Source: NORTH CAROLINA STATE UNIV submitted to
INVESTIGATIONS OF MECHANISMS MEDIATING CONTAMINATION OF PRODUCE BY LISTERIA, AND PRE-HARVEST RISK FACTORS FOR CAMPYLOBACTER IN TURKEYS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1001619
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 6, 2014
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Project Director
Kathariou, SO.
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Food, Bioprocessing, and Nutrition Sciences
Non Technical Summary
Listeria monocytogenes remains a major contributor to severe illness and death due to foodborne infection in the United States. In 2011, the largest foodborne outbreak in the United States to date involved whole cantaloupe contaminated with Listeria, and several additional recent outbreaks have involved fresh produce. In spite of the clear public health and economic burden associated with Listeria-contaminated produce, our understanding of the underlying mechanisms is currently minimal. Further understanding is critically needed for development of novel intervention strategies to prevent colonization of produce with this pathogen. In this project we will follow microbiological and molecular approaches to elucidate these mechanisms for contamination of produce. The project will result in currently lacking baseline data and knowledge needed to reduce Listeria-associated hazards of fresh produce. Campylobacter is a leading bacterial cause of human foodborne disease, with an estimated 0.8 million cases of illness each year in the United States alone. In addition to acute gastroenteritis, it is associated with potentially severe autoimmune complications. Poultry consumption is a major risk factor for human infections and farm-level control of Campylobacter colonization of poultry will be critical for reductions in human disease burden. However, pre-harvest control of Campylobacter will remain challenging and incomplete as long as we lack a good understanding of how Campylobacter is transmitted to poultry pre-harvest. It is critical that farm-level practices associated with high prevalence of Campylobacter become identified, and that effective training programs are made available to the poultry industry. The current project will combine microbiological surveys, identification of potential pre-harvest risk practices, training and outreach in order to address these needs with a focus on the turkey industry. Turkeys have been understudied in terms of their colonization with Campylobacter, but may contribute importantly to Campylobacter-related food safety risks. Industry practices leading to turkeys free of Campylobacter, or with significantly reduced loads, would promote compliance with government-mandated Campylobacter standards for poultry and also result in a product with reduced potential to compromise food safety and human health.
Animal Health Component
25%
Research Effort Categories
Basic
60%
Applied
25%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The long term goals of the research are (I) to characterize mechanisms mediating produce colonization with Listeria and to design novel control strategies for safer produce and (II) to identify and characterize routes and mechanisms of pre-harvest transmission of Campylobacter to poultry. in order to identify intervention strategies towards poultry with reduced Campylobacter burden. To contribute to these long term goals, the following specific objectives will be pursued: Objective 1: To assess relative produce colonization ability of a panel of strains of Listeria monocytogenes, including representatives of different lineages and sources. Objective 2: To identify and characterize genes of L. monocytogenes required for produce colonization (adherence, survival, growth). Objective 3: To assess the role of the genes on biofilm formation by L. monocytogenes on stainless steel and on produce. Objective 4. Identify farm-level practices that contribute to either consistently low or consistently high prevalence of Campylobacter in turkey flocks pre-harvest. Objective 5. Based on Campylobacter prevalence data and farm management assessments, develop training and outreach materials for the turkey industry. Objective 6. Evaluate impact of research, training and outreach.
Project Methods
Objective 1: To assess relative produce colonization ability of a panel of strains of L. monocytogenes, including representatives of different lineages and sources. To determine possible associations between the ability of Listeria monocytogenes (LM) to contaminate produce and attributes such as lineage, serotype, and strain source, a carefully chosen panel of diverse will be employed. Three different produce types will be used: cantaloupe (rind and flesh), leafy greens (spinach and lettuce) and celery. The spot inoculation approach will be used for inoculations and inoculated produce will be kept at 5oC (28 days), 10oC (ten days) or 25oC (72 h). CFUs from three inoculated pieces at each time point will be determined on selective media (MOX). Data will be analyzed using SAS for analysis of variance and Duncan's multiple range tests to determine significance of differences in regard to serotype, lineage and temperature. Objective 2: To identify and characterize genes of L. monocytogenes required for produce colonization (adherence, survival, growth). We will combine DNA sequencing and the screening of mariner-based mutant libraries to identify genes of LM required for (1) adherence and (2) survival and growth on produce. We will employ two different LM strains in these studies. Bioinformatics analysis of the sequence data (transposon junction sequences) will be performed to identify insertionally inactivated genes over-represented in non-adherent mutants relatively to the pool of mixed mutant libraries used as inoculum ("inoculum pool"). These genes will be candidates for adherence-required genes. Similar bioinformatics analysis will identify mutants in for genes essential for survival and growth on the produce. Choice of genes for further analysis will be based on the statistical strength of the over-representation or under-representation of the insertions relative to their abundance in the inoculum pool. Objective 3: To assess the role of the genes on biofilm formation by L. monocytogenes on stainless steel and on produce. The deletion mutants and their wildtype parental counterparts will be analyzed in biofilms on stainless steel and on produce. Mutant and wild type strains will be grown at 25oC and used to inoculate washed, sterilized stainless steel coupons. Coupons will be incubated (5, 10 and 25oC) for variable lengths of time and bacteria will be enumerated. For biofilms on produce, LM will be inoculated onto cantaloupe rind and spinach leaves as described earlier. To determine the levels of LM in biofilms versus solitary cells, bacteria will be washed off the produce and filtered through a 5 µm polycarbonate filter. Solitary cells will be measured by plating the filtrate onto MOX, and biofilm-associated cells will be determined by sonication of the filter to remove and disperse aggregates, followed by plating onto MOX. Objective 4. Identify farm-level practices that contribute to either consistently low or consistently high prevalence of Campylobacter in turkey flocks pre-harvest We will enroll 10 Campylobacter-negative and 10 Campylobacter-positive farms, each with two successive flocks per year. Each flock will be sampled for Campylobacter (1) at the brooder stage (approx. 5 wks), (2) shortly after placement at the grow-out farm, (3) during the week immediately prior to processing, and (4) after evisceration at the processing plant. Farms will be identified upon consultation with company veterinarians who have precise data on date of flock placement and location of the farms. Campylobacter in fecal droppings will be enumerated and characterized for species (C. jejuni or C. coli), antibiotic susceptibility profiles, and strain type (via PFGE and MLST). Upon our first visit to the farm we will perform a GAPS-like audit with special attention to practices that may place the farm at enhanced risk for contamination of the birds with Campylobacter. Statistical analysis using multivariable logistic regression will be done to identify relationships between high Campylobacter prevalence and specific risk practices identified by the GAPS-like audit of the farms. Similarly, practices with protective effect (associated with low prevalence or absence of Campylobacter) will be identified. Statistical analysis will also be done to assess the relationship between presence and numbers pre-harvest and at the processing plant post-evisceration. Suggestions based on these analyses will be communicated to company veterinarians, farmers and other stakeholders as detailed below. Objective 5. Based on Campylobacter prevalence data and farm management assessments, develop training and outreach materials for the turkey industry. Training materials: Practices enhancing or reducing the risk for Campylobacter will be placed in an order of priority based on their odds ratio scores. These practices will be communicated to stakeholders (company veterinarians, flock technicians, workers and farmers) through training materials and relevant demonstrations. Training material will be placed at all extension offices in the state and will be also mailed to all turkey growers from the company veterinarians' office. In addition, they will be placed on the open-access web page constructed in association with the project. Visits to the individual farms will be done by an Area Extension Agent, who has direct knowledge of the farms and the communities involved. During these visits specific risk practices will be pointed out and possible mitigations will be demonstrated. The Area Extension Agent will also schedule training sessions to groups of growers. Training sessions will include clarification of the risks involved in specific practices, and "how-to" discussions on implementation of the changes in the day-to-day-operation of the farm and the turkey house. Objective 6. Evaluate impact of research, training and outreach Evaluation of project effectiveness. During the early stages, the focus will be on monitoring and formative evaluation efforts. Program records will be used to assess whether key activities and interventions are implemented as intended (e.g., are targets enrolling in workshops). Furthermore, qualitative methods (e.g., interviews) will be used to collect information from stakeholders/targets for the purpose of refining and improving the outreach and extension activities. Near the end of the project, the emphasis will shift to impact evaluation. More structured and quantitative methods (e.g., questionnaires) with appropriate controls will be used to assess effectiveness of the outreach and extension activities. Three main sets of data will be employed for the evaluation: (i) Evaluations (surveys) administered after each training session; (ii) GAPS-like farm audit data before and after administration of training materials and outreach efforts; and (iii) laboratory data pre-harvest and at the processing plant, pre- and post- the training materials and outreach. Additional tools will involve a mail survey and assessment of web page use. Research impact will be evaluated by metrics that include numbers of graduate and undergraduate students who obtain research experience; numbers of undergraduate honors projects, theses or dissertations; numbers of presentations at scientific conferences; numbers of publications and citations in the literature.

Progress 01/06/14 to 09/30/18

Outputs
Target Audience:• Produce industry: contamination of fresh produce remains a major food safety hazard. Relevant stakeholders include farmers, farmworkers, packing plant owners, managers and operators. • The turkey and generally the integrated poultry industry: Campylobacter constitutes a leading food safety risk for poultry. Antimicrobial resistance in Campylobacter is a major public health threat. The poultry industry is interested in preharvest reductions of Campylobacter especially since the implementations of performance standards in carcass rinses and raw poultry parts. There is especially high interest in minimizing use of antimicrobials and reducing antimicrobial resistance in Campylobacter, Salmonella and other zoonotic pathogens. • Agriculture and rural communities: Implication of agricultural food production in disease (Listeria/produce) or through exceeding performance standards in poultry production (Campylobacter, Salmonella) adversely impacts the economic vitality of US agriculture, and can compromise the wellbeing and sustainability of rural communities. • Food safety and public health community: Both Campylobacter and Listeria are associated with severe burden of disease. • Community-consumers: Listeria and Campylobacter contamination of food creates serious health hazards to US consumers. Consumers show keen interest in acquisition of knowledge on how to minimize their pathogen exposure through the food supply. • Knowledge transfer and food safety literacy: High School, undergraduate and graduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including research on (1) molecular genetic and bacteriological attributes mediating Listeria's ability to colonize fresh produce and (2) Campylobacter colonization of turkeys, antimicrobial resistance and ecology. Students in Food Microbiology classes benefited from case studies pertaining to Listeria and Campylobacter. Visiting scientists, postdocs and technical staff furthered their professional development via further experience with molecular and microbiological components of diverse projects involving these foodborne pathogens, and accompanying knowledge dissemination methods. • Scientific community: Project resulted in new knowledge on the microbial ecology and genetics mediating contamination of fresh produce by Listeria; risk factors for colonization of turkey flocks with Campylobacter; emergence, dissemination and persistence of antimicrobial resistance in foodborne pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunities for training and professional development to one technical staff, several High School students from under-represented minority groups, numerous undergraduate and graduate students, and one postdoctoral scholar. Training included sample collection and analysis, pathogen isolations, enumerations, genotyping and molecular characterization, determination of resistance profiles and statistical analysis of microbiological data. Furthermore, the graduate students and the postdoctoral scholar obtained training in whole genome sequence analysis. Efforts to enhance professional development of these individuals included their involvement in the preparation and presentation of findings at laboratory meetings, local, regional and international conferences and workshops, preparation of manuscripts for peer review as well as blogs and website entries for outreach. Students in the classroom acquired experience in case report preparation and video development and delivery. Graduate students and technical staff acquired experience in mentoring and supervision of junior laboratorians working on the project. How have the results been disseminated to communities of interest?Poultry industry: Publications in research journals; Presentations at conferences with heavy industry participation; Blogs and online articles on Campylobacter and associated food safety challenges. Fresh produce Industry: Publications in research journals; Presentations at conferences with heavy industry participation. Research Community: Presentations of posters and peer-reviewed articles; submission of strain genotype (MLST) data to international, open-access databases; Establishment and preservation of strain collections as resource to the scientific community; Submission of whole genome sequence data from Campylobacter and Listeria to open databases such as NCBI; New protocols that can be applied with Listeria, Campylobacter, Salmonella and other bacterial foodborne pathogens. Academic community: Preparation and delivery of classroom modules, videos and other materials; Seminar on Grand Challenges in Food Systems. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The originally-proposed work under Objective 1 was previously completed. Under Objectives 2 and 3, genes implicated in growth of Listeria on fresh produce (cantaloupe rind) were further characterized regarding their roles in adherence and persistence of Listeria on fresh produce, as well as for their potential roles in biofilm formation and virulence, using the Galleria mellonella model. Additional genes were identified by enrichment of transposon mutant libraries and next-generation sequencing in collaboration with Lisa Gorski and William G Miller at USDA-ARS. The findings from these investigations have been published in peer-review journals and presented at conferences, with one manuscript currently under review at FEMS Lett. Microbiol. and one additional manuscript being prepared in collaboration with co-PDs Gorski and Miller. Objectives 3-6 focused on work with Campylobacter and were previously completed. Relevant findings were published in peer-review journals (Applied & Environ. Microbiol., Foodborne Pathogens and Disease) and presented at conferences and industry meetings. One manuscript is being prepared for resubmission to Applied & Environ. Microbiol, and one additional manuscript is being planned for Applied & Environ. Microbiol. We have undertaken and completed the whole genome sequence analysis of a large panel of Listeria and Campylobacter strains related to the work under these objectives. The data are currently being analyzed and the results from the bioinformatic analysis of these genomes will be presented in manuscripts and conferences.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Crespo, MD , JL Grimes, S Kathariou , Cox, NA, Buhr, RJ, Smith, DP. 2018. Turkey Breeder Pathogens-Evaluation of Vertical Transmission of Salmonella and Campylobacter in Breeder Turkeys. Journal of Animal Science and Research. Volume 2 - Issue 2 | DOI: http://dx.doi.org/10.16966/2576-6457.112
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Niedermeyer JA, Ring L, Miller WG, Genger S, Parr Lindsey C, Osborne J, Kathariou S. 2018. Proximity to other commercial turkey farms affects colonization onset, genotypes and antimicrobial resistance profiles of Campylobacter in turkeys: suggestive evidence from a paired-farm model. Appl Environ Microbiol AEM.01212-18.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Price R, Jayeola V, Niedermeyer J, Parsons C, Kathariou S. 2018. The Listeria monocytogenes key virulence determinants hly and prfa are involved in biofilm formation and aggregation but not colonization of fresh produce. Pathogens 7. . pii: E18. doi: 10.3390/pathogens7010018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Bolinger HK, Zhang Q, Miller WG, Kathariou S. Lack of Evidence for erm(B) Infiltration Into Erythromycin-Resistant Campylobacter coli and Campylobacter jejuni from Commercial Turkey Production in Eastern North Carolina: A Major Turkey-Growing Region in the United States. Foodborne Pathog Dis. 2018 Nov;15(11):698-700. doi: 10.1089/fpd.2018.2477.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Parsons C, Lee S, Kathariou S.. Heavy Metal Resistance Determinants of the Foodborne Pathogen Listeria monocytogenes. Genes (Basel). 2018 Dec 24;10(1). pii: E11. doi: 10.3390/genes10010011.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Haddad N, Johnson N, Kathariou S, M�tris A, Phister T, Pielaat A, Tassou C, Wells-Bennik MHJ, Zwietering MH. Next generation microbiological risk assessment-Potential of omics data for hazard characterisation. Int J Food Microbiol. 2018 Dec 20;287:28-39. doi: 10.1016/j.ijfoodmicro.2018.04.015.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lee S, Chen Y, Gorski L, Ward TJ, Osborne J, Kathariou S. Listeria monocytogenes Source Distribution Analysis Indicates Regional Heterogeneity and Ecological Niche Preference among Serotype 4b Clones. MBio. 2018 Apr 17;9(2). pii: e00396-18. doi: 10.1128/mBio.00396-18.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Rantsiou K, Kathariou S, Winkler A, Skandamis P, Saint-Cyr MJ, Rouzeau-Szynalski K, Am�zquita A. Next generation microbiological risk assessment: opportunities of whole genome sequencing (WGS) for foodborne pathogen surveillance, source tracking and risk assessment. Int J Food Microbiol. 2018 Dec 20;287:3-9. doi: 10.1016/j.ijfoodmicro.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parsons, CP, Chen Y, Kucerova Z, Kathariou S. 2018. Emerging IVb-v1 clone (CC554) of Listeria monocytogenes is highly prevalent among strains from suburban black bears (Ursus americanus). American Society for Microbiology General Meeting, Atlanta, GA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kathariou S. 2018 Survival and Virulence of Salmonella in Model Low-Moisture Foods. International Association for Food Protection Annual Conference
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sicun Fan, Derek Foster, Jeffrey Niedermeyer, Sophia Kathariou. 2018. Impact of antimicrobial treatment of cattle onCampylobacter spp. NCSU CALS Graduate Student Poster Symposium. June 26, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Stewart C, Saheli P, Niedermeyer J, Kathariou S. Impact of Plastic Pollution on the Bacterial Pathogen Listeria monocytogenes in a Local Stream, Rocky Branch Creek. NCSU Undergraduate Research Symposium. April 18, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jayeola V, Stone B, Porwollik S, Chu W, McClelland M, Kathariou S. 2018. Identification of novel genes mediating survival of Salmonella under low-moisture conditions. International Association for Food Protection Annual Conference , July 08  11.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parsons, C. Emerging IVb-v1 clone (CC554) of Listeria monocytogenes is highly prevalent among strains from suburban black bears (Ursus americanus). Presented at NCSU Program in Genetics 9th annual Fall Retreat
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parsons, C. Listeria in the wild: insights into a foodborne pathogen. Presented at the NCSU Biolunch series
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parsons, C. Chen, Y., Kucerova, Z. Kathariou, S. Emerging IVb-v1 clone (CC554) of Listeria monocytogenes is highly prevalent among strains from suburban black bears (Ursus americanus). American Society for Microbiology General Meeting, Atlanta GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parsons, C. Chen, Y., Kucerova, Z. Kathariou, S. Emerging clone of the foodborne pathogen Listeria monocytogenes is highly prevalent among strains isolated from suburban black bears (Ursus americanus). Presented at annual NCSU Postdoctoral Research Symposium.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Produce industry: contamination of fresh produce remains a major food safety hazard. Relevant stakeholders include farmers, farmworkers, packing plant owners, managers and operators. The turkey and generally the integrated poultry industry: Campylobacter constitutes a leading food safety risk for poultry. Antimicrobial resistance in Campylobacter is a major public health threat. The poultry industry is interested in pre-harvest reductions of Campylobacter especially since the implementations of performance standards in carcass rinses and raw poultry parts. There is especially high interest in minimizing use of antimicrobials and reducing antimicrobial resistance in Campylobacter, Salmonella and other zoonotic pathogens. Agriculture and rural communities: Implication of agricultural food production in disease (Listeria/produce) or through exceeding performance standards in poultry production (Campylobacter, Salmonella) adversely impacts the economic vitality of US agriculture, and can compromise the wellbeing and sustainability of rural communities. Food safety and public health community: Both Campylobacter and Listeria are associated with severe human disease burden. Community-consumers: Listeria and Campylobacter contamination of food creates serious health hazards to US consumers. Consumers show keen interest in acquisition of knowledge on how to minimize their pathogen exposure through the food supply. Knowledge transfer and food safety literacy: High School, undergraduate and graduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including research on (1) molecular genetic and bacteriological attributes mediating Listeria's ability to colonize fresh produce and (2) Campylobacter colonization of turkeys, antimicrobial resistance and ecology. Students in Food Microbiology classes benefited from case studies pertaining to Listeria and Campylobacter. Visiting scientists, postdocs and technical staff furthered their professional development via further experience with molecular and microbiological components of diverse projects involving these foodborne pathogens, and accompanying knowledge dissemination methods. Scientific community: Project resulted in new knowledge on the microbial ecology and genetics mediating contamination of fresh produce by Listeria; risk factors for colonization of turkey flocks with Campylobacter; emergence, dissemination and persistence of antimicrobial resistance in foodborne pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Project provided opportunities for training and professional development to one technical staff, numerous undergraduate and graduate students, and one postdoctoral scholar. Training included sample collection and analysis, Campylobacter enumerations, speciation and characterization for resistance profiles and genotypes, statistical analysis of microbiological data as well as weather data and data from audits and surveys and weather data. Graduate students and postdoc obtained training in whole genome sequence analysis. Professional development was provided via involvement of these individuals in preparation and presentation of findings at lab meetings, conferences and workshops, preparation of manuscripts for peer review as well as blogs and other material (e.g. social media) for outreach. Graduate students and the technical staff obtained further professional development and networking skills by participation in the face to face meetings with the turkey company veterinarians, other turkey industry staff and various other professional collaborators. Students in the classroom acquired experience in case report preparation and video development and delivery. Graduate students and technical staff acquired experience in mentoring and supervision of junior laboratorians working on the project. Graduate students and technical staff acquired experience in communication of the findings to the turkey company participants and in productively receiving and soliciting feedback from the industry. Graduate students and technical staff promoted their professional development by frequent and regular interactions with the statistician at NCSU, Dr. Jason Osborne. One graduate student acquired experience in weather data analysis and promoted her professional development by close interactions with Dr. Paula Hennon and her staff at the at the Cooperative Institute for Climate and Satellites in North Carolina. How have the results been disseminated to communities of interest?Turkey industry community: Face-to-face meetings with company veterinarians; numerous exchanges with the poultry industry, with themes derived from project; articles in trade journals with wide reach to the poultry and food industry; an on-site seminar that detailed the project's findings, at the headquarters of the turkey integrator that participated in the project; blogs and online articles on Campylobacter and associated food safety challenges and opportunities; presentations at conferences with heavy industry participation. Fresh produce Industry: Publications in research journals; presentations at conferences with heavy industry participation. Research and academic Community: Presentations of posters and peer-reviewed articles, including a book chapter on Listeria virulence and minireview on antimicrobial resistance in Campylobacter; seminars describing project and relevant findings at North Carolina State University and at international conferences; submission of strain genotype (MLST) data to international, open-access databases; preservation of project-derived strain collections and availability of these strains as resource to others; submission of whole genome sequence data from campylobacter and Listeria to open databases such as NCBI. Academic community: Preparation and delivery of project-informed classroom modules, videos and other lecture materials. What do you plan to do during the next reporting period to accomplish the goals?A PhD dissertation is being prepared. Several publications are being prepared. There is continuing statistical analysis of several datasets originating from the project.

Impacts
What was accomplished under these goals? We have completed the originally-proposed work under Objective 1. Under Objective 2, genes implicated in growth of Listeria on fresh produce (cantaloupe rind) have been identified through the screening of mutant libraries by employing an ampicillin selection protocol optimized in our laboratory. Additional genes are being identified by next-generation sequencing of transposon mutant libraries, in collaboration with Lisa Gorski and William G Miller at USDA-ARS. Objective 3 has been completed by the assessment of the potential roles of selected genes on produce colonization and biofilm formation. Biofilm formation has been assessed on polystyrene. On-going work is investigating biofilm formation on stainless steel coupons. Additionally, we investigated the capacity of a bacterium (Exiguobacterium spp.) isolated from fresh, untreated cantaloupe, to inhibit Listeria growth and biofilm formation on polystyrene. The findings from several of these investigations have been published in peer review manuscripts and presented in conferences. Work with Campylobacter under Objective 4 was primarily focused on the statistical analysis of the large body of data obtained from farm surveillance and from reports submitted by farm staff. In addition, Big Data from weather monitoring datasets were collected and analyzed to assess possible impacts of weather on Campylobacter prevalence in the commercial turkey farms. In addition to prevalence data, we analyzed a large body of antimicrobial resistance data from Campylobacter derived from the turkey farms, including isolates from fecal droppings and from flies, which were investigated as a potential insect vector. DNA sequence-based genotypes were determined for a panel of isolates of both fecal and fly origin. Outreach materials under Objective 5 were developed in the form of publications in trade magazines with wide reach to the poultry and food industry. One of the graduate students presented an hour-long seminar of her thesis work to a meeting with four executives and several staff from the major turkey integrator that participated in the thesis project and provided reagents from the farms. In this presentation, which was made at the industry headquarters, focus was placed on the relevance of the findings for the industry and farm-level operations. In addition, the Campylobacter-related website which we designed and continue to maintain has contributed significantly to outreach and dissemination of the findings from our work. The evaluation of the project's research, education and outreach accomplishments (Objective 5) has been completed through the involvement of Dr. Drew Rivers, who served as consultant for the evaluation components.

Publications

  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Kathariou S, Evans P, Dutta. 2017. Strain-Specific Virulence Differences in Listeria monocytogenes: Current Perspectives in Addressing an Old and Vexing Issue. In: Gurtler J., Doyle M., Kornacki J. (eds) Foodborne Pathogens. Food Microbiology and Food Safety. Springer, Cham. DOIhttps://doi.org/10.1007/978-3-319-56836-2_3
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bolinger HK, Carver DK, Kathariou, S. 2017. Reducing Campylobacter: A look at turkey industry management practices. Poultry Times, 63(8), 15-19.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bolinger HK, Mann KM, Carver, D.K., Kathariou, S. 2017 Biosecurity practices in Campylobacter-negative & -positive commercial turkey farms. Meating Place
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: BOLINGER, HANNAH K. 2017. Emerging Trends of Antibiotic Resistance and Risk Factors for Campylobacter spp. in Commercially Produced Turkey Flocks (Under the direction of Dr. Sophia Kathariou). Ph.D. Dissertation, North Carolina State University
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: KIRCHNER, MARGARET K. 2017. Response of the Turkey Gut Microbiome, Campylobacter, and Salmonella to Health-Disease Transitions and Antimicrobial Interventions. M. S. Thesis, North Carolina State University
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Rantsiou K, Kathariou S, Winkler A, Skandamis P, Saint-Cyr MJ, Rouzeau-Szynalski K, Am�zquita A. 2017. Next generation microbiological risk assessment: opportunities of whole genome sequencing (WGS) for foodborne pathogen surveillance, source tracking and risk assessment. Int J Food Microbiol. pii: S0168-1605(17)30500-7. doi: 10.1016/j.ijfoodmicro.2017.11.007
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Parsons C, Costolo B, Brown P, Kathariou S. 2017. Penicillin-binding protein encoded by pbp4 is involved in mediating copper stress in Listeria monocytogenes. FEMS Microbiol Lett. 364(20). doi: 10.1093/femsle/fnx207.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lee S, Ward TJ, Jima DD, Parsons C, Kathariou S. 2017. The arsenic resistance-associated Listeria genomic island LGI2 exhibits sequence and integration site diversity and a propensity for three Listeria monocytogenes clones with enhanced virulence. Appl Environ Microbiol. 83(21). pii: e01189-17. doi: 10.1128/AEM.01189-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Dutta V, Lee S, Ward TJ, Orwig N, Altermann E, Jima DD, Parsons C, Kathariou S. 2017. Genome sequences of Listeria monocytogenes strains with resistance to arsenic. Genome Announc. 5(19). pii: e00327-17. doi: 10.1128/genomeA.00327-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bolinger H, Kathariou S. 2017. The current state of macrolide resistance in Campylobacter spp.: Trends and Impacts of resistance mechanisms. Appl Environ Microbiol. 83(12). pii: e00416-17. doi: 10.1128/AEM.00416-17.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jahanafroozi, M., Parsons, C., Jayeola, V., Kucerova, Z., Kathariou, S. 2017. Tetracycline resistance in outbreak-associate Listeria monocytogenes Environmental Dimensions of Antimicrobial Resistance-4.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Dutta V, Altermann E, Crespo MD, Olson JW, Siletzky RM, Kathariou S. 2017. Identification of a Campylobacter coli methyltransferase targeting adenines at GATC sites. FEMS Microbiol Lett. 364(7). doi: 10.1093/femsle/fnw268.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Gkana EN, Giaouris ED, Doulgeraki AI, Kathariou S, Nychas G-N. 2017. Biofilm formation by Salmonella Typhimurium and Staphylococcus aureus on stainless steel under either mono- or dual-species multi-strain conditions and resistance of sessile communities to sub-lethal chemical disinfection. Food Control 73: 838-846
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Brown, P., Parsons, C., Kathariou , S. 2017. Phenotypic characterization of a putative lantibiotic biosynthesis genomic island in Listeria monocytogenes. American Society for Microbiology, North Carolina Branch.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Parsons, C., Niedermeyer, J., Brown, P., Gould, N., DePerno, C., Strules, J., Mesa, B., Kelly, M., Hooker, M., Chamberlain, M., Olfenbuttel, C., Kathariou, S. 2017. Prevalence and characteristics of L. monocytogenes isolated from black bears, Ursus americanus. American Society of Microbiology Microbe 2017 General Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jayeola, V., Farber, J., Kathariou, S. 2017. Assessment of survival and differential expression of stress response genes of Salmonella in low-moisture foods. International Association for Food Protection (IAFP) Annual Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bolinger, H.K., Kirchner, M., Kathariou, S. 2017. Investigation and outreach to increase public awareness of campylobacteriosis. International Association for Food Protection (IAFP) Annual Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lu, Z., Breidt, F., Niedermeyer, J., Kathariou, S. 2017. Isolation and characterization of a bacteriophage against Salmonella enterica serovar Montevideo. FEMS Microbiology Congress
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sai, K., Parsons, C. Kathariou, S., Tsuji, J. 2017. Host defense against Listeria monocytogenes through RIPK3-MLKL signaling in non-immune cells. Cell Death Meeting, Cold Spring Harbor Laboratory, August 15 - 19, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Niedermeyer, J., Bolinger, H., Li, Y., Kathariou, S. 2017. Challenges and opportunities in class discussions on complex and controversial issues: a case study on ethics disclosure in the food science graduate curriculum. Teaching and Learning Symposium, North Carolina State University


Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Produce industry: The produce industry iskeen on science-based strategies to control Listeria on fresh produce, especially since the 2011 cantaloupe outbreak and subsequent produce-related outbreaks (stone fruit, caramel apple, salad greens, frozen veggies). Relevant stakeholders include farmers, farmworkers, packing plant owners, managers and operators, and persons in the produce farm-to-retail chain. Numerous, economically devastating recalls result from detection of Listeria-contaminated produce. The turkey and generally the integrated poultry industry: This industry is interested in pre-harvest reductions of Campylobacter especially since the implementations of performance standards in carcass rinses and raw poultry parts. There is especially high interest in minimizing use of antimicrobials and reducing antimicrobial resistance in Campylobacter, Salmonella and other zoonotic pathogens. Agriculture and rural communities: Implication of agricultural food production in disease (Listeria/produce) or through exceeding performance standards in poultry production (Campylobacter, Salmonella) adversely impacts the economic vitality of US agriculture. It also severely compromises the wellbeing and sustainability of agricultural communities. Food safety and public health: The mission of these stakeholders includes reducing prevalence of human listeriosis and campylobacteriosis for which food products and production practices investigated in our research program play major roles. Community-consumers: Listeria and Campylobacter contamination of food creates serious health hazards to US consumers. Consumers show keen interest in acquisition of knowledge on how to minimize their pathogen exposure through the food supply. Knowledge transfer and food safety literacy: High School, undergraduate and graduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including research on (1) molecular genetic and bacteriological attributes mediating Listeria's ability to colonize fresh produce and (2) Campylobacter colonization of turkeys, antimicrobial resistance and ecology. Students in Food Microbiology classes benefited from case studies pertaining to Listeria and Campylobacter. Visiting scientists, postdocs and technical staff furthered their professional development via further experience with molecular and microbiological components of diverse projects involving these foodborne pathogens, and accompanying knowledge dissemination methods. Scientific community: Project resulted in new knowledge on the bacteriology, microbial ecology and genetics mediating contamination of fresh produce by Listeria; risk factors for colonization of turkey flocks with Campylobacter; emergence and dissemination of antimicrobial resistance in these foodborne pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Six undergraduate and 12 graduatestudents acquired research experienceworking on the project during the pastyear, together with 6 postdocs (one full-time). Experience included assays for adherence and growth on produce, screening of cecal, fecal and fly samples for Campylobacter and corresponding enumerations, assessments of antimicrobial resistance gene dissemination, genetic characterization and manipulation of Listeria and Campylobacter, the Galleria melonella virulence model, analysis of bioinformatics data, construction and screening of mutant libraries, biofilm assays, antimicrobial susceptibility determinations, and statistical analysis of microbiological data. Students also acquired experience in presentation of findings for conferences and workshops and preparation of manuscripts for peer review as well as in blogs and other material for outreach. In addition, three high school students participated in a summer research immersion program in the lab, working with Listeria. Technical staff acquired further experience in microbiology, food safety andmolecular biology. Technicians were trained on biofilm assays, virulence assays employing Galleria, produce colonization assays anddatabase maintenance. Several of the technical staff also acquired experience in presentation of findings for conferences and workshops andpreparation of manuscripts for peer review. One technician received further training in library selection, DNA library construction, and deep sequencing with the Illumina Mi-Seq. Graduate students, postdocs and technical staff acquired experience in mentoring and supervision of junior laboratorians working on the project. How have the results been disseminated to communities of interest?Food Industry: Findings were presented and also informally discussed at industry-attended conferences e.g. International Association for Food Protection, Turkey Days, Food Safety and Defense Task force meetings etc. Research and Academic Community: Findings were presented at conferences viaposters and presentations, as well as through informal networking events. Findings from the project were also regularly presented via case study formatsin the Food Microbiology class that Kathariou taught at North Carolina State University. Findings were also disseminated via the web site. Community at-large: Themes and findings derived from project were shared informally on numerous occasions in meetings with community groups. What do you plan to do during the next reporting period to accomplish the goals?We intend to further characterize Listeria mutants that were impaired in growth on produce (cantaloupe rind). We intend to continue screening via Next-Generation Sequencing the enriched transposon mutant libraries in order to identify additional mutants impaired in adherence or growth on produce (cantaloupe rind). Risk identification for Campylobacter colonization of turkeys will be further pursued by statistical analysis of the data from the GAP-like audits and the detailed survey forms that the turkey industry partners have been completing for us. We intend to develop additional materials (pamphlets, blogs) useful for outreach to the food industry. Materials relevant to the project will be posted on the web site. New case studies pertinent to the project will be developed in the context of classroom teaching. Data will be further disseminated via publications, presentations, the web site, a food safety blog and other venues. Undergraduate and graduate students will further pursue research, together with postdocs and a visiting scientist expected next year. Metrics from the research effort will be analyzed by the project evaluator.

Impacts
What was accomplished under these goals? Objective 1: We tested 30 strains of Listeria monocytogenes (LM) for adherence and produce on produce (cantaloupe rind, celery, lettuce).These included strains from produce-related outbreaks (celery, cantaloupe, caramel apple, stone fruit); from outbreaks involving other vehicles; sporadic human listeriosis strains; strains from food and environmental samples. Significant source-dependent differences in adherence or growth were not noted. We developed a Listeria seedling model using inoculation of surface-sterilized turnip seeds. LM populations levels onto the inoculated seeds were maintained over several days post-germination in roots and shoots of seedlings. Five strains and several transposon mutants were tested in this assay. Objective 2: We optimized protocols utilizing ampicillin selection to enrich for mutants unable to grow on the cantaloupe rind. In this approach, cantaloupe rind fragments were inoculated with the mutant library and incubated over several days (4 weeks at 4 degrees C and 72h at room temperature) in the presence of ampicillin. The approach is based on the fact that ampicillin kills only growing cells, thus allowing those unable to replicate to remain viable. Surviving Listeria populations were recovered at specific time intervals on selective media (modified oxford Agar, MOX) and individual colonies tested for growth on the produce. Several mutants were identified, with four being especially promising. In addition, we have utilized a repeat-rinse model to enrich for mutants that are unable to adhere. In this approach, the fragments were inoculated with a mutant library and then rinsed. The rinsate should be enriched in mutants unable to tightly adhere on the fragment. The rinsate was used as inoculum for new fragments, with the process repeated 4 times. Experiments used mutant libraries from two different produce outbreak-derived strains (2858, a serotype 1/2b strain from the 2011 cantaloupe outbreak and 1723, a serotype 1/2a strain from the 2010 celery outbreak). With both mutant libraries, populations in the rinsate declined to ca. 300 CFU/ml by the 4th successive rinse. Objective 3: We tested a panel of LM strains for biofilm formation on polystyrene. We did not detect a direct correlation between adherence or growth on cantaloupe rind and biofilm formation on polystyrene. Furthermore, transposon mutants in the listeriolysin O gene hly, the virulence regulator gene, prfA and the metal tolerance determinant cadA4v were found significantly impaired for biofilm formation but were not affected in adherence or growth on cantaloupe. Objective 4: We addressed whether certain actions and farm practices enhanced risk for Campylobacter colonization, whether risk was affected by specific weather trends and regional impacts, and further investigated the role of flies as reservoir and vehicle for Campylobacter in turkey farms. We constructed surveys requesting information about farm layout and practices and distributed them to commercial turkey farms in North Carolina (NC). On-farm GAP-like audits were also performed to gather flock-level data and to further assess the typical on-farm operations. During each on-farm visit both fecal (n=12) and fly (n=10) samples were collected, transported to the lab on ice, and enumerated for Campylobacter). Each month between 12/12 and 07/16 we also received from the turkey company veterinarians ceca from 10-26 differentturkey brooder flocks, approx. 4-5 wks of age. We determined Campylobacter CFU/g cecal content, and characterized the isolates. Selected farms differing in positive vs. negative status for Campylobacter were identified andwith company coordination were visited to (1) obtain fecal droppings, flies, and litter samples (2) perform the on-farm audit for potential risk factors (3) Place fly speck cards (each speck indicates a fecal spot from a fly landing on the card). We collected audit data from 42 on-farm visits, in addition to farm survey data from 27 farms. Audit and survey data were complemented by Campylobacter data from 178 flocks. Certain potential risk or protective factors were identified: (1) Farms that were consistently Campylobacter-negative were maintained a single grower who did not bring in workers from the outside and who practiced high biosecurity in general; (2)A fence at the outside perimeter of the farm correlated with lower levels of Campylobacter; (3) Well-maintained footbaths at the entrance to each turkey house correlated with lower levels of Campylobacter; (4) Low fly density and absence of Campylobacter-positive flies correlated with Campylobacter-free flocks. Flies and fecal samples were analyzed and enumerated for Campylobacter (Campylobacter CFU/g feces or CFU/fly). A subset of isolates from each positive sample was characterized for species (C. jejuni or C. coli) by multiplex PCR and for antimicrobial resistance (AMR) to a panel of antibiotics. At least one isolate of each AMR profile type was genetically subtyped by multilocus sequence typing (MLST). Strain prevalence in fecal and fly samples was statistically analyzed. Flies were Campylobacter-negative in all farms with Campylobacter-negative feces, while in farms with Campylobacter-positive flocks feces the flies were also positive. Three predominant multidrug-resistant strains predominated both in feces and flies, but strain diversity was higher among fly-derived isolates, suggesting the potential for flies to serve as sensitive sampling device for Campylobacter in turkey farms. Detection of several strains in flies was a significant predictor for their presence in the birds. Taken together, the data point to the importance of effective fly control regimes on turkey farms to reduce risk of flock colonization by Campylobacter. To gather weather data, we partnered with Dr. Paula Hennon at the Cooperative Institute for Climate and Satellites in NC. A 6-month period of unusually low prevalence of Campylobacter was identified and weather data retroactively gathered from the closest municipal airport. A logistic regression was performed, with response being the presence or absence of Campylobacter on the farm. Currently, the best statistical model includes average daily high and low temperature and average humidity. Objective 5: we had one face-to-face meeting with company veterinarians of the turkey industry participating company. At this meeting data were shared, new results were discussedand feedback was received. In addition, updates of prevalence data from cecal analyses were routinely made available to the participating company via email on a monthly basis.We also provided electronic copies of data and summary reports to the turkey industry veterinarians. Outreach included two articles in trade magazines with wide outreach to the poultry industry. We continued to update our website ("Kathariou Lab Campylobacter Research and Extension") (https://campylobacter.fbns.ncsu.edu). Objective 6: We had several electronic and conference call exchanges with Dr. Drew Rivers, whose expertise is in project evaluation. Data (publications, other communication efforts, numbers of students involved in research, contacts with various stakeholders etc.) were made available to the project evaluator and included as metrics in project evaluation. Material presented in classroom instruction was also evaluated via pre and post-semester questionnaires, a mid-semester feedback questionnaire and an end-of-semester solicitation of student self-assessment and learning objective achievement.

Publications

  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Azizoglu R.O., and S. Kathariou. 2016. Listeria: Properties and Occurrences. In: Caballero, B., Finglas, P., and Toldr�, F. (ed). The Encyclopedia of Food and Health vol. 3, pp. 567-570. Oxford: Academic Press.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Garner D, Kathariou S. 2016. Fresh produce-associated listeriosis outbreaks, sources of concern, teachable moments and insights. J. Food Prot. 79(2):337-44.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Martinez MR, Osborne J, Jayeola VO, Katic V, Kathariou S. 2016. Capacity of Listeria monocytogenes strains from the 2011 cantaloupe outbreak to adhere, survive and grow on cantaloupe. J. Food Prot. 79(5): 757-763.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pritchard J C, Jacob M E, Ward T J, Parsons C T , Kathariou S, Wood M W. 2016. Listeria monocytogenes septicemia in an immunocompromised dog. Vet. Clin Pathol. 45(2): 254-259.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Crespo MD, Altermann E, Olson J, Miller WG, Chandrashekhar K, Kathariou S. 2016. Novel plasmid conferring kanamycin and tetracycline resistance in the turkey-derived Campylobacter jejuni strain 11601MD. Plasmid. 86:32-7. doi: 10.1016/j.plasmid.2016.06.001.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Dutta V, Lee S, Ward T, Orwig N, Altermann E, Jima D, Parsons C, Kathariou S. 2016. Draft genome sequences of two historical Listeria monocytogenes strains from human listeriosis cases in 1933. Genome Announc. 4(6). pii: e01364-16. doi: 10.1128/genomeA.01364-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Dutta V, Altermann E, Crespo MD, Olson JW, Siletzky RM, Kathariou S. I 2016. dentification of a Campylobacter coli methyltransferase targeting adenines at GATC sites. FEMS Microbiol Lett. 364(7). doi: 10.1093/femsle/fnw268.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Miller WG, Huynh S, Parker CT, Niedermeyer JA, Kathariou S. 2016. Complete Genome Sequences of Multidrug-Resistant Campylobacter jejuni Strain 14980A (Turkey Feces) and Campylobacter coli Strain 14983A (Housefly from a Turkey Farm), Harboring a Novel Gentamicin Resistance Mobile Element. Genome Announc. 4(5). pii: e01175-16. doi: 10.1128/genomeA.01175-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Dutta V, Altermann E, Olson J, Wray GA, Siletzky RM, Kathariou S. 2016. Whole-Genome Sequences of Agricultural, Host-Associated Campylobacter coli and Campylobacter jejuni Strains. Genome Announc. 4(4). pii: e00833-16. doi: 10.1128/genomeA.00833-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Crespo MD, Kathariou S, Grimes JL, Cox NA, Buhr RJ, Frye JG, Miller WG, Jackson CR, Smith DP. 2016. Routes of transmission of Salmonella and Campylobacter in breeder turkeys. J. Appl. Poultry Res. doi: 10.3382/japr/pfw035
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Palerme JS, Pan PC, Parsons CT, Kathariou S, Ward TJ, Jacob ME. 2016. Isolation and characterization of atypical Listeria monocytogenes associated with a canine urinary tract infection. J Vet Diagn Invest. 28(5):604-7. doi: 10.1177/1040638716661381.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ebrahimi, H., S. Rahimi, P. Khaki, J. K. Grimes and S. Kathariou. 2016. The effects of probiotics, organic acid, and a medicinal plant on the immune system and gastrointestinal microflora in broilers challenged with Campylobacter jejuni. Turk. J. Vet. Anim. Sci. 40: 329-336.
  • Type: Other Status: Published Year Published: 2016 Citation: Kathariou S, Nychas GJ. 2016. SYMPOSIUM: Can whole genome sequencing guide and inform intra-species virulence rankings ? International Association for Food Protection European Symposium on Food Safety, Athens, Greece.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Parsons P, Brown P, Kathariou S. 2016. Metal homeostasis in Listeria monocytogenes: Which metals? Which genes? BioLunch, North Carolina State University
  • Type: Other Status: Published Year Published: 2016 Citation: Kathariou, S. 2016. Food Safety: the academy, industry, regulation and public health nexus. Eisenhower School Agribusiness Study group site visit presentation
  • Type: Other Status: Published Year Published: 2016 Citation: Kathariou S, Jayeola V, Bolinger H. 2016. Microbial pathogens typical to food. Math/Science Education Network (MSEN), North Carolina State University, Raleigh, NC August 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jayeola, V O, Parsons, C, Miller G W, Gorski L, Kathariou S. 2016. Ampicillin selection of Listeria monocytogenes mutants unable to replicate on rind of fresh cantaloupe. International Association for Food Protection Annual Meeting, St. Louis, MO.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kathariou, S. 2016. The Listeria surface at the host-food-environment interface. EMBO Conference on Problems of ListeriosisISOPOL XIX. Paris, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Parsons C, Brown P, Kathariou S. 2016. Novel locus mediating metal homeostasis in Listeria monocytogenes. Proceedings of 115th General Meeting of American Society for Microbiology.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kathariou S. 2016. Intra-species virulence differences: Insights and opportunities from outbreak investigations in the era of Whole Genome Sequencing. International Association for Food Protection European Symposium on Food Safety, Athens, Greece.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Parsons P, Niedermeyer JN, Brown P, Kathariou S, Gould N, Strules, J, Mesa B, Kelly M, Hooker M, Chamberlain M, Olfenbuttel C, and DePerno, C. 2016. Black bears as novel vectors/reservoirs for potential human and animal pathogens. The Wildlife Society, Annual Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Parsons C, Brown B, Kathariou S. 2016. Functional genomics of heavy metal resistance in the foodborne pathogen Listeria monocytogenes. National Institute of Environmental Health Sciences (NIEHS) Environmental Health Sciences FEST
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jayeola, V. O., Parsons, C., Miller, G. W., Gorski and S. Kathariou. 2016. Ampicillin selection of Listeria monocytogenes mutants unable to replicate on rind of fresh cantaloupe. USDA PIs Conference, St Louis, MO.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kathariou S. 2016. The complexity of antibiotic resistance  The need for multi-system approaches (Roundtable) International Association for Food Protection Annual Meeting, St Louis, MO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jayeola VO, Parsons C, Martinez MR, Kathariou S. 2016. Persistence of Listeria monocytogenes on Fresh Produce. North Carolina Governors Food Safety Task Force Meeting. Salisbury, NC. March, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Niedermeyer J, Foster D, Kirchner M, Bolinger H, Miller GM, Kathariou S. 2016. Impact of enrofloxacin treatment on fecal populations of Campylobacter spp. in calves. International Association for Food Protection European Symposium on Food Safety, Athens, Greece.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Niedermeyer, JN, Melander C, Kathariou S. 2016. Novel compounds that inhibit biofilm formation by Listeria monocytogenes. Center for Advanced Processing and Packaging Studies (CAPPS) Conference, Columbus, OH
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bajwa A, Chandrashekhar K, Kathariou S. 2016. Study of adaptive responses in multi-drug resistant strains of Campylobacter jejuni. Undergraduate student Research conference, NCSU
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jahanafroozi M, Fan S, Jayeola V, Parsons, C, Kathariou S. 2016. Characterization of the genetic basis and stability of tetracycline resistance in an outbreak strain of Listeria monocytogenes. North Carolina State University CBE Biolunch, May 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bollinger H, Kirchner M, Chandrasekhar K, Miller W, Niedermeyer J, Kathariou S. 2016. Investigation of erythromycin-resistant Campylobacter jejuni from turkey farms in North Carolina. International Association for Food Protection Annual Meeting. 2016 July 31- August 1. St Louis, MO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kirchner M, Carver D, Badgley B, Kathariou S. 2016. Turkey Triangle: A look into the cecal microbiota of young turkeys. CALS: Stewards of the Future Seminar 2016, October 20. Raleigh, NC.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Parsons, Cameron Tyler 2016. Functional Genomics of Heavy Metal Resistance in the Foodborne Pathogen Listeria monocytogenes PhD Dissertation, North Carolina State University
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Foster D, Jacob M, Kathariou S, Ferguson K, Papich MG. 2016. Impact of enrofloxacin dosing regimens on intestinal pharmacokinetics and the selection for resistant enteric bacteria. World Buitrics Congress, July 2016.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Price, Robert E. 2016. Characterization of Selected Transposon-mediated Mutants of Listeria monocytogenes Regarding Survival and Growth on Cantaloupe. MS Thesis, North Carolina State University
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Anekella, Kartheek. 2016. Characterization of L. plantarum/pentosus for Starter Cultures in Cucumber Fermentation and Conjugative Transferability of Antibiotic Resistance in Lactic Acid Bacteria. PhD Dissertation, North Carolina State University
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. What Do Food Microbiology Students Know About Campylobacter? Campylobacter.fbns.ncsu.edu, 28, Nov. 2016, < https://campylobacter.fbns.ncsu.edu/2016/11/28/what-do-students-know-about-campylobacter/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. 2014 NARMS Data Released! Campylobacter.fbns.ncsu.edu, 25 Nov. 2016, < https://campylobacter.fbns.ncsu.edu/2016/11/25/2014-narms-data-released/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. Campylobacter and WGS: The Review Weve Been Waiting For. , campylobacter.fbns.ncsu.edu, 9 Oct. 2016, < https://campylobacter.fbns.ncsu.edu/2016/10/09/campylobacter-wgs-review/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. IAFP Reflection 1: Metagenomics and Microbiomes. campylobacter.fbns.ncsu.edu, 20 Sept. 2016, < https://campylobacter.fbns.ncsu.edu/2016/09/20/metagenomics-and-microbiomes/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. Throwback Thursday (1 Day Late), campylobacter.fbns.ncsu.edu, 16 Sept. 2016, < https://campylobacter.fbns.ncsu.edu/2016/09/16/throwback-thursday-1-day-late/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. IFT Reflections: Understanding FSMA and How it Relates to Campylobacter. campylobacter.fbns.ncsu.edu, 15 Aug. 2016, < https://campylobacter.fbns.ncsu.edu/2016/08/15/ift-reflections-understanding-fsma-relates-campylobacter/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Why Isnt Campylobacter Better Known? campylobacter.fbns.ncsu.edu, 25 July, 2016, < https://campylobacter.fbns.ncsu.edu/2016/07/25/campylobacter-better-known/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M, WANTED: Thanksgiving Ruiners. Campylobacter.fbns.ncsu.edu, 21 Nov. 2016, < https://campylobacter.fbns.ncsu.edu/2016/11/21/wanted-thanksgiving-ruiners/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Franek, P. My Experience as an Undergraduate Lab Researcher. campylobacter.fbns.ncsu.edu, 21 July, 2016, < https://campylobacter.fbns.ncsu.edu/2016/07/21/undergraduate/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. Know Your Lingo: How to Read Microbiome and Metagenomic Articles. Campylobacter.fbns.ncsu.edu, 18 Nov. 2016, < https://campylobacter.fbns.ncsu.edu/2016/11/18/know-your-lingo-microbiome-and-metagenomic-article-terminology/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. CALS Stewards of the Future: Collaboration and Microbiomes. Campylobacter.fbns.ncsu.edu, 28 Oct. 2016,
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. The Economic Burden of Foodborne Illnesses- How Much Does Campylobacter Contribute? campylobacter.fbns.ncsu.edu, 20 Oct. 2016, < https://campylobacter.fbns.ncsu.edu/2016/10/20/economic-burden-foodborne-illnesses-much-campylobacter-contribute/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK, Carver, D, Kathariou, S. What Are Poultry Producers Doing About Campylobacter? campylobacter.fbns.ncsu.edu, 14 July, 2016, < https://campylobacter.fbns.ncsu.edu/2016/07/14/poultry-producers-campylobacter/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Chandrashekhar, K. Campylobacter Biofilms: A Super Resistant Army. campylobacter.fbns.ncsu.edu, 7 July, 2016, < https://campylobacter.fbns.ncsu.edu/2016/07/07/biofilms/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. Microbiomes and Campylobacter : How They Fit Together and Why Theyre Important. campylobacter.fbns.ncsu.edu, 27 June, 2016, < https://campylobacter.fbns.ncsu.edu/2016/06/27/microbiomes/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Scotland, Thank You for the Big Pink Chicken. campylobacter.fbns.ncsu.edu, 22 June, 2016, < https://campylobacter.fbns.ncsu.edu/2016/06/22/big-pink-chicken/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Latest on the May 2016 Campylobacter Outbreak in California. campylobacter.fbns.ncsu.edu, 21 June, 2016, < https://campylobacter.fbns.ncsu.edu/2016/06/21/151/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Link to 32 Sickened by Campylobacter Outbreak in California, campylobacter.fbns.ncsu.edu, 10 June, 2016, < https://campylobacter.fbns.ncsu.edu/2016/06/10/32-sickened-by-campylobacter-outbreak-in-california/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Kirchner, M. IAFP Abstract: Erythromycin Resistant C. jejuni Information. campylobacter.fbns.ncsu.edu, 2 June, 2016, < https://campylobacter.fbns.ncsu.edu/2016/06/02/iafp-abstract-erythromycin-resistant-c-jejuni-information/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Link to More about how houseflies can serve as vectors of campylobacteriosis. campylobacter.fbns.ncsu.edu, 4 May, 2016, < https://campylobacter.fbns.ncsu.edu/2016/05/04/91/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Link to FSA suspends sampling of Campylobacter from retail chicken in UK. campylobacter.fbns.ncsu.edu, 2 May, 2016, < https://campylobacter.fbns.ncsu.edu/2016/05/02/fsa-suspends-sampling-of-campylobacter-from-retail-chicken-in-uk/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK. Link to 280,000 Sickened With Campylobacter in the UK Last Year, campylobacter.fbns.ncsu.edu, 29 April, 2016, < https://campylobacter.fbns.ncsu.edu/2016/04/29/last-year-280000-people-were-sickened-by-campylobacter-in-the-uk/>
  • Type: Websites Status: Published Year Published: 2016 Citation: Bolinger, HK, Link to USDA targets reduction of Salmonella and Campylobacter in poultry by Joe Whitworth, campylobacter.fbns.ncsu.edu, 30 March, 2016, < https://campylobacter.fbns.ncsu.edu/2016/03/30/71/>


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences for the Listeria-related research program included the produce industry, keen on science-based strategies to control Listeria on fresh produce. Relevant stakeholders in this target audience include farmers, workers, packing plant owners and operators, and other individuals in the fresh produce distribution chain from farm to retail. The food safety and public health sector were relevant target audiences, since their mission includes reducing prevalence of human listeriosis, for which contaminated produce has emerged as major vehicle. The community at large were important stakeholders, as Listeria contamination remains a major health threat for populations at high risk, especially pregnant women and the elderly. Graduate and undergraduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including research on molecular genetic and bacteriological attributes mediating Listeria's ability to adhere to, survive and persist on fresh produce. Technical staff participating in project was served by development of additional expertise including produce sampling, microbiological enumerations and characterization. The scientific community was served by acquisition of further knowledge on contamination of fresh produce by Listeria. Target audiences for the Campylobacter-related research program included the turkey and generally the integrated poultry industry which is interested in pre-harvest reductions of Campylobacter, especially since the relatively recent implementations of performance standards in carcass rinses and raw poultry parts. The food safety and public health sector in the US were relevant target audiences, since their mission includes reducing prevalence of Campylobacter infections, for which contaminated poultry are extensively recognized as major vehicle. In addition, antimicrobial resistance (AMR) of bacterial pathogens is widely recognized as a leading public health threat, and government agencies are keen on further understanding of the AMR issue in zoonotic pathogens such as Campylobacter. The poultry industry is also interested in the AMR issue, as current market pressures and public health data suggest the need for eliminating or minimizing pre-harvest antimicrobial uses in poultry and other animals. Poultry company veterinarians are especially interested in further understanding the complex interactions between antimicrobial use, health status of the birds and potential correlations with Campylobacter numbers and resistance profiles. Graduate and undergraduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including analysis of risk factors associated with Campylobacter levels and antimicrobial resistance, potential transmission vehicles such as flies and bacteriological characterization of Campylobacter via species designations, AMR profiles and genetic determinants mediating selected resistance attributes. Technical staff participating in project was served by development of additional expertise in sample collection, GAP-like audits, sample analysis. Graduate and undergraduate students in the classroom were served by modules and other didactic activities informed by the project and integrated in Food Microbiology and Food Safety courses. The scientific community was served by acquisition of further knowledge on transmission pathways, farm ecosystem ecology, pre-harvest colonization of food animals, and bacteriological characteristics of Campylobacter in the food chain. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four undergraduate and 11 graduate students acquired research experience working on the project during the past year, together with two postdocs. Experience included assays for adherence and growth on produce, screening of cecal, fecal and fly samples for Campylobacter and corresponding enumerations, genetic characterization and manipulation of Listeria and Campylobacter, analysis of bioinformatics data, construction and screening of mutant libraries, biofilm assays, antimicrobial susceptibility determinations, and statistical analysis of microbiological data. Students also acquired experience in presentation of findings for conferences and workshops and preparation of manuscripts for peer review as well as in blogs and other material for outreach. In addition, two high school students participated in a summer research immersion program in the lab, working with Listeria. Technical staff acquired further experience in microbiology, food safety and molecular biology. Technicians were trained on biofilm assays, virulence assays employing Galleria, produce colonization assays and database maintenance. Several of the technical staff also acquired experience in presentation of findings for conferences and workshops and preparation of manuscripts for peer review. One technician received further training in library selection, DNA library construction, and deep sequencing with the Illumina Mi-Seq. Graduate students, postdocs and technical staff acquired experience in mentoring and supervision of junior laboratorians working on the project. How have the results been disseminated to communities of interest?Food Industry: Findings were presented and also informally discussed at industry-attended conferences e.g. International Association for Food Protection, Turkey Days, Food Safety and Defense Task force meetings, FDA workshop, etc. Research and Academic Community: Findings were presented at conferences via posters and presentations, as well as through informal networking events. Findings from the project were also regularly presented via case study formats in the Food Microbiology class that Kathariou taught at North Carolina State University. Community at-large: Themes and findings derived from project were shared informally on numerous occasions in meetings with community groups. What do you plan to do during the next reporting period to accomplish the goals?We intend to further characterize the Listeria mutants that appear to be impaired in produce adherence or growth on produce. We intend to screen via Next-Generation Sequencing the enriched transposon mutant libraries in order to identify additional mutants impaired in adherence or growth on produce (cantaloupe rind). Risk identification for Campylobacter colonization of turkeys will be further pursued by statistical analysis of the data from the GAP-like audits and the detailed survey forms that the turkey industry partners have been completing for us. Campylobacter prevalence data will also be analyzed for seasonal and weather impacts, in collaboration with experts in weather data analysis. We intend to develop additional materials (pamphlets, blogs) useful for outreach to the food industry. Materials relevant to the project will be posted on the web site. New case studies pertinent to the project will be developed in the context of classroom teaching. Data will be further disseminated via publications, presentations, the web site, a food safety blog and other venues. Undergraduate and graduate students will further pursue research, together with postdocs and a visiting scientist expected next year. Metrics from the research effort will be analyzed by the project evaluator.

Impacts
What was accomplished under these goals? Objective 1. We tested additional strains of Listeria monocytogenes (LM) for adherence and produce on fresh produce (cantaloupe rind). These included strains from recent produce-related outbreaks (caramel apple, stone fruit). Significant differences in adherence or growth among strains were not noted. Objective 2. We optimized protocols utilizing ampicillin selection to enrich for mutants unable to grow on the cantaloupe rind. In this approach, cantaloupe rind fragments were inoculated with the mutant library and incubated over several days (4 weeks at 4 degrees C and 72h at room temperature) in the presence of ampicillin. The approach is based on the fact that ampicillin kills only growing cells, thus allowing those unable to replicate to remain viable. Several mutants were identified, with four being especially promising. In addition, we have utilized a repeat-rinse model to enrich for mutants that are unable to adhere. In this approach, the fragments were inoculated with a mutant library and then rinsed. The rinsate should be enriched in mutants unable to tightly adhere on the fragment. The rinsate was used as inoculum for new fragments, with the process repeated 4 times. Objective 3. We tested a panel of LM strains for biofilm formation on PVC (hydrophobic surface), borosilicate glass (hydrophilic surface), and stainless steel. The strains were previously tested for both adherence (after a 90 minute exposure to LM) and growth on lettuce at 4 and 25 degrees C. PVC and glass biofilm assessments have been completed. PVC and glass biofilms were stained with crystal violet and measured by A580 determinations. Preliminary data suggest strain-dependent differences in biofilm formation between PVC and glass. However, we did not detect a direct correlation between adherence or growth on lettuce and biofilm formation on PVC. Similar findings have been obtained with certain transposon mutants (in the listeriolysin O gene hly and in the virulence regulator gene, prfA) which were impaired for biofilm formation but were not affected in adherence or growth on cantaloupe. Confocal microscopy was done with L. monocytogenes strain 2387 harboring a GFP plasmid (GFP-LM). The GFP-labeled bacteria were allowed to adhere to romaine lettuce leaves for 90 min and then allowed to grow on the leaf surface for 24 hours at 25 degrees C . Confocal microscopy showed that a few GFP-LM cells were scattered over the lettuce leaf while most were congregated at the cut edge of the leaf and around the stomatal openings. This may reflect preferential adherence to sites with higher nutrient levels or sites not already occupied by resident microbiota. We saw no evidence of GFP-LM internalization into the lettuce leaf. We have isolated and preserved apanel of bacterial strains (including numerous cultures of lactic acid bacteria) from fresh cantaloupe rind. These will be utilized to assess Listeria colonization in mixed cultures with bacteria indigenous to the produce. Objective 4. To further characterize risk factors for Campylobacter colonization of turkeys, each month we received from the veterinarians of the turkey company participating in the project intestinal samples (ceca) from 10-26 different turkey flocks in the brooder stage, approx. 4-5 weeks of age; a total of 168 flocks were thus tested during the year. From each flock we received 6 individual ceca, which were combined into one composite sample that was employed for enumeration of Campylobacter CFU/g cecal content, and for subsequent isolation and characterization of isolates. We found that 25-100% of the flocks were Campylobacter-positive. Prevalence varied markedly by season, with the lowest noted in February (33% positive) and March (25% positive). These trends were much more pronounced than in previous years. For instance, in 2014 prevalence in February and March was 85 and 50%, respectively. The severe drop in positive flocks during winter-spring of the past year made it difficult to identify control flocks. Starting with April, increasing prevalence trends were noted, with 97-100% of the flocks being Campylobacter-positive after May. Data from farm GAP-like audits and surveys, together with Campylobacter data from each farm, were coded for statistical analysis. The first assessments of the data suggest the need to examine certain potential risk or protective factors in more detail, specifically: (1) Number of workers at the farm. Farms that were consistently Campylobacter-negative tended to be maintained a single grower who did not bring in other workers and practiced high biosecurity in general ; (2) Presence of a fence at the outside perimeter of the farm. Presence of the fence appeared to correlate with lower levels of Campylobacter; (3) Well maintained footbaths at the entrance to each turkey house. These appeared to correlate with lower levels of Campylobacter; (4) fly control measures and low density of flies. We analyzed isolates of Campylobacter from feces and flies of the same turkey farm.Determination ofspecies and antimicrobial resistance (AMR) analysis revealed that isolates from flies tended to exhibit a greater diversity of Campylobacter than suggested by analysis of isolates from feces. Only 25% of the flocks yielded the same strains from feces and from flies. In flocks where isolates from flies and from feces did not completely overlap, the non-overlapping strain types were more likely to be recovered from the flies than from feces. Data suggest that flies may serve as a highly sensitive sampling device, valuable in supplementing strain type data derived from analysis of fecal droppings. We also investigated Campylobacter colonization (timing, levels) in two farms initiated at the same time and region and from the same hatchery but operating with different management. Farms were stocked with birds of two different breeds (Hybrid, Nicholas). Even though both flocks became colonized, the flock exhibiting better biosecurity and litter management lagged by about 2 weeks. Strains were different between the flocks. The farm with better biosecurity and litter management became colonized largely by strains only resistant to tetracycline whereas the other was colonized by multidrug resistant strains. Data suggested profound impact of flock management in colonization and strain types of Campylobacter. Objective 5. We held two face-to-face meetings with company veterinarians of the turkey industry participating company. At these meetings data were shared, new trends were discussed and feedback was received. In addition, updates of prevalence data from cecal analyses were routinely made available to the participating company via email on a monthly basis. Data were informally discussed with company veterinarians, service staff and farmers during the Turkey Days event in Wilmington, NC. Outreach has included an on-line article highlighting work based on the project and an article on the biology and antimicrobial resistance of Campylobacter has been published New Foods, a magazine with wide reach to the poultry and generally the food industry. A website ("Kathariou Lab Campylobacter Research and Extension") has been established (https://campylobacter.fbns.ncsu.edu). Objective 6. We have met twice in person and also had several electronic and conference call exchanges with Dr. Drew Rivers, whose expertise is in project evaluation. Data (publications, other communication efforts, numbers of students involved in research, contacts with various stakeholders etc.) were made available to the project evaluator.

Publications

  • Type: Other Status: Published Year Published: 2015 Citation: Garner, D. 2015 Listeriosis and Produce: Whats the Connection? Food Safety News (April 01, 2015). http://www.foodsafetynews.com/2015/04/listeriosis-and-produce-whats-the-connection/#.VcPguSpVikq
  • Type: Other Status: Published Year Published: 2015 Citation: Bolinger, H. 2015. Food Safety 101: What is Campylobacter (And what are we doing about it)? [blog] (2015, April 2). Retrieved from:https://news.ncsu.edu/2015/03/campylobacter-bolinger/
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bolinger, H B, Kathariou, S. 2015. The drugs and bugs in your birds: Food safety implications of antibiotic resistance in Campylobacter. New Food, Issue 1 2015. http://www.newfoodmagazine.com/16159/supplements/food-safety-supplement-2015/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Price, R, Parsons C, Jaeyola, V, Kathariou, S. 2015. Investigation of the role of hemolysin and prfA of Listeria monocytogenes on adherence and growth on produce. USDA PIs Conference, Portland, OR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Gorski, L. 2015. Molecular interactions between Listeria monocytogenes and produce. Western Food Safety Summit, Hartnell College
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Gorski, L, Liang, A, Romanolo, K, Walker, S. 2015. Examination of Gorski, L, Liang, A, Romanolo, K, Walker, S. 2015. Examination of environmental isolates of Listeria monocytogenes indicates that their inla genotypes are intact and the strains potentially virulent. International Association for Food Protection, July 25-28, Portland, OR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: DDutta,V, Price, R, Parsons, C, Jayeola, V, Ryan, G, Evans, P, Kathariou, S. 2015. Development of restriction-modification isogenic mutants to study global methylation patterns in Listeria monocytogenes. Proceedings of 114th General Meeting of American Society for Microbiology, New Orleans, LA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dutta, V, Ward, T J, Lee, S, Parsons, C, Kathariou, S. 2015. Diverse genomic location and sequence content of a Listeria monocytogenes chromosomal island harboring heavy metal resistance and other genes. Proceedings of 114th General Meeting of American Society for Microbiology, New Orleans, LAProceedings of 114th General Meeting of American Society for Microbiology, New Orleans, LA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kathariou, S. 2015. Listeria and produce. Annual Conference of the North Carolina Food Safety & Defense Task Force, Raleigh, NC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kathariou, S. 2015. Listeria Virulence. FDA workshop on Listeria, FDA-JFSAN, Washington, DC June 15-16, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kathariou, S. 2015. Listeria ecology. FDA workshop on Listeria, FDA-JFSAN, Washington, DC June 15-16, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ring, L, Niedermeyer, JA, Kathariou, S. 2015. The antibiotic profiling and strain characterization of Campylobacter in turkey flocks raised in different environments. 2015. North Carolina State University Undergraduate Research Spring Symposiums.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Bolinger, HB, Niedermeyer, JA, Mann KM, Carver, DK, Kathariou, S. Characterization of Campylobacter spp. from flies sourced from conventional turkey farms. International Association for Food Protection, July 25-28, Portland, OR.


Progress 01/06/14 to 09/30/14

Outputs
Target Audience:Target audiences for the Listeria-related research program included the produce industry, keen on science-based strategies to control Listeria on fresh produce. Relevant stakeholders in this target audience include farmers, workers, packing plant owners and operators, and other individuals in the fresh produce distribution chain from farm to retail. The food safety and public health sector were relevant target audiences, since their mission includes reducing prevalence of human listeriosis, for which contaminated produce has emerged as major vehicle. The community at large were important stakeholders, as Listeria contamination remains a major health threat for populations at high risk, especially pregnant women and the elderly. Graduate and undergraduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including research on molecular genetic and bacteriological attributes mediating Listeria's ability to adhere to, survive and persist on fresh produce. Technical staff participating in project was served by development of additional expertise including produce sampling, microbiological enumerations and characterization. The scientific community was served by acquisition of further knowledge on contamination of fresh produce by Listeria. Target audiences for the campylobacter-related research program included the turkey and generally the integrated poultry industry which is interested in pre-harvest reductions of Campylobacter, especially since the relatively recent implementations of performance standards in carcass rinses and raw poultry parts. The food safety and public health sector in the US were relevant target audiences, since their mission includes reducing prevalence of Campylobacter infections, for which contaminated poultry are extensively recognized as major vehicle. In addition, antimicrobial resistance (AMR) of bacterial pathogens is widely recognized as a leading public health threat, and government agencies are keen on further understanding of the AMR issue in zoonotic pathogens such as Campylobacter. The poultry industry is also interested in the AMR issue, as current market pressures and public health data suggest the need for eliminating or minimizing pre-harvest antimicrobial uses in poultry and other animals. Poultry company veterinarians are especially interested in further understanding the complex interactions between antimicrobial use, health status of the birds and potential correlations with Campylobacter numbers and resistance profiles. Graduate and undergraduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including analysis of risk factors associated with Campylobacter levels and antimicrobial resistance, potential transmission vehicles such as flies and bacteriological characterization of Campylobacter via species designations, AMR profiles and genetic determinants mediating selected resistance attributes. Technical staff participating in project was served by development of additional expertise in sample collection, GAP-like audits, sample analysis. Graduate and undergraduate students in the classroom were served by modules and other didactic activities informed by the project and integrated in Food Microbiology and Food Safety courses. The scientific community was served by acquisition of further knowledge on transmission pathways, farm ecosystem ecology, pre-harvest colonization of food animals, and bacteriological characteristics of Campylobacter in the food chain. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five undergraduate and 16 graduate students acquired research experience working on the project during the past year, together with two postdocs. Experience included assays for adherence and growth on produce, screening of cecal, fecal and fly samples for Campylobacter and corresponding enumerations, genetic characterization and manipulation of Listeria and Campylobacter, analysis of bioinformatics data, construction and screening of mutant libraries, biofilm assays, antimicrobial susceptibility determinations, and statistical analysis of microbiological data. Students also acquired experience in presentation of findings for conferences and workshops and preparation of manuscripts for peer review as well as in blogs and other material for outreach. In addition, two high school students participated in a summer research immersion program in the lab, working with Listeria. Technical staff acquired further experience in microbiology, food safety and molecular biology. Technicians were trained on biofilm assays, virulence assays employing Galleria, produce colonization assays and database maintenance. Several of the technical staff also acquired experience in presentation of findings for conferences and workshops and preparation of manuscripts for peer review. One technician received training in library selection, DNA library construction, and deep sequencing with the Illumina Mi-Seq. Graduate students, postdocs and technical staff acquired experience in mentoring and supervision of junior laboratorians working on the project. How have the results been disseminated to communities of interest?Food Industry: Findings were presented and also informally discussed at industry-attended conferences e.g. Poultry Science Association Meeting, Turkey Days, Fresh Produce Task force meetings, etc. Research and Academic Community: Findings were presented at conferences via posters and presentations, as well as through informal networking events. Findings from the project were also regularly presented via case study formats in the Food Microbiology class that Kathariou taught at North Carolina State University. Community at-large: Themes and findings derived from project were shared informally on numerous occasions in meetings with community groups. What do you plan to do during the next reporting period to accomplish the goals?We intend to further characterize the genetic basis of Listeria's interactions with fresh produce by a combination of confocal microscopy, targeted screening of the mutant libraries (e. g. for non-adherent mutants, or for mutants unable to grow on produce) and characterization of new mutants of special interest. We also intend to analyze the Campylobacter data from cecal, fecal and fly samples in order to better access the Campylobacter risk associated with flies in the turkey farms. Risk identification will be further done by statistical analysis of the data from the GAP-like audits and the detailed survey forms that the turkey industry partners have been completing for us. We intend to develop materials (pamphlets, blogs) useful for outreach to the food industry. A web site will be developed with special focus on Campylobacter. New case studies pertinent to the project will be developed in the context of classroom teaching. Data will be further disseminated via publications, presentations, the web site, a food safety blog and other venues. Undergraduate and graduate students will further pursue research, together with postdocs and a visiting scientist expected next year. Metrics from the research effort will be analyzed by the project evaluator.

Impacts
What was accomplished under these goals? Objective 1. A panel of strains was examined for adherence and colonization on leaves of romaine lettuce leaves purchased at retail. A subset of this panel was also tested for adherence and colonization on cantaloupe rind and flesh, as well as celery stalks. A total of 24 LM strains were tested, representing diverse serotypes (1/2a, 1/2b, 4b) and sources (environmental, clinical, and food) and including several from produce and produce-related outbreaks. Internalin mutants with premature stop codons in inlA were also included. Overall strains did not differ significantly in adherence or subsequent growth, regardless of serotype, source, or inlA mutation. The best attaching strains exhibited a 2 log increase after 90 minutes post-inoculation and grew further thereafter. The best-attaching strain was chosen to introduce a GFP plasmid in order to observe microscopically how the L. monocytogenes cells interact with lettuce tissue. Construction of chromosomal GFP fusions was also pursued with two of the cantaloupe outbreak strains, but was unsuccessful, in spite of repeated efforts. Objective 2. Mutant libraries with the mariner-based transposon were constructed in four different strains of serotypes 4b, 1/2a and 1/2b. The mutant libraries were validated for representation by screening for mutants impaired in specific phenotypes (hemolysis, motility, cold growth). Relevant mutants were identified from all constructed libraries, suggesting that the mutant libraries were indeed representative. Selected mutants from two strains, 2858 (serotype 1/2b, 2011 cantaloupe outbreak) and 1723, 2010 celery outbreak) were tested for their ability to adhere to and grow on produce. These included non-hemolytic mutants with single insertions in the gene for listeriolysin (hly) or prfA and two motility-impaired mutants. Mutants with insertions in hly or prfA were not impaired in ability to adhere to and grow on cantaloupe at either 25 or 37oC but were impaired in biofilm formation on PVC microtiter plates. Findings suggest that produce colonization cannot be predicted by relative capacity of the strains for biofilm formation on abiotic surfaces. Both of the two tested non-motile mutants adhered on the cantaloupe more efficiently than the wildtype parental strain, suggesting that adherence was not impaired and may in fact be enhanced. One mutant , with a single insertion in a motility and chemotaxis gene cluster was not impacted in subsequent growth or fitness. The other mutant, with a single insertion in a gene encoding an RNA helicase, was not impacted for growth when inoculated alone, but its competitive fitness in comparison to the wildtype was markedly reduced in mutant + wildtype (1:1) co-inoculations. Genetic complementation with the intact RNA helicase gene restored competitive fitness. Data suggested that the RNA helicase is required for competitive fitness of LM on fresh produce. A panel of outbreak strains was characterized for virulence using the insect model, Galleria mellonella. As with produce adherence and growth, strain specific differences were noted but overall the strains did not differ significantly from genetically unrelated strains implicated in other outbreaks (deli meats, cheese). Objective 3. A total of 34 strains were tested for biofilm formation on PVC, using the 96-well microtiter plate biofilm assay. Strains represented diverse serotypes (1/2a, 1/2b, 4b) and sources (environmental, clinical, and food) and included several from produce and produce-related outbreaks. Of these, 24 strains were also tested for biofilms on stainless steel and glass. The levels of biofilm formation on those surfaces varied by strain. The data to date suggest that attachment on PVC did not correlate with attachment to lettuce tissue. Objective 4. Each month in the course of the past year we received from the veterinarians of the turkey company participating in the project intestinal samples (ceca) from 10-26 different turkey flocks in the brooder stage, approx. 4-5 weeks of age. From each flock we received 6 individual ceca, which were combined into one composite sample that was employed for enumeration of Campylobacter CFU/g cecal content, and for subsequent isolation and characterization of isolates. A pronounced decrease in Campylobacter-positive samples was noted between January and March, corresponding to the coldest period. Selected farms sampled at approximately the same time and differing in positive vs. negative status were identified and visited to collect fecal and fly samples and perform a GAP-like assessment of the farm. A detailed survey regarding practices at these farms was provided to the turkey company in order to be filled out by the service staff assigned to the particular farm.We collected and analyzed for Campylobacter both fecal samples and flies from 13 brooder farms. were visited and feces as well as flies analyzed for Campylobacter. Flies were Campylobacter-negative in all farms with Campylobacter-negative feces, while in flocks with Campylobacter-positive feces the flies were also positive. Prevalence of Campylobacter-positive flies in these Campylobacter-positive flocks ranged from 30 to 100% (average, 58 %). All flocks with Campylobacter-positive feces also had Campylobacter-positive flies. Campylobacter counts ranged from 10 to >1x104 CFU/fly (average, 3.7x103 CFU/fly), while positive feces ranged from 1.4x104 to 8.0x107 CFU/g with an average prevalence of 8.7x106 CFU/g. Objective 5. We had two face-to-face meetings with company veterinarians of the turkey industry participating company. At these meetings data were shared, new trends were discussed and feedback was received. In addition, updates of prevalence data from cecal analyses were routinely made available to the participating company via email on a monthly basis. Data were informally discussed with company veterinarians, service staff and farmers during the Turkey Days event in Wilmington, NC in the fall of 2014. Objective 6. We have met twice in person and also had several electronic and conference call exchanges with Dr. Drew Rivers, whose expertise is in project evaluation. Data (publications, other communication efforts, numbers of students involved in research, contacts with various stakeholders etc.) were made available to the project evaluator.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Isolation of Listeria monocytogenes from Food and Water: Official and Experimental Protocols. Azizoglu RO, Gorski L, Kathariou S. Curr Protoc Microbiol. 2014 May 1;33:9B.5.1-19. doi: 10.1002/9780471729259.mc09b05s33.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mutant construction and integration vector-mediated gene complementation in Listeria monocytogenes. Azizoglu RO, Elhanafi D, Kathariou S. Methods Mol Biol. 2014;1157:201-11. doi: 10.1007/978-1-4939-0703-8_17.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Genetic characterization of plasmid-associated triphenylmethane reductase in Listeria monocytogenes. Dutta V, Elhanafi D, Osborne J, Martinez MR, Kathariou S. Appl Environ Microbiol. 2014 Sep;80(17):5379-85. doi: 10.1128/AEM.01398-14. Epub 2014 Jun 20.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: R. O. Azizoglu, L. Gorski and S. Kathariou. 2014. Listeria and Produce: a troublesome Liaison! New Food (October 2014 issue). http://www.newfoodmagazine.com/15048/new-food-magazine/past-issues/issue-5-2014/listeria-produce-troublesome-liaison/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Thanissery R, Kathariou S, Smith D P. Rosemary oil, clove oil, and a mix of thyme-orange essential oils inhibit Salmonella and Campylobacter in vitro.2014 J Appl Poult Res doi: 10.3382/japr.2013-00888
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: R. Price, S. Kathariou, C. Parsons and B. Costolo. 2014. Attachment and survival of motility-Impaired Listeria monocytogenes mutants on fresh cantaloupe. 113th American Society for Microbiology General Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Lee and S. Kathariou. 2014. Multiple chromosomal insertion sites of a L. monocytogenes genomic island harboring arsenic resistance genes. 113th American Society for Microbiology General Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: M. D. Crespo, N. A. Cox, S. Kathariou, J. L. Grimes, R. J. Buhr and D. P. Smith. 2014. Evaluation of transmission of Salmonella and Campylobacter through eggs and offspring through artificial insemination of Breeder Turkeys . Poultry Science Association Meeting.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: V. Dutta, P. Evans and S. Kathariou. 2014. Comparative analysis of gene synteny for adaptive molecular determinants utilizing sequenced genomes of Listeria spp. 113th American Society for Microbiology General Meeting.