Progress 12/15/16 to 12/14/18
Outputs
Target Audience:Target audience: Purdue University undergraduate and graduate food science students, including two international graduate students and one undergraduate international exchange student who audited the course. Effort: Developed and taught one-credit upper level undergraduate/graduate level discussion and writing-based course which focused on developing skills in literature analysis and scientific writing through peer review of published literature on food safety and development of an original pre-proposal on a food safety research area. This course was attended by five students (three auditing unofficially); three students completed all assignments for the course. The materials for this course have been compiled and placed on the Oliver lab server for future re-use. The PI has also proposed to teach this course in faculty applications. Target audience:Food science community at a major food safety conference Effort: helped develop and convene symposium "A Paradigm Shift in Understanding and Controlling Salmonella of the Future" submitted by the Meat and Poultry PDG for the International Association for Food Protection Annual Meeting 2017. Target audience: Food science students in an international food safety association Effort:Helped develop and present a webinar on "Starting Bioinformatics from Zero" in June 2018. Changes/Problems:1) PI opted to teach a course on proposal writing in food safety vs a course on pathogen adaptation to and survival in stressful conditions, as proposal writing was a more pressing curriculum gap in the food science curriculum. What opportunities for training and professional development has the project provided?This project provided the PI with extensive opportunity to mentor undergrads, leading to increased mentoring capabilities for her, as well as advancing the skills and career prospects of three undergraduate researchers who were involved in research for this proposal during 2017-2018. One undergraduate researcher completed the sanitizer tolerance assays under the PI's supervision during spring 2017. Another undergraduate researcher extended previously completed attachment work by completing nine-day attachment assays during summer 2017. A third undergraduate researcher currently continued in vitro invasion studies for this project through early 2018. Additionally, the PI used the bioinformatics skills developed for this project to train a visiting scholar in RNA-seq data processing and analysis. Additionally, the research from this project allowed the PI to give a research talk at the 2017 International Association for Food Protection Annual Meeting which wonthe Developing Scientist Awards Technical Talk Competition Award for best graduate student oral presentation at IAFP's Annual Meeting. This award and $1500 cash prize recognizes excellent graduate student research and presentation ability. The third aim of this research project provided the PI with the opportunity to develop and teach a course under the mentorship of her adviser. This experience prepared her well for a developing and teaching courses as a faculty member. This project also provided the PI the opportunity to networkand share her research with members of the USDA and CDC, providing invaluable contacts for research going forward in her academic career. How have the results been disseminated to communities of interest?The results of this project were presented to the USDA-FSIS and to the CDC-DFWED via online webinars and in-person seminars. It was also communicated at the International Association for Food Protection via a poster. Titles are as follows: Etter, A.J., Oliver, H.F. July 2018. Outbreak-associated Salmonella Heidelberg have higher baseline expression of genes encoding heat shock proteins, stress tolerance mechanisms, & virulence Systems at 37°C. Poster Presentation. International Association for Food Protection Annual Meeting, Salt Lake City, UT Etter, A.J. May 2018.Salmonella Heidelberg food isolates associated with the 2013-2014 Foster Farms outbreak have enhanced stress tolerance capabilities and increased expression of stress tolerance systems, MDR efflux pumps, and virulence genes. CDC-DFWED seminar series (web presentation). Etter, A.J. and West, A.M. December 2017. Outbreak-associated Salmonella Heidelberg have increased tolerance to stress. Remote USDA-FSIS presentation. Ray, A. J., Oliver, H.F. Virulence genes and multi-drug efflux pumps are differentially expressed in Salmonella Heidelberg exposed to heat shock. Technical talk. International Association for Food Protection Annual Meeting, Tampa, FL. West, A. M., Ray, A.J., and H. F. Oliver. Outbreak-associated Salmonella Heidelberg food isolates have enhanced biofilm formation under stress conditions. Poster presentation. International Association for Food Protection Annual Meeting, Tampa, FL. Ray, A.J. February 2017. Transcriptome of outbreak-associated Salmonella Heidelberg in stationary phase and during heat shock. Talk. USDA-ARS, FSIS Food Safety Meeting. Shepherdstown, WV What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Ultimately, we determined that (i) six Salmonella Heidelberg isolates associated with the foodborne outbreak had enhanced heat tolerance, (ii) one outbreak isolates with enhanced heat tolerance also had enhanced biofilm-forming ability under stressful conditions, (iii) exposure to heat stress increased expression of Salmonella Heidelberg multidrug efflux and virulence genes, and (iv) outbreak-associated isolates are likely transcriptionally primed to better survive processing stresses and potentially to cause illness. This study provides deep analysis of the intrinsic stress tolerance and virulence capabilities of Salmonella Heidelberg that may have contributed to the length and severity of a recent salmonellosis outbreak. Additionally, itprovides a comprehensive analysis of the transcriptomic response to heat stress in Salmonella Heidelberg and compares baseline stationary phase gene expression among outbreak and non-outbreak-associatedisolates. Objective 1:Determine transcriptional basis ofSalmonellaHeidelberg ORFIs' (Outbreak-Related Food Isolate) enhanced heat resistance 1) Activities completed:We evaluated the total gene expression profile of three isolates during stationary phase (control conditions) and heat shock to investigate potential mechanisms of enhanced heat tolerance and compare the baseline level of gene expression in the outbreak-associated isolates compared to SL476. 2) Data collected:Total gene expression profile of threeSalmonellaHeidelberg isolates at 37°C and 56°C, collected in biological triplicates. 3) Summary statistics and results:We analyzed changes in gene expression using DESeq2, with significance set at padj< 0.05 for genes with ≥ 2-fold change in expression between comparison groups. We determined that all testedSalmonellaHeidelberg isolates had significantly higher expression of multiple multi-drug efflux pumps and some virulence genes during heat stress. Heat shock and stress genes were not consistently higher expressed among isolates. Under control conditions, heat-resistantSalmonellaHeidelberg isolates had significantly higher expression of genes encoding stress-resistance and heat-shock proteins, multi-drug resistance genes, and virulence genes during stationary phase than reference strain SL476 did. This suggests that these two heat-tolerant isolates are better prepared to encounter stress than a "normal" isolate like SL476.In summary, the data suggests someoutbreak-associatedSalmonellaHeidelberg isolates may be uniquely primed to survive stress in order to cause illness in humans. 4) Key outcomes:To our knowledge, this is the first such study forSalmonellaHeidelberg (previous RNA-sequencing studies of heat tolerance have been done in less virulent serovars) and will inform future government risk assessments and industry food safety best practices for this serovar. Objective 2:Compare attachment capability, sanitizer tolerance, and invasiveness ofS. entericaORFIs and non-outbreak -related isolates. 1) Major activities completed:Sanitizer tolerance (peroxyacetic acid-based sanitizer) and attachment capacity evaluation of all nineSalmonellaHeidelberg outbreak-associated isolates as well asSalmonellaHeidelberg and Typhimurium reference strains and eight food isolates of Typhimurium and Newport was completed. We completed invasion assays in Caco-2 mammalian cells to determine thein vitroinvasiveness of 4/9SalmonellaHeidelberg outbreak-associated isolates and several reference isolates. We were unable to assess5/9 outbreak-associated isolates due to their extreme gentamicin resistance (>250 µg/ul) and resistance to multiple other antibiotics. 2) Data collected: Attachment was testedat 21°C, 4°C, and 37°C under nutrient rich and nutrient poor conditions at 1, 3, and 5 days. 9-day data was also collected for nutrient-poor conditions at 21°C. Peroxyacetic acid tolerance data was assessed under nutrient rich and nutrient poor conditions at concentrations ranging from 25-250ppm. Invasion assay data was collected for log-phase bacteria in confluent Caco-2 cells. 3) Summary statistics and results: Sanitizer tolerance: None of theSalmonellaisolates grew consistently in greater than 25ppm peroxyacetic acid sanitizer, indicating none of the isolates had enhanced tolerance to this sanitizer. Attachment: Statistical analyses were performed in SAS 9.4. OD600 was log10 transformed to fit a generalized linear mixed model with Proc Glimmix. Analysis of attachment capacity was conducted at 4°Cand 21°C, at both serovar and isolate level (α=0.05). At 21°C in 1/20X TSB, Salmonella Heidelberg attached significantly less than Salmonella Typhimurium, Enteritidis, and Newport (padj < 0.05). At "isolate" level, Salmonella Enteritidis isolate R1-0010 had the highest attachment. Salmonella Heidelberg R1-0002 had the lowest attachment and was significantly lower than all the isolates except SL476 and R1-0005 (pad j<0.05). Heat tolerant Salmonella Heidelberg isolates R1-0006 and R1-0007 did not attach significantly less than Salmonella Enteritidis R1-0010. At 4°C in 1/20X TSB, Salmonella Typhimurium had the highest attachment among the serovars. Salmonella Heidelberg had significantly lower attachment than Salmonella Typhimurium, but it was not significantly different from Salmonella Enteritidis or Newport (padj < 0.05). Additional findings at "isolate" level at 4°C included heat tolerant isolate Salmonella Heidelberg R1-0006 had the highest attachment among all the isolates, while SL476 had the lowest attachment, and Salmonella Typhimurium 14028 had comparable attachment with most Salmonella Heidelberg and Newport isolates. (padj<0.05). Invasion: Data were analyzed with ANOVA with Tukey's test for strain means comparisons in SAS (v. 9.4); significance was defined as padj < 0.05. There were no significant differences in mean invasiveness among the six strains after adjusting for multiple comparisons. Invasiveness ranged from approximately 3% in R1-0009 to just over 1.2% for R1-0006. 4) Key outcomes: The data suggests isolates with high tolerance to one stress are likely to possess increased tolerance to other stresses. Objective 3:Develop and publish a course on mechanisms of bacterial survival and persistence; 1) Major activities completed:The PI developed and taught one-credit upper level undergraduate/graduate level discussion and writing-based course which focused on developing skills in literature analysis and scientific writing through peer review of published literature on food safety and development of an original pre-proposal on a food safety research area. 2) Data collected:student feedback from mid-semester and end of semester evaluations. 3) Summary statistics andresults:This course was attended by five students. Three students completed all assignments for the course; another completed all but the final revision and presentation of his proposal. At the end of the course, the three students who submitted all assignments "strongly agreed" they felt more confident in their ability to write a proposal than they had at the start of the course and that they felt they had a strong understanding of what makes for a strong research proposal. 2/3 students "strongly agreed" they felt confident in their ability to assess the quality of a research proposal and all students "agreed" or "strongly agreed" that their writing had improved from taking the course. 4) Key outcomes: Five students gained increased skill in reading and reviewing scientific literature; four students gained increased skill in reviewing and writing scientific research proposals. The materials for this course have been compiled and placed on a Purdue server forre-use. The PIhas gained invaluable experience and mentoring in how to design a course from the ground.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Etter, A.J., West, A.M., Burnett, J.L., Wu, S.T., Veenhuizen, D., Ogas, R.A., Oliver, H.F. Salmonella Heidelberg food isolates associated with a foodborne outbreak have enhanced stress tolerance capabilities. (Submitted)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Etter, A.J., Oliver, H.F. Outbreak-Associated Salmonella Heidelberg Have
Higher Baseline Expression of Genes Encoding Heat Shock Proteins, Stress
Tolerance Mechanisms, & Virulence Systems at 37°C. Poster Presentation.
International Association for Food Protection Annual Meeting, Salt Lake City, UT
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Ray, A. J., Oliver, H.F. Virulence Genes and Multi-Drug Efflux Pumps Are Differentially Expressed in Salmonella Heidelberg Exposed to Heat Shock. Technical talk. International Association for Food Protection Annual Meeting, Tampa, FL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
West, A. M., Ray, A.J., and H. F. Oliver. Outbreak-associated Salmonella Heidelberg food isolates have enhanced biofilm formation under stress conditions. Poster presentation. International Association for Food Protection Annual Meeting, Tampa, FL.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Etter, A. J. 2017. Determining variation in virulence, persistence, and stress tolerance among Salmonella enterica and Listeria monocytogenes from food and food-associated environments. Department of Food Science. Doctorate. Purdue University, West Lafayette.
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Progress 12/15/16 to 12/14/17
Outputs
Target Audience:Target audience: Purdue University undergraduate and graduate food science students, including two international graduate students and one undergraduate international exchange student who audited the course. Effort: Developed and taught one-credit upper level undergraduate/graduate level discussion and writing-based course which focused on developing skills in literature analysis and scientific writing through peer review of published literature on food safety and development of an original pre-proposal on a food safety research area. This course was attended by five students (three auditing unofficially); three students completed all assignments for the course. The materials for this course have been compiled and placed on the Oliver lab server for future re-use. The PI has also proposed to teach this course in faculty applications. Target audience: Food science community at a major food safety conference Effort: helped develop and convene symposium "A Paradigm Shift in Understanding and Controlling Salmonella of the Future" submitted by the Meat and Poultry PDG for the International Association for Food Protection Annual Meeting 2017. Changes/Problems:PI opted to teach a course on proposal writing in food safety vs a course on pathogen adaptation to and survival in stressful conditions, as proposal writing was a more pressing curriculum gap in the food science curriculum. What opportunities for training and professional development has the project provided?Three presentations, training four undergraduate researchers, learning bioinformatics How have the results been disseminated to communities of interest?Selected results from Aims 1 &2 of this project were presented at the International Association for Food Protection's annual meeting in 2017. Additionally, results from Aim 1were presented at the USDA-ARS, FSIS Food Safety Meeting. Shepherdstown, WV in February 2017. Key results from this project will be presented at an online internal webinar for the USDA FSIS on December 6th in collaboration with Stephanie Defibaugh-Chavez and Alice Green.? What do you plan to do during the next reporting period to accomplish the goals?Invasion assays will be completed and project data submitted for publication
Impacts
What was accomplished under these goals?
Our data suggest that many of our outbreak-associatedSalmonellaHeidelberg are uniquely primed to survive stress in order to cause illness in humans. Additionally,SalmonellaHeidelberg with high tolerance to one stress are likely to tolerate other stresses better. Importantly, antibiotic-resistant isolates are not more likely to be stress tolerant than susceptible isolates.This data will contribute to future risk assessments and regulatory decisions forSalmonellaHeidelberg and may contribute to the development of assays to identify isolates more likely to survive processing stresses and cause outbreaks. In addition to increasing our understanding of outbreakSalmonella enterica, this project employed and trained four undergraduate research assistants. One student was a finalist for an undergraduate poster competition at a major food safety conference for her research from this project. The course in food safety proposal writing taught peer review and proposal writing to five students, included two graduate students and two undergraduate students. Proposal writing is a vital skill for success in science and student feedback indicates this course was successful in building student competency in this skill. ? Objective 1:Determine transcriptional basis ofSalmonellaHeidelberg ORFIs' enhanced heat resistance 1) Activities completed:We evaluated the total gene expression profile of three isolates during stationary phase (control conditions) and heat shock to investigate potential mechanisms of enhanced heat tolerance and compare the baseline level of gene expression in the outbreak-associated isolates compared to SL476. 2) Data collected:Total gene expression profile of threeSalmonellaHeidelberg isolates at 37°C and 56°C, collected in biological replicates. 3) Summary statistics and results:We analyzed changes in gene expression using DESeq2, with significance set at padj< 0.05 for genes with ≥ 2-fold change in expression between comparison groups. We determined that all testedSalmonellaHeidelberg isolates had significantly higher expression of multi-drug efflux pumps, attachment genes, and some virulence genes during heat stress. Under control conditions, heat-resistantSalmonellaHeidelberg isolates had significantly higher expression of genes encoding stress-resistance and heat-shock proteins, attachment genes, and virulence genes during stationary phase than reference strain SL476 did, which would make them more likely to survive exposure to stress and potentially more invasive.In summary, the data suggests our outbreak-associatedSalmonellaHeidelberg isolates may be uniquely primed to survive stress in order to cause illness in humans, but that considerable variation exists among the isolates. 4) Key outcomes:To our knowledge, this is the first such study forSalmonellaHeidelberg (previous RNA-sequencing studies of heat tolerance have been done in less virulent serovars) and will inform future government risk assessments and industry food safety best practices for this serovar. Objective 2:Compare attachment capability, sanitizer tolerance, and invasiveness ofS. entericaORFIs and non-outbreak -related isolates. 1) Major activities completed:Sanitizer tolerance (peroxyacetic acid-based sanitizer) and attachment capacity evaluation of all nineSalmonellaHeidelberg outbreak-associated isolates as well asSalmonellaHeidelberg and Typhimurium reference strains and eight food isolates of Typhimurium and Newport was completed. We arecompleting invasion assays in Caco-2 mammalian cells to determine thein vitroinvasiveness of 4/9SalmonellaHeidelberg outbreak-associated isolates and several reference isolates. We are unable to assess5/9 outbreak-associated isolates due to their extreme gentamicin resistance (>250 µg/ul) and resistance to multiple other antibiotics. 2) Data collected: Attachment was testedat 21°C (room temperature), 4°C (refrigeration temperature), and 37°C (idealSalmonellaconditions) under nutrient rich and nutrient poor conditions at 1, 3, and 5 days. 9-day data was also collected for nutrient-poor conditions at 21°C. Peroxyacetic acid tolerance data was assessed under nutrient rich and nutrient poor conditions at concentrations ranging from 25-250ppm, representing sub-lethal concentrations to approved industry usage. 3) Summary statistics and results:Differences in attachment were analyzed in SAS (v. 9.4) using ANOVA with significance defined at α=0.05. When grown in nutrient poor conditions at 21°C, six outbreak-associatedSalmonellaHeidelberg isolates had significantly higher attachment than reference strain SL476 (p< 0.0001). Further, two outbreak-associatedSalmonellaHeidelberg isolates had significantly higher attachment than SL476 (p<0.0001) when grown in nutrient poor conditions at 4°C. However, outbreak-associatedSalmonellaHeidelberg strains had significantly lower attachment thanSalmonellaTyphimurium strains in nutrient rich conditions at 21°C; (p< 0.0001).Outbreak-associated Salmonella Heidelberg with significantly greater heat tolerance than SL476 also attached significantly better than the reference strain SL476 under stressful conditions. None of theSalmonellaisolates grew consistently in greater than 25ppm peroxyacetic acid sanitizer under either nutrient rich or nutrient poor conditions, indicating none of the isolates had enhanced tolerance to this sanitizer. However, it is important to note that these were ideal conditions for sanitizer functionality and are unlikely to represent actual processing conditions. 4) Key outcomes: The data suggests isolates with high tolerance to one stress are likely to possess increased tolerance to other stresses. Ultimately, many of our outbreak associatedSalmonellaHeidelberg isolates may be uniquely primed to survive stress in order to cause illness in humans. Objective 3:Develop and publish a course on mechanisms of bacterial survival and persistence; 1) Major activities completed:The PI developed and taught one-credit upper level undergraduate/graduate level discussion and writing-based course which focused on developing skills in literature analysis and scientific writing through peer review of published literature on food safety and development of an original pre-proposal on a food safety research area. 2) Data collected:student feedback from mid-semester and end of semester evaluations. 3) Summary statistics andresults:This course was attended by five students (three auditing unofficially). Three students completed all assignments for the course; another completed all but the final revision and presentation of his proposal. At the end of the course, the three students who submitted all assignments "strongly agreed" they felt more confident in their ability to write a proposal than they had at the start of the course and that they felt they had a strong understanding of what makes for a strong research proposal. 2/3 students "strongly agreed" they felt confident in their ability to assess the quality of a research proposal and all students "agreed" or "strongly agreed" that their writing had improved from taking the course. 4) Key outcomes: Five students gained increased skill in reading and reviewing scientific literature; four students gained increased skill in reviewing and writing scientific research proposals. The materials for this course have been compiled and placed on a Purdue server for future re-use. The PI has also intends to teach this course as a faculty member.
Publications
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
West, A. M., Ray, A.J., and H. F. Oliver. Outbreak-associated Salmonella Heidelberg food isolates have enhanced biofilm formation under stress conditions. Poster presentation. International Association for Food Protection Annual Meeting, Tampa, FL.
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2017
Citation:
Etter, A. J. 2017. Determining variation in virulence, persistence, and stress tolerance among Salmonella enterica and Listeria monocytogenes from food and food-associated environments. Department of Food Science. Doctorate. Purdue University, West Lafayette.
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
Ray, A. J., Oliver, H.F. Virulence Genes and Multi-Drug Efflux Pumps Are Differentially Expressed in Salmonella Heidelberg Exposed to Heat Shock. Technical talk. International Association for Food Protection Annual Meeting, Tampa, FL.
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