ADAPTATIONS AND CONTROL OF BACTERIAL FOODBORNE PATHOGENS IN FOOD AND AGRICULTURAL ECOSYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017316
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Food, Bioprocessing, and Nutrition Sciences

Non Technical Summary
The bacterial foodborne pathogens Salmonella, Shiga toxin-producing Escherichia coli, Campylobacter and Listeria monocytogenes are associated with severe human disease burden and compromise the vitality and sustainability of the food industry. Here we will address special challenges posed by these pathogens in the food chain. In Objective 1, we will develop and validate novel microelectronics tools which can harness volatile organic compounds to sensitively, rapidly and non-invasively detect Salmonella, Shiga toxin-producing Escherichia coli, and Listeria monocytogenes on leafy greens. In Objective 2, we will investigate a novel food safety hazard, i.e. contamination of fresh fruit with Listeria monocytogenes. We will employ unique strain constructions and next-generation sequencing to investigate the fate of different Listeria strains on apples, and will also assess the impact of apple variety and production region on contamination. Whole genome sequence-based tools will be utilized in Objective 3 to elucidate the diversity of Listeria in natural reservoirs and identify reservoirs and genomic attributes especially associated with virulence and prevalence in human listeriosis. A major public health issue, antimicrobial resistance in the food chain, will be addressed in Objective 4, utilizing as model Campylobacter, a leading zoonotic foodborne pathogen. We will characterize emergence, spread and persistence of antimicrobial resistance in Campylobacter. Lastly, in Objective 5 we will employ bacteriological and molecular tools to elucidate Salmonella's capacity to survive and persist in low-moisture environments. Salmonella in such environments exhibits pronounced tolerance to inactivation, and we will assess the effectiveness of novel nanomaterials for inactivation. Collectively, these research efforts will provide novel tools and information critical for design of improved mitigation strategies to reduce food safety and public health risks associated with these major bacterial pathogens.

Animal Health Component

10%

Research Effort Categories

Basic

70%

Applied

10%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4099	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella

listeria monocytogenes

shiga toxin-producing escherichia coli

campylobacter

volatile organic compounds (vocs)

microelectronics

sensors

leafy greens (romaine lettuce, spinach)

apples

biofilms

strain bar-coding

next-generation sequencing

antimicrobial resistance

macrolide resistance

transformation

conjugation

lateral gene transfer, horizontal gene transfer

resistance mutations

fitness

water microcosms

reservoirs

wildlife

whole genome sequencing

clones

virulence

genomic regions

water

low-moisture foods

food contact surface

dehydration

persistence

survival

deletion mutants

transcriptome

galleria mellonella

Goals / Objectives
The long-term goal of the research is to enhance our understanding of the ecology and adaptations of bacterial foodborne pathogens in order to further guide the development of science-based intervention strategies. To contribute to this long-term goal we will pursue the following specific objectives:Objective 1. To develop and validate the utilization of volatile organic compounds (VOCs) for electronic-sensor-based detection of foodborne bacterial pathogens on leafy greensObjective 2: To characterize the fate of Listeria on apples and other fresh produceObjective 3: To characterize genetic diversity, unique genomic attributes and virulence of Listeria in natural reservoirsObjective 4: To assess emergence, spread and persistence of antimicrobial resistance in Campylobacter Objective 5: To characterize survival and virulence of Salmonella in low-moisture foods and on dry abiotic surfaces

Project Methods
Objective 1. We will investigate the capacity of electronic sensors to sensitively, rapidly and non-invasively detect bacterial foodborne pathogens contaminating leafy greens. The sensors will be developed by Dr. Omer Oralkan and Dr. Gregory Parsons at the College of Engineering at North Carolina State University. Dr. Ralph Dean will be a collaborator in this objective. Leafy greens (romaine lettuce, spinach) will be spot-inoculated with individual strains and cocktails of Salmonella, shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes (Lm). Inoculated produce will be kept at specific relative humidity levels and temperatures. Populations of the pathogens will be enumerated on selective media. At specific time points, the inoculated produce will be made available to collaborators who will analyze the profile of volatile organic compounds emitted by the produce. Uninoculated controls will be inlcuded at all time points. Non-pathogenic Salmonella and Lm strains will be tested in parallel, and evaluated for their potential to serve as non-pathogenic surrogates.Objective 2. We will monitor the fate of a panel of eight Lm strains on fresh apples of different variety, produced at different regions, and stored under different conditions. The strains will be bar-coded with 30-bp sequence tags following established protocols. They will include strains of different serotypes from recent produce-associated outbreaks of listeriosis, including the strains implicated in the 2014 apple and stone fruit outbreaks. Inoculum will include planktonic cells as well as cells from biofilms established on stainless steel coupons. Populations of Lm will be monitored over several months on selective and non-selective media. Strain construction and inoculum preparation will be done at North Carolina State University. Inoculations and storage of the apples will be done by collaborators Elliot Ryser and Randy Beaudry at Michigan State university. At specific timepoints the inoculated apples will be swabbed, and the rinsates from the swab used for community DNA extractions. Next-generation sequencing by collaborator Yi Chen at the FDA will be employed to determine the relative abundance and populations of each strain.Objective 3. We will investigate the genotypes and diversity of Lm from two types of potential natural reservoirs: wildlife and surface water. This Objective will be pursued in collaboration with Dr. Christopher DePerno at North Carolina State University, Dr. Rob Dungan at USDA-ARS and Dr. Yi Chen at the FDA. A library of approx. 400 strains from wildlife and surface water has been established and is preserved at -80oC. The wildlife strains originated from feces, rectal swabs and nasal swabs of black bears in the urban and suburban region of Ashville, NC. The water strains were from sewage effluent, creeks and ponds in North Carolina, 2003-2018, and from the Snake River Watershed in Idaho, 2017-onwards. In addition, all available human clinical isolates from North Carolina from 2017-2017 are available and will be included for comparisons. Multiplex PCR will be employed to determine serotype designations for the strains and at least one representative of each serotype from each sample will be identified for whole genome sequencing (WGS). The WGS data will be analyzed to derive whole-genome MLST (wgMLST) and core-genome MLST (cgMLST) designations. Phylogenetic trees will be constructed to assess relationships among strains. Comparative WGS analysis will determine potential relationships among the isolates from the different sources (human, wildlife, water).The WGS of predominant clones will be analyzed for unique genomic islands. Novel genomic islands found to be harbored by predominant clones will be characterized further in silico to assess conservation among different strains and experimentally, via deletion construction and phenotypic characterizations. Mutants and genetically complemented derivatives will be analyzed for virulence in the Greater Wax Moth (Galleria mellonella) model.Objective 4. We will investigate the emergence and transfer of antimicrobial resistance (AMR) in Campylobacter. We will also characterize the potential fitness impacts of AMR, as these can importantly inform the stability and persistence of AMR in agricultural ecosystems. This objective will be pursued in collaboration with Dr. Jesse Grimes (Poultry Science, North Carolina State University) who will collaborate on experiments using the day-old chick model, Dr. Craig Parker and Dr. William G. Miller (both at USDA-ARS), who will collaborate on WGS analysis. Spontaneous mutations that become selected upon exposure to macrolides (erythromycin) will be detected by WGS of the resistant strains. Bioinformatic analysis will be employed to identify compensatory mutations that accompany resistance-associated substitutions. Lateral gene transfer (LGT) involving transformation will be investigated in vivo, using the day-old chick model inoculated with specific strains to be used as donor and recipient for LGT-mediated AMR. Resistant derivatives will undergo WGS to identify potential compensatory mutations. LGT involving both transformation and conjugation will be studied in laboratory media, in poultry feces and water microcosms, in the presence or absence of sub-inhibitory levels of antimicrobials. Fitness impacts and stability of AMR will be investigated in poultry feces and water microcosms stored at 4oC in the dark, to maximize the window where potential fitness differences can be detected.Objective 5. We will investigate the survival and global transcriptional profile of Salmonella contaminating low-moisture foods (LMS) for varying lengths of time. We will also determine genomic regions important for survival of Salmonella on dry abiotic surfaces, and the capacity of novel nanomaterials to inhibit Salmonella in this persistent state. A panel of six LMFs (chocolate; cornflakes, three types of dry fruit; pistachios) will be inoculated with a cocktail of five Salmonella strains, brought to their original Aw and subsequently stored in the dark at 25oC. Three of these food types (pistachios, dry apples, and cornflakes) will be also inoculated with Salmonella Enteritidis (SE) PT30 and similarly incubated. At specific time intervals Salmonella will be enumerated on selective xylose lysine deoxycholate agar (XLD) and non-selective (TSA-YE) agar. Cells will be also tested with LIVE/DEAD stains and direct viable counts to assess the possibility that they may have converted to a viable but non-culturable state. For transcriptome assessments, the three LMFs inoculated with SE PT30 will processed both for enumerations of Salmonella and for extraction of total RNA. RNA samples pf adequate quality and concentration will be made available to the Genomic Sciences Laboratory Core Facility at North Carolina State University for establishment of RNASeq libraries, and the RNAseq data will be analyzed in consultation with Dr. Dereje Jima, bioinformaticist at the Center for Human Health and the Environment at North Carolina State University. Salmonella eluted from the inoculated products following varying lengths of time will be examined for virulence using the Greater Wax Moth (Galleria mellonella) model. Dry nitrocellulose will be employed as model abiotic surface. A deletion mutant library obtained in collaboration with Dr. Michael McClelland (University of California, Irvine) will be screened to identify mutants impaired for survival on this and other (plastic, stainless0 abiotic surfaces. In collaboration with Dr. Liju Yang (North Carolina Central University) and Dr. Ya-Ping Sun (Clemson University) we will investigate the effectiveness of a family of "Carbon Dots" nanoparticles to inactivate Salmonella on the dry surfaces.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Food Safety Professionals Food Science professionals Public Health and One Health professionals Microbiology professionals Food Industry (produce, processed foods, food animals) Academia-postdoctoral scientists, graduatestudents, undergraduate researchers, research technicians, faculty, other researchers Academia- classroom outreach for graduate and undergraduate classes Community at large-science communication to the public, citizen science initiatives? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project contributed to the research training and novel method development and optimization skills for several graduate and undergraduate students and one postdoctoral scientist . In addition to new laboratory skills, the graduate students and the postdoctoral scientist acquired novel bioinformatics experience through this project. Students in classroom settings became exposed to food safety challenges in the food chain and to the opportunities for mitigation, as well as to the procedures and tools involved in investigation of foodborne disease outbreaks. Development opportunities included the preparation and presentation of posters, manuscripts and other materials, and the attendance and presentations in scientific conferences and in regular within-lab meetings. Major contributions to professional development and networking were mediated by regular and frequent interactions and exchanges with collaborators at North Carolina State University (Poultry Science, Microbiology, Applied Ecology), the FDA, CDC and USDA-ARS. In addition, the students participated in collaborations that were established with scientists at other universities, i.e., Michigan State University, university of California-Irvine. How have the results been disseminated to communities of interest?The project's findings have been disseminated via presentations and exchanges at professional conferences and via peer-reviewed publications in highly-regarded journals. Findings have also been presented during regular meetings of the PI's lab that include former lab members now in diverse locations and professional affiliations in the US and internationally. The project and its results were shared with students in Food Microbiology classes. The PI has frequently described the thrust and objectives of this project in seminars and workshops dedicated to citizen science and science communication.? What do you plan to do during the next reporting period to accomplish the goals?Objective 1. To develop and validate the utilization of volatile organic compounds (VOCs) for electronic-sensor-based detection of foodborne bacterial pathogens on leafy greens. In the next reporting period, we will continue to investigate the potential for optimal imaging sensors to detect Listeria monocytogenes on fresh apples. Objective 2: To characterize the fate ofListeriaon apples and other fresh produce. In the next reporting period, the samples from the second full year of the two-year assessment will be examined. The inoculum will consist of planktonic and biofilm-derived cells. Cells will be investigated for their virulence in the Galleria mellonella model. We will in addition investigate the changes in the native microbiota during the storage period, to further assess potential impacts of these microorganism on Listeria recovery. Objective 3:To characterize genetic diversity, unique genomic attributes and virulence ofListeriain natural reservoirs. In the next reporting period, the genomes of the wildlife and surface water-derived strains will be compared with those of the same genotype but from other sources, specifically food processing environments, foods, and cases of human listeriosis. Virulence assessments will be performed for more strains using Galleria mellonella. The antimicrobial resistance determinants will be further analyzed to determine their potential location on mobile genetic elements, and those on such elements will be investigated for their transmission potential to other strains. Objective 4: To assess emergence, spread and persistence of antimicrobial resistance inCampylobacter. In the next reporting period, transformation-mediated spread of antimicrobial resistance will be further investigated. Much of the previous work involved a common reference strain as recipient. In the next reporting period, a panel of contemporary strains from chickens in the US poultry supply will be investigated for their capacity to acquire antimicrobial resistance via transformation, and to determine the impact of resistance marker on transformation frequency. The panel of transformants and mutants with specific resistance traits will be expanded for analysis via whole genome sequencing. Objective 5:To characterize survival and virulence ofSalmonellain low-moisture foods and on dry abiotic surfaces. In the next reporting period, the analysis of the transcriptome of Salmonella from the low-moisture foods will be completed and prepared for publication. In addition, analysis of Salmonella on the dry abiotic surface model will be completed and prepared for publication. Lastly, the findings pertaining to the viable but not culturable state of Salmonella on dried fruit will be prepared for publication. The data pertaining to virulence of Salmonella from the inoculated low-moisture foods with the Galleria mellonella model will be further analyzed.

Impacts
What was accomplished under these goals? Objective 1. To develop and validate the utilization of volatile organic compounds (VOCs) for electronic-sensor-based detection of foodborne bacterial pathogens on leafy greens. To further pursue this objective, the Kathariou laboratory participated in the development of collaborations with the engineer Dr. Michael Kudenov to examine the suitability of optical image systems for real-time, in situ detection of foodborne pathogens on fresh apples. Objective 2: To characterize the fate ofListeriaon apples and other fresh produce. To further pursue this objective, the panel of eight barcoded Listeria monocytogenes strains and their parental counterparts were investigated via whole genome sequence analysis. The findings revealed the presence of single nucleotide polymorphisms in a small number of loci. Fresh, unwaxed apples from three different regions (Michigan, Pennsylvania, Washington) were spot-inoculated with the panel of the barcoded strains for the year 1 of the two-year project. The inoculum was derived planktonic cultures as well as from 48-h biofilms on polystyrene surfaces. Objective 3:To characterize genetic diversity, unique genomic attributes and virulence ofListeriain natural reservoirs. To further pursue this objective, the whole genome sequence data of 156 Listeria monocytogenes and 25 other Listeria spp. from wildlife and surface water was determined. The whole genome sequence data were analyzed to determine genotypes, presence of pathogenicity islands and antimicrobial resistance determinants. The analysis of the genetic relatedness of these strains with strains from food or clinical sources was initiated. Objective 4: To assess emergence, spread and persistence of antimicrobial resistance inCampylobacter. To further pursue this objective, we investigated transformation-mediated transfer of different antimicrobial resistance genes in Campylobacter coli and Campylobacter jejuni. Such transfer involves uptake of naked DNA by a living. recipient cell. We investigated resistance traits based on substitutions in pre-existing genes as well as traits based on acquisition of specific determinants. Certain resistance traits, e.g., to quinolone resistance, were significantly more prone to be transferred than others. Emergence of antimicrobial resistance via mutation was also investigated in control experiments without donor DNA in each of these transformation assays. Panels of transformants and mutants were established and preserved for future analysis of fitness traits, whole genome sequencing and bioinfomatic analysis. Objective 5:To characterize survival and virulence ofSalmonellain low-moisture foods and on dry abiotic surfaces. To further pursue this objective, we investigated the transcriptome of Salmonella enterica serovar Enteritidis upon inoculation of a panel of low-moisture foods, specifically dry cereal, dry fruit, chocolate and pistachios, at several time points post-inoculation. In addition, survival was monitored for correlations between levels of surviving populations and expression of specific genes. Analysis of the transcriptome of Salmonella inoculated on cornflakes, pistachios and dried apples identified several up-regulated and down-regulated genes, with most pronounced shits in transcriptome profiles being noted in the first 24h. Analysis of the data identified a panel of genes with pronounced differential expression. Deletion mutants in several of these genes were assessed for their survival on pistachios. Analysis of Salmonella on dried fruit revealed that recovery of Salmonella was noticeably lower than from pistachios or corn flakes. Confocal microscopy indicated that a significant fraction of the salmonella population on dried apples were viable but not culturable.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Rahimi S, Kathariou S, Fletcher O, Grimes JL. 2019. Effect of a direct-fed microbial and prebiotic on performance and intestinal histomorphology of turkey poults challenged with Salmonella and Campylobacter. Poult Sci. pii: pez436. doi: 10.3382/ps/pez436.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Good L, Miller WG, Niedermeyer J, Osborne J, Siletzky RM, Carver D, Kathariou S. 2019. Strain-specific differences in survival of Campylobacter spp. in naturally contaminated turkey feces and water. Appl Environ Microbiol. 85(22). pii: e01579-19. doi: 10.1128/AEM.01579-19. Print 2019 Nov 15
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Foster DM, Jacob ME, Farmer KA, Callahan BJ, Theriot CM, Kathariou S, Cernicchiaro N, Prange T, Papich MG. 2019. Ceftiofur formulation differentially affects the intestinal drug concentration, resistance of fecal Escherichia coli, and the microbiome of steers. PLoS One. 14(10):e0223378. doi: 10.1371/journal.pone.0223378. eCollection 2019.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sai K, Parsons C, House JS, Kathariou S, Ninomiya-Tsuji J. 2019. Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing. J Cell Biol. 2019 Apr 11. pii: jcb.201810014. doi: 10.1083/jcb.201810014.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Parsons C, Jahanafroozi M, Kathariou S. 2019. Requirement of lmo1930, a gene in the menaquinone biosynthesis operon, for esculin hydrolysis and lithium chloride tolerance in Listeria monocytogenes. Microorganisms. 7(11). pii: E539. doi: 10.3390/microorganisms7110539.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Jayeola V, Parsons C, Gorski L, Kathariou S. 2019. Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce. FEMS Microbiol Lett. 2019 Apr 1;366(7). pii: fnz076. doi: 10.1093/femsle/fnz076.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Parsons C, Niedermeyer J, Gould N, Brown P, Strules J, Parsons AW, Bernardo Mesa-Cruz J, Kelly MJ, Hooker MJ, Chamberlain MJ, Olfenbuttel C, DePerno C, Kathariou S. 2019. Listeria monocytogenes at the human-wildlife interface: black bears (Ursus americanus) as potential vehicles for Listeria. Microb Biotechnol. doi: 10.1111/1751-7915.13509.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Parsons C, Chen Y, Niedermeyer J, Hernandez K, Kathariou S. 2019. Draft genome sequence of multidrug resistant Listeria innocua strain UAM003-1A, from a wild black bear (Ursus americanus). Microb Resource Announc. 8(47). pii: e01281-19. doi: 10.1128/MRA.01281-19.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Musser ML, Berger EP, Parsons C, Kathariou S, Johannes CM. 2019. Vaccine strain Listeria monocytogenes abscess in a dog: a case report. MC Vet Res. 15(1):467. doi: 10.1186/s12917-019-2216-y.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Food Safety Professionals Food Science professionals Public Health and One Health professionals Microbiology professionals Food Industry (produce, processed foods, food animals) Academia-postdoctoral scientists, graduatestudents, undergraduate researchers, research technicians, faculty, other researchers Academia- classroom outreach for graduate and undergraduate classes Community at large-science communication to the public, citizen science initiatives Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project contributed to the research training and novel method development and optimization skills for three graduate students, one postdoctoral scientist and one undergraduate student. In addition to new laboratory skills, two of the graduate students acquired novel bioinformatics experience through this project. Students in classroom settings became exposed to the challenges associated with AMR in the food chain and to the opportunities for mitigation. Professional development opportunities included the preparation and presentation of posters, manuscripts and other materials, and the attendance and presentations in scientific conferences and in regular within-lab meetings. Major contributions to professional development and networking were mediated by regular and frequent interactions and exchanges with collaborators at North Carolina State University (Poultry Science, Microbiology), the FDA and USDA-ARS. How have the results been disseminated to communities of interest?The project's findings have been disseminated via presentations and exchanges at professional conferences and via peer-reviewed publications in highly-regarded journals. Findings have also been presented during regular meetings of the PI's lab that include former lab members now in diverse locations and professional affiliations in the US and internationally. The project and its results were shared with students in Food Microbiology classes. The PI has frequently described the thrust and objectives of this project in seminars and workshops dedicated to citizen science and science communication. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. To develop and validate the utilization of volatile organic compounds (VOCs) for electronic-sensor-based detection of foodborne bacterial pathogens on leafy greens. In the next reporting period, and with funding from the proposals that were submitted, survival of the pathogens will be investigated in specific model systems. The collaborating engineers will monitor correlations between signal intensity and pathogen concentration to assess specificity and sensitivity of the detection system. Objective 2: To characterize the fate ofListeriaon apples and other fresh produce. In the next reporting period, the samples from the first full year of the two-year assessment will be examined. The genome of the bar-coded strains will be determined and analyzed to confirm similarity with the parental counterparts. Each bar-coded strain will be assed for its survival on apples, in comparison to its parental counterpart. Biofilms will be investigated as source of inoculum for the apples, and the findings will be compared with those from planktonic cells used as inoculum. Objective 3:To characterize genetic diversity, unique genomic attributes and virulence ofListeriain natural reservoirs. In the next reporting period, the genomes of the wildlife and water-derived strains will be further analyzed to determine transmission pathways and elucidate the ecology of Listeria in its natural reservoirs. Virulence assessments will be performed for more strains using Galleria mellonella. Objective 4: To assess emergence, spread and persistence of antimicrobial resistance inCampylobacter. In the next reporting period, transformation-mediated spread of antimicrobial resistance will be investigated in depth. Special attention will be paid on determining potential differences in dissemination of different resistance traits that are mediated by different antimicrobial resistance determinants. A panel of transformants and mutants with specific resistance traits will be constructed for analysis via whole genome sequencing. Objective 5:To characterize survival and virulence ofSalmonellain low-moisture foods and on dry abiotic surfaces. In the next reporting period, the transcriptome of Salmonella from the low-moisture foods will be analyzed over the entire incubation period. Genes that are found to be upregulated on the low-moisture foods will be analyzed by assessments of the survival of mutations harboring deletions in the respective genes. Virulence of Salmonella from the inoculated low-moisture foods will be assessed in Galleria mellonella.

Impacts
What was accomplished under these goals? Objective 1. To develop and validate the utilization of volatile organic compounds (VOCs) for electronic-sensor-based detection of foodborne bacterial pathogens on leafy greens. To further pursue this objective, the Kathariou laboratory participated in the preparation of research proposals with focus on rapid and sensitive in situ detection of foodborne pathogens on fresh produce. Objective 2: To characterize the fate ofListeriaon apples and other fresh produce. To further pursue this objective, a panel of eight barcoded Listeria monocytogenes strains were constructed and characterized for general traits and their capacity to form biofilms and to be virulent in larvae of the invertebrate animal model Galleria mellonella (Greater Wax Moth). Fresh, unwaxed apples from three different regions (Michigan, Pennsylvania, Washington) were inoculated with the panel of the barcoded strains to initiate survival studies. Objective 3:To characterize genetic diversity, unique genomic attributes and virulence ofListeriain natural reservoirs. To further pursue this objective, a panel of Listeria strains from natural reservoirs, specifically surface water and wildlife, were characterized for species, serotype, and resistance to quaternary ammonium sanitizer, the heavy metal cadmium and arsenic, and the antibiotic tetracycline. Water samples from Hurricane Florence were also analyzed for Listeria, yielding a panel of strains that were then characterized. The whole genome sequences of 185 strains were determined. Bioinformatics analysis was pursued on several of the sequenced genomes. Genes mediating tetracycline resistance were identified and localized in the genomes of tetracycline-resistant strains from wildlife. Objective 4: To assess emergence, spread and persistence of antimicrobial resistance inCampylobacter. To further pursue this objective, we investigated transfer of antimicrobial resistance genes in Campylobacter coli and Campylobacter jejuni. Transfer was investigated between pairs of living strains in growth media and in chicken cecal contents. In addition, genomic DNA from C. jeuni and C. coli with specific antimicrobial resistance markers was used to examine the capacity of the resistance traits to be disseminated via transformation, i.e., the uptake of naked DNA by living recipient cells. Emergence of antimicrobial resistance via mutation was also investigated in control experiments without donor DNA in each of these transformation assays.

Publications

• 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. 2018 Feb 1;7(1). pii: E18. doi: 10.3390/pathogens7010018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Lee S, Chen Y, Gorski L, Ward TJ, Osborne J, Kathariou S. 2018. Listeria monocytogenes source distribution analysis indicates regional heterogeneity and ecological niche preference among serotype 4b clones. MBio. 9(2). pii: e00396-18. doi: 10.1128/mBio.00396-18.
• 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. 2018. Next generation microbiological risk assessment-Potential of omics data for hazard characterisation. Int J Food Microbiol. pii: S0168-1605(18)30166-1. doi: 10.1016/j.ijfoodmicro.2018.04.015.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Niedermeyer JA, Lynde R, 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. 84(18). pii: e01212-18. doi: 10.1128/AEM.01212-18.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bolinger HK, Zhang Q, Miller WG, Kathariou S. 2018. 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. doi: 10.1089/fpd.2018.2477.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Parsons C, Lee S, Kathariou S. 2018. Heavy metal resistance determinants of the foodborne pathogen Listeria monocytogenes. Genes (Basel). 10(1). pii: E11. doi: 10.3390/genes10010011.