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
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