serovars poultry samples [Year 1]Objective 1.1 - Establish optimal conditions for CRISPR-SeroSeq PCR (two Students; Quarter 1-2).Objective 1.2 - Development of the CRISPR-SeroSeq bioinformatics pipeline (one Student; Quarter 2-3).Objective 1.3 - Test CRISPR-SeroSeq on poultry samples (three Students; Quarter 3-4).

Specific Aim 2: Use CRISPR-SeroSeq to determine antibiotic resistance in background Salmonella serovars in chickens [Year 2]Objective 2.1 - Use CRISPR-SeroSeq to evaluate Salmonella serovar diversity in chickens during processing (three Students; Quarter 5-6).Objective 2.2 - Determine differential antibiotic resistance profiles in background and dominant serovars (three students; Quarter 6-8).

More information
Salmonella is a leading cause of bacterial foodborne illness in the United States. Since 2012, 31% of outbreaks have been poultry-related, accounting for 46% of salmonellosis cases. Thus Salmonella in poultry poses a major health concern to consumers, plus substantial economic loss for farmers and processors. The presence of antibiotic resistant strains further contributes to the medical financial burden (~$3.6 billion/year). Salmonella enterica, subspecies enterica can be separated into >1500 different serovars, which can differ in pathogenicity and host colonization. Importantly, different serovars can also exhibit varied patterns of antibiotic resistance. Current routine surveillance for Salmonella during poultry processing only identifies the dominant serovar - that is the serovar that is in greater abundance - due to sampling limitations that include enrichment and isolation of one or two Salmonella-positive colonies. Such a singular approach lacks the resolution to detect rare/background serovars. Subsequently, antimicrobial resistance is only analyzed in dominant serovars. Since different serovars can exhibit different patterns of antimicrobial resistance, without recognizing the entire population of Salmonella in poultry samples, we are unable to determine the full extent of antimicrobial resistance. To effectively mitigate antimicrobial resistance, it is crucial to discern the extent to which elements that confer resistance exist within the Salmonella reservoir. Without a clear picture of serovar diversity plus corresponding profiles of antimicrobial resistance, current mitigation strategies may be futile. The extent of multiple serovars present throughout poultry processing is not known, though recent studies have showed that there are links between Salmonella found on the farm and in processing facilities and, furthermore, that multiple serovars have been detected in some broiler flocks. CRISPRs are dynamic genetic loci present in all Salmonella analyzed to date that, at the sequence level, show distinct serovar-specific patterns. CRISPR-SeroSeq is a molecular technology that exclusively combines the resolution and sensitivity of next-generation sequencing with the ability to identify individual Salmonella serovars based on their unique CRISPR DNA profiles. Thus, this approach will be able to detect background serovars present at several orders of magnitude lower than the dominant serovar. In this proposal, we intend to use CRISPR-SeroSeq to determine the population of serovars in samples at two different steps during poultry processing (before and after intervention). From a Salmonella surveillance perspective this molecular tool will provide the ability to scan complex samples and determine Salmonella diversity, at a higher resolution than has previously been possible. CRISPR-SeroSeq could also potentially be used to monitor to success of serovar-specific intervention techniques that may be used. Though such detection is useful in and of itself, our goal in this proposal extends further: we will utilize the sequence-specific serovar information contained within CRISPR sequences to reveal the antimicrobial resistance profiles of background serovars. Ultimately, we intend to determine population-wide antimicrobial resistance profiles by examining several samples during poultry processing. We expect our findings to be valuable when considering implementation of serovar-specific interventions as well as mitigation strategies for antimicrobial resistance in the poultry industry.
Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Natural Toxins
Meat, Poultry, Game