USE OF WHOLE GENOME SEQUENCING AND OTHER APPROACHES TO INVESTIGATE A POTENTIAL WILDLIFE RESERVOIR FOR THE FOODBORNE PATHOGEN, LISTERIA MONOCYTOGENES.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1011225
• Grant No.: 2017-67012-26001
• Project No.: NC09830
• Proposal No.: 2016-04709
• Program Code: A7201
• Project Start Date: Dec 1, 2016
• Project End Date: May 31, 2019
• Grant Year: 2017

Project Director
Parsons, C.

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

Performing Department
Food, Bioprocess & Nutrition S

Non Technical Summary
Listeria monocytogenes is a facultative intracellular pathogen responsible for listeriosis, a severefoodborne illness in humans and animals. Listeria can be found ubiquitously in nature, having beenisolated from humans, animals, plants, water, and soil. While much is known about the ability ofListeria to cause disease, its behavior in the wild is still reasonably unknown. A recent survey ofwild and peri-urban bears revealed that they are asymptomatic carriers of L. monocytogenes. Thisstudy would seek to characterize the virulence potential of these nature derived strains, thensequence their genomes. These data could then be correlated with geospatial data obtained fromGPS collars that are used to track the bear's movement to obtain information about the movement ofthe pathogen in the wild. The genomes of these strains would also provide an insight into theadaptation of Listeria in nature, enabling a comparative genomic analysis between these strains andthe genomes of strains derived from a variety of outbreak sources. The genetic data from these wildbears offers the opportunity to not only be able to view the movement of this pathogen in bothnatural and urban settings, but also identify the mechanisms by which Listeria is able to causedisease, colonize an animal host, and contaminate food / processing environments.

Animal Health Component

20%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
722	4010	1100	100%

Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

listeria

monocytogenes

zoonosis

ursus

americanus

comparative

genomics

food

safety

Goals / Objectives
1) To employ whole genome sequencing and sequencing informed analysis to gain an understanding ofthe characteristics of L. monocytogenes in an environmental reservoir.a) To characterize the virulence potential of wild isolates for comparison against those of food/ processing / outbreak origins.b) Whole genome sequencing and phylogenetic analysis of bear isolates.2) Environmental survival and movement of L. monocytogenes in relation to an animal host.a) Discern regionality of L. monocytognes isolated from bearsb) Examine known factors of animal host and environment to assess impacts on L.monocytogenes in the wild.3) To identify and characterize genes that are involved in the adaptation of L. monocytogenes to ananimal host.a) Comparative genomic analysis to identify genes unique to L. monocytogenes isolated frombears.b) Comparative genomic analysis to identify genes from food outbreaks that may or may notbe lacking in L. monocytogenes isolated from wild bears.

Project Methods
The bear isolates would first be characterized for antibiotic resistance, as describedpreviously (Srinivasan et al., 2005). Virulence assessments would then be carried out in the Galleriamellonella model as described previously (Mukherjee et al., 2010), the mentor's lab has used this modelextensively and successfully in the past. This would involve both observation for mortality of thelarvae, but also enumeration of bacterial load over time after infection to assess asymptomatic carriageas described previously (Joyce & Gahan, 2010). At least two isolates from the same animal, with thesame serotype and antibiotic resistance profile would be selected for whole genome sequencing.Genome sequencing would focus preferentially on L. monocytogenes strains, though all Listeria spp.isolated would be tested for antibiotic resistance to assess its prevalnce. Genome sequencing would becarried out by collaborators at the Food and Drug Administration (FDA) Center for Food Safety andApplied Nutrition (see letter of collaboration by Dr. Marc Allard, FDA). The FDA has implementedtheir Whole Genome Sequencing (WGS) Program(http://www.fda.gov/Food/FoodScienceResearch/WholeGenomeSequencingProgramWGS/) tosequence isolates collected from foodborne outbreaks, contaminated food products and environmentalsources. They are making these sequences publicly available through a database called GenomeTrakr.This allows access to a large pool of strains from diverse origins all of significance to food safety,which include strains isolated from outbreaks involving vehicles of animal origin, from outbreaksinvolving produce as vehicles. WGS of the bear isolates will be accompanied by in-silicodeterminationof multi locus sequence typing (MLST) to facilitate assessment of these strains in thecontext of the extensive MLST database, and phylogenetic analysis would be done utilizing WGS andMLST data as described previously (Lee et al., 2013). Gene targets identified through comparativegenome assessment, specific to the wild bear strains, would then be tested in vivo through eithercomplementation or deletion to validate gene function. All of these techniques are feasible to execute,within the two year project timeline. Collaborators at both the North Carolina Museum of NaturalSciences, and the Cooperative Institute for Climate and Satellites (see letters of support from ArielleParsons and Paula Hennon) have offered data and analysis assistance for the construction of geospatialand statistical models related to the movement and instance of the pathogen in the environment. Asubstantial amount of the material required is already in place, collaborators with the necessarymaterial and expertise have already expressed their enthusiasm and support of this project, the mentor'slab is adequately equipped with resources (including the strain library) and expertise for the work, andthe program director already has experience performing a large number of the techniques required forthe execution of this project.Outcomes: Outcomes of virulence assessments will give us an understanding of the nature of thestrains inhabiting the wild bear population. Knowing their ability to cause disease will inform if thesestrains pose a health risk to humans and other animals, or if these are stains that have become adaptedto life as commensal organisms inside bears with attenuated or reduced virulence potential. In eithereventuality, whole genome sequencing and MLST data should provide valuable insight to therelatedness and predicted functional capabilities of the wild bear derived strains. Knowing theirrelatedness to strains that have caused public health problems will also elucidate the degree to whichwild animals may serve as transmission routes for L. monocytogenes into the food supply.

Progress 12/01/16 to 05/31/19

Outputs
Target Audience:The audiences reached by the efforts in this reporting period have been largely the scientific community, though given the interdisciplinary nature of this project the scientific audiences addressed were rather diverse. There were numerousposter presentations of work associated with the project, as well as several talks and published papers. These mediahave spanned several different disciplines and scientific societies, so the variety of the scientific audience reached has been rather broad. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project was a collaborative research effort involving at least three universities, and two different federal government agencices. Given the large amount of share holders and collaborative input there was a considerable amount of networking and professional development for many of the parties invovled, most especially the project director. Additionally these same collaborators were able to furnish a great deal of training to the project director involving a wide array of technical and professional skills applicable not just to performing bioinformatic analysese, but also best practices for implementing and executing a collaborative research project spanning multiple universities and agencies. How have the results been disseminated to communities of interest?The project has so far resulted in two peer reviewed publications, of which two more are currently in preparation for submission and there are plans for another three beyond that. There were also three oral presentations and six poster presentations at eigth different conferences, presenting this work. These audiences have spanned microbiologists, wildlife biologists, those working in the broader field of genetics as a whole, and the entire campus community at North Carolina State University. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project isolated, genome sequenced, and functionally characterized Listeria isolated from black bears in the southeastern United States. While Listeria from clinical and food-associated sources has been well characterized, much of Listeria's ecology in the natural environment is still poorly understood. Our study was able to determine the prevalence of both pathogenic and non-pathogenic Listeriain black bears, and get a relative measure of its relation toListeria monocytogenesfound in clinical cases and in the food supply. Our study found a close relation between some isolates from the study and some clinical cases, suggesting a common source, and also suggesting that wildlife, particularly black bears, are functioning in the circulation ofListeriabetween humans and the natural environment. We also identified a large number of novel genotypes of this pathogen, one of which was later identified in a clinical case of listeriosis, this along with the isolation of several emerging clones ofL. monocytogenessuggests that not only do these animals harbor novel variants of this pathogen, but many of them are of an emerging nature, suggesting continued wildlife surveillance would have a beneficial impact on food safety. Lastly, our functional assessments of both virulence potential and antibiotic resistance of these isolates revealed these strains to be very capable of causing disease, and while pathogenic strains were unlikely to harbor antibiotic resistance there was a considerable amount of resistance among non-pathogenic species altering us to their potential to transmit these determinants between pathogenic and non-pathogenic specices. 1) To employ whole genome sequencing and sequencing informed analysis to gain an understanding of the characteristics ofL. monocytogenesin an environmental reservoir. a) To characterize the virulence potential of wild isolates for comparison against those of food/ processing / outbreak origins. Genome sequence data and virulence model data showed these wild isolates are fully virulent, possessing the knownL. monocytogenesvirulence factors, and demonstrating full virulence in aGalleria melonellavirulence model. In some cases wildlife isolates showed greater virulence than outbreak strains, even of the same genotype. b) Whole genome sequencing and phylogenetic analysis of bear isolates. Phylogenetic analysis of bear isolates revealed a large proportion of novel genotypes that had not been previously reported, as well as a notable lack of many of the genotypes previously identified in other large scale genetic surveys ofL. monocytogenes. 2) Environmental survival and movement ofL. monocytogenesin relation to an animal host. a) Discern regionality ofL. monocytognesisolated from bears Samples were collected from three different geographic locations (NC, GA and VA), with roughly equivalent proportions of recovery at all three locations, there were no significant distinctions found in either temporal or genotypic recovery between locations. b) Examine known factors of animal host and environment to assess impacts onL. monocytogenesin the wild. A multivariate model was applied to animal movement and biometric data, and the only factor that was determined to be significant was that there was more spatial overlap between bears that were positive than those that were negative, suggesting a social aspect to the circulation ofL. monocytogenes?among wild black bears. 3) To identify and characterize genes that are involved in the adaptation ofL. monocytogenesto an animal host. a) Comparative genomic analysis to identify genes unique toL. monocytogenesisolated from bears. Comparative analysis suggested that among the genotypes tested there were no genes differentially associated with wildlife when compared against strains from other isolation sources. b) Comparative genomic analysis to identify genes from food outbreaks that may or may not be lacking inL. monocytogenesisolated from wild bears. All genotypes tested failed to indicated separate gene content associated with different sources, again supporting the assertion that those strains recovered from wildlife were fully capable of causing disease and are of a signficant public health concern.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Parsons, Cameron, Niedermeyer, Jeff; Gould, Nicholas; Brown, Phillip; Strules, Jennifer, Parsons, Arielle W, Bernardo Mesa?Cruz, J, Kelly, Marcella J, Hooker, Michael J, Chamberlain, Michael J, DePerno, Christopher, & Kathariou, Sophia. 2020. Listeria monocytogenes at the humanwildlife interface: black bears (Ursus americanus) as potential vehicles for Listeria Microbial Biotechnology 13 (3), 706-721
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Parsons, Cameron, Chen, Yi, Niedermeyer, Jeffrey, Hernandez, Kevin, Kathariou, Sophia. 2019. Draft Genome Sequence of Multidrug-Resistant Listeria innocua Strain UAM003-1A, Isolated from a Wild Black Bear (Ursus americanus). Microbiology resource announcements 8 (47)
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: DePerno, C., C. Parsons, J. Niedermeyer, P. Brown, N. Gould, J. Strules, B. Mesa-Cruz, M. Kelly, M. Hooker, M. Chamberlain, C. Olfenbuttel, and S. Kathariou,. Black bears (Ursus americanus) are novel vehicles for Listeria monocytogenes. 24th Eastern Black Bear Workshop, YMCA of the Ozarks Trout Lodge, Potosi, Missouri.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Kathariou,S., C. Parsons, J. Niedermeyer, P. Brown, N. Gould,V. Dutta, D. Elhanafi. Listeria Clones in the Anthropocene: Insights from Urban Wildlife and Water. International Symposium on Problems of Listeria and Listeriosis (ISOPOL) 2019
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Parsons, Cameron, Niedermeyer, Jeff; Gould, Nicholas; Brown, Phillip; Strules, Jennifer, Parsons, Arielle W, Bernardo Mesa?Cruz, J, Kelly, Marcella J, Hooker, Michael J, Chamberlain, Michael J, DePerno, Christopher, & Kathariou, Sophia. Emerging IVb-v1 clone (CC554) of Listeria monocytogenes is highly prevalent among strains from suburban black bears (Ursus americanus)9th annual NC State Program in Genetics Retreat

Progress 12/01/16 to 11/30/17

Outputs
Target Audience:Thus far the audiences reached by the efforts in this reporting period have been largely the scientific community. While there have been several poster presentations of work associated with the project, they have been at conferences and meetings frequented primarily by scientists. These meeting have spanned several different disciplines and scientific societies, so the variety of the scientific audience reached has been rather broad. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided me the opportunity to attend training seminars and access to several different kinds of software for performing genomic comparisons and analyses further my technical as well as biological knowledge. I have also had the opportunity to guest lecture in several classes pertaining to both bacterial pathogens, genomic analysis, and wild animal pathogen vectors. I have also been able to attend several conferences and symposia that have allowed me to expand my professionial network and set up potentially fruitful future collaborations. How have the results been disseminated to communities of interest?Thus far results have been disseminated primarily through presentations at academic conferences. What do you plan to do during the next reporting period to accomplish the goals?There are plans to continue presentations at academic conferences. There has been discussion of potentially three different journal articles associated with this project. The first is in preparation presently and should be submitted in the next coming weeks with the second to follow soon after and the third requiring a greater degree of completion of the project before it can be completed. We have ideas for two different social media platforms that should also be up and running within the next few weeks.

Impacts
What was accomplished under these goals? 1) Sequencing of L. monocytogenes isolates from wild reservoirs is commencing and results to date have been and continue to be analyzed. Novel mobile genetic elements have been identified, as well as several novel resistance determinants. a) virulence potential of these isolates has been and continues to be assessed in both a lepidopteran and cell culture model. Thus far all strains tested appear to be fully equivalently virulent with known outbreak strains, with several demonstrating higher virulence potential than known outbreak strains. b) phylogentic analysis of sequenced strains has shown several sequence types that have been shown to cause disease in North Carolina, but also there are a large portion of strains that belong to previously uncharacterized sequence types. Work is ongoing to submit these novel sequence types to the Pasteur institute in Paris France for assignment. 2) Collaborators at both ORACLE and NC State University are working to assist with correlating genomic data with available bear movement data to assist in forming a model that incorporates climate data as well to allow us to asses strain movement and environmental factors that might be associated with L. monocytogenes colonization. We have been able to establish that several sequnce types of strains isolated from bears were identical to those found in individuals within North Carolina sick with Listeriosis, work is ongoing to establish relatedness between the strains isolated from the wild and those found in clinical patients. 3) As more genomes are made available work can now begin on the comparative analysis now that enough data is availble.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Parsons, C., Niedermeyer, J., Brown, P., Kathariou, S., Gould, N., Strules, J., Mesa, B., Kelly, M., Hooker, M., Chamberlain, M., Olfenbuttel, C., and C. S. DePerno. Black bears as novel vectors/reservoirs for potential human and animal pathogens. 23rd Annual Conference of The Wildlife Society, Raleigh Convention Center, Raleigh, North Carolina.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Parsons, C., Niedermeyer, J., Brown, P., Kathariou, S., Gould, N., Strules, J., Mesa, B., Kelly, M., Hooker, M., Chamberlain, M., Olfenbuttel, C., and DePerno, C. Prevalence and characteristics of Listeria monocytogenes isolated from black bears, Ursus americanus. American Society for Microbiology Microbe 2017 Conference. June 1-5; New Orleans, LA winner of Outstanding Abstract Award
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Brown P., Parsons C., Kathariou S. Phenotypic characterization of a putative lantibiotic biosynthesis genomic island in Listeria monocytogenes. North Carolina branch of the American Society for Microbiology annual meeting 2017. October 21 2017 Raleigh NC.
• Type: Journal Articles Status: Other Year Published: 2018 Citation: Parsons, C., Niedermeyer, J., Brown, P., Kathariou, S., Gould, N., Strules, J., Mesa, B., Kelly, M., Hooker, M., Chamberlain, M., Olfenbuttel, C., and DePerno, C. Prevalence and characteristics of Listeria monocytogenes isolated from black bears, Ursus americanus.