Source: GETTYSBURG COLLEGE submitted to
USING CRISPR-SEROSEQ TO INVESTIGATE SALMONELLA ECOLOGY AND THE PREVALENCE OF ANTIBIOTIC RESISTANCE DURING POULTRY PROCESSING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1008086
Grant No.
2016-69003-24615
Project No.
PENW-2015-07836
Proposal No.
2015-07836
Multistate No.
(N/A)
Program Code
A4171
Project Start Date
Jun 1, 2016
Project End Date
May 31, 2019
Grant Year
2016
Project Director
Shariat, N.
Recipient Organization
GETTYSBURG COLLEGE
300 N WASHINGTON ST
GETTYSBURG,PA 17325
Performing Department
Biology Department
Non Technical Summary
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.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101040100%
Goals / Objectives
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)
Project Methods
All Salmonella investigated to date (>150 serovars) contain CRISPR loci, which comprise two sequence elements - invariant direct repeats and variant spacers, each ~30 nucleotides in length - that form an array. The majority of arrays contain several spacers, each flanked by a direct repeat on either side. Conservation of some CRISPR elements in all serovars will allow the use of a single primer set to PCR amplify portions of the CRISPR array from a complex sample that contains multiple Salmonella serovars. Since the spacers are variant among serovars, they can be used for serovar identification. Novel to this approach is that the resulting PCR products will be analyzed by next-generation sequencing, which will provide increased sensitivity and depth to our sampling. Resulting sequences will be run through our non-redundant database to distinguish which serovars are present. Importantly, this approach should provide us the ability to detect serovars present at low frequencies within a sample. Initial approaches will test the sensitivity of CRISPR-SeroSeq in samples that contain varying amounts of different serovars (for which we know the sequence of the entire CRISPR array). This will allow us to optimize our PCR and sequencing protocols and to develop our bioinformatics pipeline. These initial approaches will enable us to evaluate the potential success of CRISPR-SeroSeq and is essential before progressing. Following this, we will collect poultry samples from two different stages in processing (immediately after slaughter and in packaged meat) and perform CRISPR-SeroSeq on these samples. To ascertain differential antimicrobial resistance in different serovars, the CRISPR sequence information derived above will allow us to use qPCR approaches to track the survival of each serovar in the presence of a panel of 15 antibiotics routinely analyzed in Salmonella by NARMS.

Progress 06/01/17 to 05/31/18

Outputs
Target Audience: Our target audience includes state and federal agencies, poultry producers and processors, academic agriculture and microbiology research groups, trainees, and the general public Changes/Problems:Obj 2.2 has been a challenege. In our poultry samples, success of this objective has depended on the assumption that the background serovars have different antibiotic resistance profiles to the dominant serovar. We have tried this with multiple probes and conditions without success. As an alternative, we are now using samples collected as part of a different project in cattle where distinct and measurable differences in antibiotic resistance are known between different serovars that are present in different frequencies. What opportunities for training and professional development has the project provided?Two students funded by this project in Year 2 presented research posters at the Allegheny Branch of the American Society for Microbiology (Nov 2017). The student funded in Year 1 and who has worked through the academic year on this project also presented a talk at this meeting and won 2nd place in the Undergraduate Environmental Microbiology section. How have the results been disseminated to communities of interest?This work was accepted for publication in Applied and Environmental Microbiology, therefore is being disseminated to the broad microbiology community. Dr. Shariat, lead PI on this project, has given seminars at multiple universities desribing CRISPR-SeroSeq as a powerful tool over the last year What do you plan to do during the next reporting period to accomplish the goals?Continue assaying the qPCR approach to examine antibiotic resistance Over the next year, we will also be developing a web-based tool to allow users to use CRISPR-SeroSeq.

Impacts
What was accomplished under these goals? Object 2.2 - currently in progress, nothing to report.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Thompson CP, Doak A, Schroeder EA, Amirani N, Wright J, Kariyawasam SK, Lamendella R, and Shariat NW. High-resolution identification of multiple Salmonella serovars in a single sample using CRISPR-SeroSeq (In Press Applied and Environmental Microbiology. Vol 84, issue 21)


Progress 06/01/16 to 05/31/18

Outputs
Target Audience: Our target audience includes state and federal agencies, poultry producers and processors, academic agriculture and microbiology research groups, trainees, and the general public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project was accomplished by undergraduate students. Six undergraduates are co-authors on the papers resulting from this work. And all have presented posters and/or given talks at regional conferences (ASM-Branch), plus one student presented at a national meeting (ASM-Microbe): David Medina: Salmonella populations vary by enrichment broth; Nov 2018 2018 Allegheny Branch of the American Society for Microbiology, Gettysburg College, PA Won 2nd Placein the UndergraduateEnvironmental Microbiology Talks Cameron Thompson: CRISPR-SeroSeq: A new tool for resolving Salmonella serotype diversity; Nov 2017 2017 Allegheny Branch of the American Society for Microbiology, Juniata College, PA Won 2nd Place in the Undergraduate Environmental Microbiology Talks Alexandra Doak: Bioinformatics to probe Salmonella CRISPRs; Nov 2018 2018 Allegheny Branch of the American Society for Microbiology, Gettysburg College, PA Erin Schroeder: Developing a molecular assay for determining antibiotic resistance in low abundance Salmonella; Nov 2017 2017 Allegheny Branch of the American Society for Microbiology, Juniata College, PA Naufa Amirani: Genome assembly and population of a CRISPR Spacer Database for the CRISPR-Seroseq pipeline; Nov 2017 2017 Allegheny Branch of the American Society for Microbiology, Juniata College, PA Dorothy Vosik: CRISPR Typing and antibiotic resistance correlates with polyphyletic distribution in human isolates of Salmonella Kentucky;Jun 2017 2017 American Society for Microbiology-Microbe Meeting, New Orleans, LA Cameron Thompson; CRISPR-SeroSeq: A New Tool for Salmonella Serotyping; Nov 2016 2016 Allegheny Branch of the American Society for Microbiology, Penn State Behrand, Erie, PA. Won joint 1st Placein the Environmental Microbiology UndergraduateResearchPoster Session Dorothy Vosik: Uncovering Salmonella Strain Diversity Using CRISPR-MVLST; Nov 2016 2016 Allegheny Branch of the American Society for Microbiology, Penn State Behrand, Erie, PA How have the results been disseminated to communities of interest?Dr. Shariat has given the following seminars: Salmonella population dynamics: can we measure them and why do we need to? Oct 2018 Seminar, Poultry Research and Diagnostic Center, University of Georgia, Athens, GA CRISPR-SeroSeq: A Novel Amplicon-based Tool For Probing Salmonella Serovar Sep 2018 Diversity Keynote Speaker, American Society for Microbiology Conference on Rapid Applied Microbial Next- Generation Sequencing and Bioinformatic Pipelines, Washington, DC Probing Salmonella serotype diversity in poultry Sep 2018 Arkansas Association for Food Protection, Fayetteville, AR Salmonella CRISPRs reveal phenotypic differences and allow high resolution population May 2018 analyses Seminar, Animal Science Department, University of Connecticut, Storrs, CT Discerning reservoirs of Salmonella in poultry using CRISPR analyses Apr 2018 Seminar, Poultry Research and Diagnostic Center, University of Georgia, Athens, GA Determining Salmonella serotype diversity in multiple environments Apr 2018 Seminar, Plant Sciences & Landscape Architecture Department, University of Maryland, College Park, MD Probing Salmonella Population Diversity using CRISPRs Mar 2018 Graduate Student invited seminar speaker, Department of Biological Sciences, Duquesne University, Pittsburgh, PA Salmonella CRISPRs reveal phenotypic differences and allow high-resolution population Mar 2018 analyses Seminar, Department of Food Science, Cornell University, Ithaca, NY What can CRISPRs tell us about Salmonella diversity and antibiotic resistance? Oct 2017 BASS Seminar, Department of Biology, Gettysburg College, PA CRISPRs: Molecular Barcodes to Trace and Decode Salmonella Diversity Aug 2017 Public Health Seminar Series, Pennsylvania Department of Health, Harrisburg, PA Resolving Salmonella Serovar Diversity Using CRISPR-SeroSeq Jul 2017 USDA-NIFA Project Directors Meeting, Tampa, FL CRISPR-SeroSeq: A New Tool for Resolving Salmonella Serovar Diversity Jun 2017 2017 American Society for Microbiology-Microbe Meeting, New Orleans, LA What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Specific Aim 1: Using CRISPR-SeroSeq to detect lesser prevalent Salmonella serovars poultry samples Objective 1.1 (Completed in Year 1) - We used genomic DNA from Salmonella serovars Enteritidis and Kentucky, which are the top two serovars found in poultry in the United States, to both optimize the CRISPR-SeroSeq PCR step and to determine the sensitivity of CRISPR- SeroSeq PCR step. These PCR products were successfully sequenced, validating the CRISPR-SeroSeq approach, and the 'background' serovar was identified as low as 1:10,000 dilution. Objective 1.2 - (Completed in Year 2-3 (given a one year, no-cost extension to 5/2019) Development of the CRISPR-SeroSeq bioinformatic pipeline. This objective has been accomplished in two major steps: 1) Building a Salmonella CRISPR spacer database 2) Developing a python-based code to parse through sequencing reads. To date, our Salmonella spacer database contains spacer information from 130 serovars. Serovars were selected based on those that are frequently associated with human illness, veterinary-associated serovars, environmental- associated serovars. Serovars for which there are a number of well-sequenced genomes available on NCBI/GenomeTrakr/Pathogen Detection were also included. Between 5-10 isolates or genomes were included for each serovar to ensure capture of CRISPR polymorphisms that exist across different subtypes. Examination of the CRISPR content in serovar Kentucky led to us further characterizing this serovar and demonstrating that its polyphyly is also visualized through the CRISPR content. This was published in our Vosik et al 2018 paper in Foodborne Pathogens and Disease. We developed a python-based bioinformatic pipeline that parses through raw sequence reads and matches perfect (100%) and less-than-perfect (>90%) hits between these sequence reads and spacers in the database. This is accomplished using a local version of BLAST, and read numbers and sequence information is written directly to an Excel file, which ranks the serovars in order of confidence, based on the number of associated sequencing reads. Accurate detection of serovars currently relies on manual curation of the data once written to Excel. This is due to the inherent CRISPR biology and shared or redundant spacers that can exist between some serovars. In continuing work, we are working with collaborators to automate this analysis, based on CRISPR-SeroSeq analyses of individual serovars. Objective 1.3 - (Completed in Year 1) We successfully performed CRISPR-SeroSeq PCR on some test poultry samples collected from Penn State's Poultry and Education Reserch Center. Since Obj. 1.1 worked so well, we did not sequence these PCR samples and instead moved on to Obj. 2.1. Findings from Specific Aim 1 were published in our Thompson et al, 2018. Applied and Environmental Microbiology that detailed the development of CRISPR-SeroSeq as a novel tool to examine Salmonella populations. Specific Aim 2: Use CRISPR-SeroSeq to determine antibiotic resistance in background Salmonella serovars in chickens Objective 2.1 (Completed Year 3) Use CRISPR-SeroSeq to evaluate Salmonella serovar diversity in chickens Successful completion of Objective 2.1 led to two publications: Thompson et al (2018) in Applied and Environmental Microbiology 96 samples were collected from multiple poultry houses (using boot socks and collecting fecal matter), and from two locations within a poultry processing facility (using chicken rinses). Those that tested positive for Salmonella (~20 samples), post enrichment, were analyzed using CRISPR-SeroSeq. All samples contained multiple serovars (up to four serovars), and the most prevalent serovars were Montevideo and Kentucky. For both these serovars, we further separated them each into two different groups, based on their CRISPR spacer content. CRISPR-SeroSeq data was directly compared to traditional serotyping and the most dominant serovar from CRISPR-SeroSeq matched those identified using culture methodology. Cox et al, 2019. Journal of Food Protection (In press) CRISPR-SeroSeq was performed on broiler carcasses from a commercial processing plant. As above, we identified mixed serovar populations in all samples tested. In this study, we directly compared Salmonella serovar profiles between different culture enrichments (TT vs. RV broth) and revealed biases that exist in use of both these broths. Serovar Schwarzengrund was preferentially enriched in RV broth, while serovar Enteritidis was enriched better in TT broth. We also compared standard USDA-FSIS carcass rinse protocols with a prolonged incubation of the carcass in pre-enrichment media and found that more serovars were detected after incubation of the whole carcass. Impactfully, our data from Objective 2.1 demonstrates that mixed serotypes are more common in poultry samples than previously demonstrated. In this work, we have demonstrated an ability to detect serovars that comprise as low as 0.003% of the population. Object 2.2 - (Completed Year 3) Determine fluctuations in serovar diversity in response to antibiotic exposure We established this assay using individual serovars with differential antibiotic resistance. However, our efforts to perform this analysis in poultry samples were unsuccessful, despite designing multiple different primer/probe sets and testing multiple serovars. We think this is likely due to 1) the minority serovar did not have a different AMR profile, or 2) we did not recovery the whole Salmonella population from frozen glycerol stocks when we set up the expt. As an alternative approach, we collaborated with a research group that has characterized Salmonella serovar populations in cattle after antibiotic treatment. CRISPR-SeroSeq demonstrated that an antibiotic resistant serovar was present prior to antibiotic treatment (this serovar was undetected using conventional culture analyses) and that it was enriched after treatment. We cultured pre-treatment mixed cultures with and without tetracycline selection and detected a robust increase in the antibiotic-resistant serovar after selection. Thus, our qPCR assay can detect background serovars with a different AMR profile to the dominant serovar. We are preparing a manuscript that details this tool development. In overcoming this hurdle, the major goal of the project (to develop a tool that can detect low abundance Salmonella with differential AMR profiles), was still addressed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Appl Environ Microbiol. 2018 Oct 17;84(21). pii: e01859-18. doi: 10.1128/AEM.01859-18. Thompson CP, Doak A, Schroeder EA, Amirani N, Wright J, Kariyawasam SK, Lamendella R, and Shariat NW. High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: In press at Journal of Food Protection Cox NA, Berrang ME, House SL, Medina M, Cook K, and Shariat NW. Population analyses reveal pre-enrichment method and selective enrichment media affect Salmonella serovars detected on broiler carcasses.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Foodborne Pathog Dis. 2018 Feb;15(2):101-108. doi: 10.1089/fpd.2017.2298. Vosik D, Tewari, D, Dettinger L, Mikanatha N, and Shariat NW CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky


Progress 06/01/16 to 05/31/17

Outputs
Target Audience:Our target audience includes state and federal agencies, poultry producers and processors, academic agriculture and microbiology research groups, trainees, and the general public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Both students funded by this project in Year 1 presented their research at a scientific conference - Allegheny Branch of the American Society for Microbiology. How have the results been disseminated to communities of interest?Since our Year 1 objectives were to establish and optimize our protocol and to develop a pipeline, and we are in the process of analyzing the data, our results have not been disseminated (beyond the two student poster presentations at a scientific conference). What do you plan to do during the next reporting period to accomplish the goals?For Year 2, we will complete analysis of the poultry Salmonella samples, refine our bioinformatic pipeline and establish a webserver for CRISPR-SeroSeq analysis by other groups. Our goal is to submit a manuscript detailing this work next year. To address Obj. 2.2, we will also design and test multiple qPCR probes to allow us to analyze antibiotic resistance profiles in different background serovars. These will then be used to test the samples collected in Obj. 2.1

Impacts
What was accomplished under these goals? Objective 1.1 - We used genomic DNA from two Salmonella serovars that are prevalent in poultry to optimize the CRISPR-SeroSeq PCR step. These PCR products were successfully sequenced, validating the CRISPR-SeroSeq approach. Objective 1.2 - A primary version of this bioinformatic pipeline is working sufficiently for us to analzye sequence data. We are currently refining and populating our database to optimize this pipeline. Objective 1.3 - We successfully performed CRISPR-SeroSeq PCR on some test poultry samples collected from Penn State's Poultry and Education Reserch Center. Since Obj. 1.1 worked so well, we did not sequence these PCR samples and instead moved on to Obj. 2.1. Objective 2.1 - 96 samples were collected from multiple poultry houses (using boot socks and collecting fecal matter), and from two locations within a poultry processing facility (using chicken rinses). Those that tested positive for Salmonella (~20 samples), post enrichment, were analyzed using CRISPR-SeroSeq. The majority of samples analyzed contained multiple serovars. Importantly, in one sample, we identified four different serovars; and in another sample we were able to detect at least two different strains of the same serovar. We are performing traditional serotyping on colonies to corroborate our apporach with serotyping analysis. Object 2.2 - currently in progress, nothing to report.

Publications