Sophia Kathariou

The US Food and Drug Administration (FDA) has recognized Salmonella as one of the main causes of food-borne outbreaks associated with shell eggs and egg products. The FDA estimates that 79,000 cases of foodborne illness and 30 deaths each year are caused by eating eggs contaminated with Salmonella (USFDA, 2016a). Over 60% of salmonellosis cases due to contaminated eggs reported by the Centers for Disease Control and Prevention (CDC) have been attributed to Salmonella Enteritidis (SE) (Cao et al., 2009; Vaninni et al., 2009; CDC, 2013). Even though SE remains a leading serovar for egg-associated salmonellosis in the United States, several additional serovars including Typhimurium and Heidelberg also make major contributions (Chousalkar et al., 2018); in the past year a major outbreak involving serovar Braenderup caused illnesses in 10 states and prompted a massive recall of eggs (CDC, 2018). Besides the obvious food safety and public health implications, massive recalls of shell eggs due to frequent outbreaks (CDC, 2018; Food Safety News, 2016; USFDA, 2016b; USFDA, 2010) are accompanied by substantial economic losses to the industry. To reduce the incidence of such outbreaks, the FDA implemented a regulation which requires shell egg producers to take measures to prevent SE contamination of eggs (USFDA, 2009). Washing and disinfecting shell eggs using chemical sanitizers is a common practice to reduce organic load and inactivate pathogens from the exterior of the eggs. These chemical sanitizers may have undesirable consequences such as chemical residues, degradation of the cuticle, development of bacterial resistance, and adverse environmental impacts (Hutchison et al., 2003; Hutchison et al., 2004; Cao et al., 2009; Gole et al., 2014).| Developing novel methods that are less detrimental to the quality of eggs and to the environment, but are effective on pathogen inactivation, is essential. These novel methods should significantly reduce microbial load on eggs and provide protection from cross-contamination without raising safety concerns or causing changes in the integrity, cuticle coverage, and porosity of the egg shell. Recently, increased attention has been paid on novel pathogen inactivation strategies employing plasma, also referred to as the fourth state of matter. Plasma is ionized gas consisting of charged species, excited atoms and molecules, and high-energy photons (Niemira, 2012; Brandenburg et al., 2018; Pankaj et al., 2018; Keener, 2018). Plasma-activated water (PAW) is a novel sanitizer that is generated by exposing water to plasma in the presence of air at atmospheric pressure. The reactive nitrogen (RNS) and reactive oxygen species (ROS) in PAW have been shown to inactivate microbes on a variety of surfaces such as fresh produce (Ma et al., 2015; Xu et al., 2016, Joshi et al., 2018). However, no published data are available on the use and effectiveness of plasma-activated water (PAW) for egg washing. The application of this novel sanitizing procedure is particularly attractive because PAW is safe and environmentally friendly, does not leave potentially harmful residues, and may not adversely impact the outer shell integrity of eggs. In this proposal we will evaluate the use of PAW as a more effective and a less damaging alternative to the conventional methods for egg washing. We hypothesize that the reactive species in PAW will substantially inactivate Salmonella on the shell egg, while not being detrimental to the cuticle layer and the structural integrity of shell egg. Metastable reactive nitrogen and reactive oxygen species in PAW will provide antimicrobial effect without leaving any chemical residue. Keeping the cuticle layer intact will provide protection from bacterial migration through the pores in egg shells during storage and transport and thus increase the shelf-life of eggs. In the long term, PAW can serve as an environmentally friendly and effective sanitizer which can be prepared on site. Our specific objectives are to: 1) Assess the efficacy of PAW for inactivation of a pan

This project will assess the use of visible/natural light-activated carbon nanomaterials, specifically carbon ����������������quantum��������������� dots or carbon dots (����������������CDots���������������), for the highly efficient inactivation of persistent bacterial pathogens on model food contact surfaces under visible/natural light. The specific aims include: (1) To evaluate three selected CDots platforms for inactivating the persistent foodborne pathogens Listeria monocytogenes and Salmonella enterica, established on model food contact surfaces including stainless steel, polyvinyl chloride (PVC), and polyethylene (PE), and (2) Assess the efficacy of CDots for inactivation of these pathogens in mixed-species biofilms, and in the presence of organic material, on stainless steel, PVC and PE surfaces. The Kathariou laboratory will contribute to this project by culturing the bacterial pathogens in planktonic cultures and on biofilms established on different model food contact surfaces, i.e. stainless steel, PVC, and PE. Biofilms will be established and quantified following methods already established in the Kathariou laboratory. Pathogen inactivation will be monitored quantitatively via culture-based enumerations, live-dead staining and quantitative real-time PCR. The impact of CDots concentration, contact time, temperature, presence of other microbes (e.g. Pseudomonas spp., lactic acid bacteria, non-pathogenic Listeria spp.) in mixed-species biofilms, and organic material will be determined.

Listeria monocytogenes (LM) has been repeatedly linked to outbreaks involving fresh vegetables and fruit. In 2014, whole apples (Granny Smith and Gala) were for the first time implicated in a listeriosis outbreak involving both caramel and green apples. LM exhibits no or minimal growth on the surface of whole, intact apples, but can survive for impressive lengths of time and grow inside apples despite the low pH. The various attributes mediating LM survival on whole apples remain poorly understood, with a dearth of data on potential strain-specific differences in survival. Hence, the fate of LM on fresh apples as potentially impacted by both LM inoculum size and physiological state, and the variety of apple remain to be further characterized. Such information is critically needed to identify the risks posed by LM contamination of apples, and to adequately design and validate LM inactivation strategies. To address these important knowledge gaps, we will pursue the following objectives: Objective 1. Determine the fate of LM on apples of three different varieties, from three major apple-producing regions, under two different simulated commercial storage conditions. Objective 2. Characterize the potential impact of inoculum state on the fate of LM fate on apples. Objective 3. Characterize the relative strain fitness of various LM strains on apples. Objective 4. Assess the impact of waxing on the fate of LM on apples. The findings will provide data needed to guide the apple industry on conditions and processes that can minimize the food safety risk from LM in the apple supply.

Hurricane Florence made landfall on the North Carolina coast on September 14, 2018, and in the subsequent days several North Carolina communities received up to 35 inches of rain. Inland rivers did not crest until a week post-landfall, but the extent of cresting was unprecedented. The heavy rainfall and flooding can massively impact water quality and safety in flooded areas, especially via runoff from agricultural and industrial operations. We propose to analyze floodwater samples collected by the hydrology and civil engineering teams led by Ryan Emanuel and Angela Harris at North Carolina State University for prevalence of the human foodborne pathogens Salmonella, Listeria and Campylobacter. These pathogens will be also characterized for species designations and, in the case of Listeria, for serotypes. The data will complement those from the other team members who will monitor source-specific fecal indicators, Escherichia coli, and chemical contaminants in these samples.

Due to a number of food safety concerns with "low moisture foods" (LMF), there has been global recognition of the need to more rigorously consider and manage the microbiological hazards associated with these products. Various serotypes of Salmonella enterica have been the main pathogens involved in outbreaks due to LMF, but currently there are also concerns about the survival of Listeria monocytogenes in LMF such as dried fruits and tree nuts. Our hypothesis is that there will be changes in gene expression profiles of Salmonella and L. monocytogenes over time with storage in LMF, and that these changes will lead to changes in stress tolerance and virulence of the organisms. Such information will inform strategies to control and inactivate pathogens in LMF.

Alternate sanitizing methods are needed to replace detrimental chemicals in produce and egg washing. Plasma-activated water is proving to be a promising and environmentally friendly sanitizer. However research is needed in ensure reproducibility on the level of inactivation by PAW. There is a need to correlate the reactive species concentration to microbial inactivation of gram positive and gram negative organisms. Hence this study will focus on 1. Characterization of PAW to determine reactive nitrogen species (RNS) concentration, electrical conductivity, pH, oxidation- reduction potential (ORP), and hydrogen peroxide (H2O2) concentration, 2. Evaluating efficacy of PAW on gram-positive bacteria (Bacillus cereus and Listeria) and gram-negative bacteria (Salmonella and E. coli) in planktonic system, and 3. Assessment of the efficacy of PAW for inactivation of a panel of pathogenic Salmonella enterica strains representing different serovars during washing of eggs and tomatoes and study its effect on quality This study will provide methods and qualification of RNS concentration, electrical conductivity, pH, ORP, and H2O2 concentration, a systematic approach for assessments of inactivation of gram negative and gram positive bacteria by PAW and its correlation with reactive species concentration and optimized conditions for inactivation of Salmonella on tomatoes and eggs using PAW.

Listeria monocytogenes is the causative agent of the disease listeriosis. Prior to the twentyfirst century the majority of listeriosis outbreaks involved vehicles of animal origin such as seafood, hotdogs, deli meats, and dairy products������������. However, since 2008 produce has become a more frequent vehicle for Listeria. 2011 saw the largest Listeria outbreak in US history, which involved cantaloupe, 2014 saw three separate produce related outbreaks, involving three separate produce vehicles������������, and as recently as January of this year there was an outbreak involving prepackaged salad������������. The ability of Listeria to colonize food processing enviornments, and contaminate a wide array of products of both plant and animal origin, makes it of particular concern for food safety. Listeria is known to be ubiquitous in the environment������������, though there is considerable variation in the ability of differing strains to cause disease. This has lead some to conclude that differing strains have become genetically adapted to differing niches, be that survival in the environment, colonizing production plants, or causing disease in human / animals . While much work has been done characterizing how L. monocytogenes is able to cause disease within the body, and how it is able to colonize food processing plants, the ecology of the pathogen is still poorly understood, particularly how it moves in the environment and comes to contaminate farms and food processing facilities������������. Over the past two years our lab in collaboration with the Department of Forestry and Environmental Resources at North Carolina State University has worked to isolate a collection of strains of L. monocytogenes from wild and peri-urban bears. These strains are unique in that they represent this pathogen in the environment. After characterizing these strains for their virulence potential, and whole genome sequencing, we plan to use these data to address several key questions. The GPS tracking data from the bear's collars will be correlated with the genomic and virulence data to create geospatial and predictive modeling. Then a comparative genomics approach, against other strains of diverse and unique origins (processing plants, produce, outbreak associated with foods of animal origins, and outbreak related to produce) would generate a list of novel gene targets to then be tested with a functional genomics approach to gain an understanding of how Listeria may be using animals as vectors/ reservoirs, as well as its ability to cause disease, colonize animal hosts, or colonization of food and/or processing facilities.

OBJECTIVES OF THIS RESEARCH ARE: (1) Identification of antibiotic resistance genes in the poultry production system through complete genomic DNA; and (2) To characterize the fitness and virulence of Campylobacter jejuni and Campylobacte coli strains analyzed in Objective 1.

Listeria monocytogenes (LM) remains a major contributor to severe illness and death due to foodborne disease in the United States. Much work has focused on LM?s colonization and persistence in the environment of food processing plants. There is, however, a dearth of information on LM?s pre-harvest associations with produce, even though produce contaminated with this pathogen has been implicated in outbreaks of listeriosis and in an increasing number of recalls. Recent findings suggest that Listeria colonizes produce pre-harvest and that removal from the colonized surface of the produce is quite difficult. It is thus imperative that we elucidate the mechanisms that Listeria employs to colonize produce pre-harvest and to persist on edible portions of the plant. The overall hypothesis is that LM employs a dedicated set of mechanisms to adhere to produce pre-harvest and to persist on edible portions of the plant, thus posing food safety risks at harvest. The goal is to employ experimental approaches to elucidate these mechanisms in the context of leafy greens (spinach, lettuce) and other produce. Strains will be screened for produce colonization ability and efficiently colonizing representatives of different lineages will be used for transposon mutant library construction. The libraries will be screened for mutants that are altered (either impaired or enhanced) in their ability to colonize produce and to persist in the edible portions of the plant. Genes of relevance will be identified and further characterized via deletion mutagenesis and genetic complementation. RNA-Seq will be employed to characterize LM transcriptional profiles during colonization of produce. Transcription of genes found to be induced will be further characterized via qRT-PCR, and transcription of promising candidates will be analyzed under different conditions of relevance to produce colonization. Biofilms on produce surfaces will be characterized via confocal microscopy and viable count determinations. Findings from the project will elucidate currently poorly understood mechanisms that LM employs for persistent colonization of produce. Understanding of molecular mechanisms mediating persistent colonization of produce will be critical for design of novel strategies to prevent or minimize contamination of produce pre-harvest.

Campylobacter is a leading bacterial cause of acute human gastroenteritis in the United States, and contaminated poultry is a leading risk factor for human infection. Campylobacteriosis is especially prevalent in children < 5 years old (approx. 23 cases/100,000). In addition to diarrheal symptoms, campyobacteriosis can have severe autoimmune sequelae. Campylobacter colonizes chickens and turkeys, and can contaminate poultry products during processing. Understanding of the mechanisms via which Campylobacter enters the poultry production system and colonizes the birds is critical to development of science-based interventions to reduce or prevent campylobacter on the meat. The overall, long-term goal of our research is to identify and characterize routes and mechanisms of transmission of Campylobacter to poultry. This will be necessary for targeted approaches to prevent or reduce colonization of poultry pre-harvest and subsequent contamination of poultry products with this pathogen. To contribute to the pursuit of this long-term goal, the following three specific objectives will be pursued in the current project: In Specific Objective 1, we will identify farm management practices that contribute to either consistently low (or below detectable levels) or consistently high prevalence of Campylobacter in turkey flocks pre-harvest. In Specific Objective 2, we will monitor Campylobacter prevalence in turkeys from flocks with low or high colonization levels, following slaughter and processing in commercial processing plants. Lastly, in specific Objective 3we will develop outreach materials customized for farms with high or low prevalence of Campylobacter in their turkey flocks, and evaluate the impact of this outreach on knowledge, farm management practices, and Campylobacter contamination levels of the birds pre-harvest and at the processing plant.