Molecular mechanisms for interactions between Listeria monocytogenes and produce

MOLECULAR MECHANISMS FOR INTERACTIONS BETWEEN LISTERIA MONOCYTOGENES AND PRODUCE

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

TERMINATED

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

0227646

Grant No.

2012-67017-30218

Project No.

NC09239

Proposal No.

2011-03603

Multistate No.

(N/A)

Program Code

A1331

Project Start Date

Feb 15, 2012

Project End Date

Feb 14, 2017

Grant Year

2012

Project Director
Kathariou, S.

Recipient Organization
NORTH CAROLINA STATE UNIV
COLLEGE OF VETERINARY MEDICINE
RALEIGH,NC 27606

Performing Department
Food, Bioprocessing, and Nutrition Sciences.

Non Technical Summary
Listeria monocytogenes is a major cause of severe foodborne illness and death in the United States. Even though several high-profile outbreaks of listeriosis have involved deli meats and soft cheeses, current epidemiological trends suggest that Listeria -contaminated produce is associated with significantly higher disease burden than previously recognized. A multistate outbreak in 2008-09 was attributed to soybean sprouts, and in 2010 an outbreak with high mortality was due to chopped celery. This past summer and fall one of the largest known outbreaks of listeriosis (133 cases, 28 deaths) involved contaminated cantaloupe. Listeria is also responsible for numerous recalls. However, in spite of the evident public health and economic burden of Listeria-contaminated produce, only rudimentary knowledge is available on mechanisms and attributes mediating Listeria's ability to adhere to and colonize produce. Under Objective 1, produce dip inoculation will be employed to assess produce adherence and colonization ability of a carefully chosen panel of strains. Strains with high adherence and colonization potential will be chosen to identify and characterize genes required for adherence and colonization, in Objective 2. In Objective 3 we will employ molecular, bacteriological and microscopy tools to characterize biofilm formation on stainless steel and on produce surfaces, taking into account the presence of produce-associated microbiota. This component of the study is especially relevant for Listeria, a bacterium which is notorious for its ability to persistently colonize the food processing plant environment. Such environmental contamination has indeed been implicated in the recent cantaloupe outbreak of listeriosis as well as in the previous celery-associated outbreak. The study will provide baseline data critical for novel targeted strategies to reduce food safety threats associated with Listeria-contaminated produce, including threats associated with persistent colonization of produce-processing facilities and with bacterial adherence and growth on the produce itself.

Animal Health Component

(N/A)

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

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;

Keywords

listeria on produce

strains from produce outbreaks

listeria strains and lineages

listeria serotypes

pathogen adherence and growth

molecular mechanisms

mutant screening

gene identification

functional genetic analysis

produce colonization assessment

stainless steel biofilms

biofilms on produce

Goals / Objectives
The long term goal of the research is to characterize the mechanisms mediating produce colonization with Listeria monocytogenes, and to design novel control strategies. To contribute to this long term goal, the following specific objectives will be pursued: Objective 1: To assess relative produce colonization ability of a panel of strains of L. 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 role of the genes on biofilm formation by L. monocytogenes on stainless steel and produce surfaces.

Project Methods
Objective 1. A carefully chosen panel of 48 strains will be employed to determine the relative ability of the bacteria to survive and grow on lettuce and on soybean sprouts, cantaloupe and celery at different temperatures. Bacteria will be enumerated from the inoculated produce incubated at different temperatures. Objective 2. We will employ two different Listeria monocytogenes strains, and two mariner mutant libraries for each strain. A pool of non-adherent mutants will be obtained from the rinsate of inoculated produce, along with an adherent pool which will remain on the surface. The mariner-based transposon-Listeria junction fragments in each pool will be processed for 454 sequencing and genes with different representation in each pool will be identified. The involvement of selected genes in colonization of produce will be characterized by deletion construction and genetic complementation analysis. Objective 3. Mutants and their wild type parental counterparts will be analyzed in biofilms on stainless steel and on produce surfaces. Stainless steel biofilms will be constructed with pure cultures as well as with Listeria in the presence of produce-derived microbiota. Viable L. monocytogenes will be enumerated by quantitative real-time PCR (qPCR) with propidium monoazide. For biofilm assessments on produce, L. monocytogenes will be inoculated onto produce and inoculated surfaces will be screened by confocal laser scanning microscopy.

Progress 02/15/12 to 02/14/17

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. Agriculture and rural communities: Discontinuation of production due to implication in disease adversely impacts the economic vitality of US agriculture and compromises the wellbeing and sustainability of agricultural communities. Food safety and public health: Their mission includes reducing prevalence of human listeriosis, for which contaminated produce has emerged as major vehicle. Community-consumers: Listeria contamination is a potentially life-threatening threat for several populations. Consumers are directly impacted by listeriosis and associated disease burden and are keenly interested in home-based procedures to minimize risk of disease from contaminated produce; in fact, our Martinez et al 2016 article, J Food Prot. 79(5): 757-763 , included a simple method that can be employed at home to reduce risk from Listeria-contaminated cantaloupe within a certain time window. Recent findings from listeriosis outbreaks, including the one via caramel apples, revealed that listeriosis risk extends beyond previously documented categories of the severely immocompromised, elders, and pregnant women: healthy children / young teens were among the Listeria meningitis patients. 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 molecular genetic and bacteriological attributes mediating Listeria's ability to colonize fresh produce. Students in Food Microbiology classes benefited from case studies are other knowledge-acquisition modules with focus on Listeria and produce. Visiting scientists, postdocs and technical staff furthered their professional development via further experience with molecular and microbiological components of interactions between Listeria and produce, 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. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Eight High School students, five undergraduate and 11 graduate students acquired research experience working on the project. Experience included assays for adherence and growth on produce, genetic characterization and manipulation of LM, analysis of bioinformatics data, construction and screening of mutant libraries, biofilm assays and statistical analysis of microbiological data. Students also acquired experience in presentation of findings for conferences, workshops and manuscripts as well as in preparation of blogs and other material for outreach. Four technical staff acquired further experience in microbiology, food safety and molecular biology. Technicians were trained on sequencer machines (Illumina Mi-Seq), biofilm assays, produce colonization assays, biofilm determinations 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. Four postdoctoral scientists and one visiting scientist acquired further experience in food safety, microbiology, molecular biology and bioinformatics. Graduate students, technical staff and postdoctoral scientists acquired experience in mentoring and supervision of junior laboratorians working on the project. How have the results been disseminated to communities of interest?Fresh Produce Industry: Findings were presented at the North Carolina Food Safety and Defense Task Force annual meeting, and NC Governor's Food Safety Task Force conference. Both conferences included strong participation by the North Carolina fresh produce industry and the food industry in general. Kathariou informally shared findings from the project during the 2015 Center for Produce Safety (CPS) Research Symposium (Atlanta, GA). Project-related information was disseminated also via an article in New Food, a journal with wide reach to the industry, and with a blog in Food Safety News. Research and Academic Community: Findings were presented via peer-reviewed manuscripts, including an invited review article in J. Food Prot. on produce-related outbreaks of listeriosis; posters and presentations at regional and international (American Society for Microbiology, International Association for Food protection) conferences; workshop participation; and seminars at North Carolina State University and elsewhere. Findings from the project were regularly presented via case study formats and "teachable moments" in the Food Microbiology class that Kathariou taught annually 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. The Food Safety News blog (http://www.foodsafetynews.com/2015/04/listeriosis-and-produce-whats-the-connection/#.VcPguSpVikq) reached numerous individuals in the community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: We tested 30 strains of Listeria monocytogenes (LM) for adherence and produce on fresh produce (cantaloupe rind, celery, lettuce). These included strains from produce-related outbreaks (celery, cantaloupe, caramel apple, stone fruit); strains from outbreaks involving other vehicles; sporadic human listeriosis strains; strains from food and environmental samples. Internalin mutants with premature stop codons in inlA were also included.Significant source-dependent differences in adherence or growth among strains were not noted. To some degree all strains attached to produce tissue, and the best-attaching strains were of serotypes 4b and 1/2b (lineage I). The best attaching strains exhibited a 2 log increase after 90 minutes post-inoculation and grew further thereafter. The best-attaching strain (strain 2387) was chosen to introduce a GFP plasmid for confocal microscopic analysis of LM interactions with lettuce tissue. Numerous efforts were made to establish a lettuce seedling model to assess ability of different LM strains to adhere and grow, and also for subsequent testing of mutant libraries. However, the lettuce seedling model proved challenging due to little / no colonization and inconsistent data. Therefore, romaine lettuce leaves available at retail were used for lettuce colonization assays. Pieces of lettuce were exposed to LM by immersing in a suspension of LM for 90 sec, and then allowing adherence for 90 minutes before vigorous washing to enumerate cells attached to the plant tissue. 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. 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 revealed that most GFP-LM cells were congregated at the cut edge of the leaf and around the stomatal openings. This may reflect preferential adherence to sites with nutrient leakage or not occupied by resident microbiota. We saw no evidence of GFP-LM internalization into the lettuce leaf. A panel of outbreak strains was characterized for virulence using the insect model, Galleria mellonella. Strain-dependent differences were noted but overall the strains did not differ significantly from genetically unrelated strains implicated in other outbreaks (deli meats, cheese). Objective 2 : We optimized protocols utilizing ampicillin selection to enrich for mutants unable to grow on cantaloupe rind. 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 was based on the fact that ampicillin kills only growing cells, 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. A panel of mutants were identified rom the 4 deg. C selection. As expected, some had impaired cold tolerance. However, mutants without noticeable cold growth impairment but still unable to grow on cantaloupe rind at 4 deg C were identified. Collections of the recovered ampicillin-selection populations were sent to the Gorski and Miller laboratories (co-PIs) for analysis by next-generation sequencing approach to identify insertions over-represented among the ampicillin-treated populations, in comparison with populations in the absence of ampicillin. In addition, we utilized a repeat-rinse model to enrich for mutants unable to adhere. The rind fragments were inoculated with a mutant library and then rinsed. The rinsate (enriched in mutants unable to tightly adhere on the fragment) was used as inoculum for new fragments, with the process repeated 4 times. Mutant libraries were derived from strain 2858, serotype 1/2b strain from the 2011 cantaloupe outbreak; strain 1723, serotype 1/2a strain from the 2010 celery outbreak; and the serotype 4b celery-derived strain F8027. With all mutant libraries, populations in the rinsate declined to ca. 300 CFU/ml by the 4th successive rinse. Targeted analysis of mutant libraries revealed multiple non-hemolytic, non-motile and cold-sensitive mutants. Two non-hemolytic mutants (insertions in hly and prfA) were found significantly impaired in biofilm formation but not in growth and relative fitness on cantaloupe rind. Two motility-impaired mutants were characterized for growth on cantaloupe. One mutant , with a single insertion in a motility and chemotaxis gene cluster was not impacted in growth or fitness. The other non-motile 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. Objective 3: we tested a panel of 34 LM strains for biofilm formation on PVC (hydrophobic surface), borosilicate glass (hydrophilic surface), and stainless steel. The strains were also tested for both adherence (after a 90 minute exposure to LM) and growth on lettuce at 4 and 25 degrees C. 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. PVC and glass biofilms were stained with crystal violet and measured by A580 determinations. There were 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. This was surprising, as found that under certain time-temperature combinations LM exhibited more growth grew more on cantaloupe rind than flesh or in cantaloupe juice, and hypothesized that biofilm formation was involved in the observed growth on the rind. A similar lack of correlation between biofilm-forming capacity and adherence/growth on fresh produce was noted with transposon mutants (in the listeriolysin O gene hly; the virulence regulator gene, prfA; the metal tolerance determinant cadA4; and the penicillin-binding protein gene pbp4) which were significantly impaired in biofilm formation but were not impaired in adherence and growth on fresh cantaloupe or celery.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Gorski L, Liang, A, Romanolo K F, 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 Annual Meeting. Portland, OR.
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dutta R, 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
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dutta V, Ward T, Lee S, Parsons P, 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.
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Niedermeyer, JN, Melander C, Kathariou S. 2015. Novel compounds that inhibit biofilm formation by Listeria monocytogenes. Center for Advanced Processing and Packaging Studies Conference, Columbus, OH
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kathariou S, Melander C. 2015. Assessment of novel synthetic and naturally occurring compounds to inhibit and disperse biofilms by spoilage and pathogenic bacteria of concern to the food industry. Center for Advanced Processing and Packaging Studies Conference, Raleigh, NC
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Rakic-Martinez M, Parsons, C, Jayeola V, Price R,. Gorski L, Kathariou S. 2015. Molecular Mechanisms for Interactions Between Listeria monocytogenes and produce. NIFA Investigators Annual meeting, Portland, OR.
Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Price R, Kathariou S, Parsons C, Costolo B. 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: Gorski L, Liang AS, Walker S, Nguyen KM, Govoni J. 2014. Prevalence and serotypes of Salmonella and Listeria monocytogenes in a public access watershed sites near a central California agricultural region. Proceedings of 113th General Meeting of American Society for Microbiology, May 17-20, 2014, Boston, MA.
Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Dutta V, Evans P, Kathariou S. 2014. Comparative analysis of gene synteny for adaptive molecular determinants utilizing sequenced genomes of Listeria spp. Proceedings of 113th General Meeting of American Society for Microbiology, May 17-20, 2014, Boston, MA.
Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Parsons C, Martinez RM, Elhanafi D, Kathariou S. 2014. Genetic evidence for confirmation of the role of a novel gene (cada4) in cadmium resistance of Listeria Monocytogenes, and virulence assessment in an insect model. Proceedings of 113th General Meeting of American Society for Microbiology, May 17-20, 2014, Boston, MA
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Parsons C, Lee S, Kathariou S. 2013. Characterization of a novel cadmium resistance gene in Listeria monocytogenes. Proceedings of 112th General Meeting of American Society for Microbiology.
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Martinez RM, Parsons P, Azizoglu RO, Siletzky RM, Kathariou S. 2013. Survival and growth of Listeria monocytogeneson cantaloupe. International Association for Food Protection Annual Meeting, July 2831, Charlotte, North Carolina
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lee S, Kathariou S. 2013. Distribution of a genomic island harboring arsenic resistance genes in Listeria monocytogenes and other Listeria spp. 112th General Meeting of American Society for Microbiology, May 18-21, 2013, Denver, CO.
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: Other Status: Other Year Published: 2015 Citation: Gorski L. 2015. INVITED SEMINAR AND EDUCATIONAL OUTREACH: Molecular Interactions between Listeria monocytogenes and Produce. Western Food Safety Summit, Hartnell College
Type: Other Status: Other Year Published: 2013 Citation: Kathariou, S. 2013 GUEST LECTURE/SEMINAR. Listeria and Produce safety; career and interests in food safety and food microbiology. MB360 class series, North Carolina State university
Type: Other Status: Published Year Published: 2013 Citation: Kathariou, S. 2013. EDUCATIONAL OUTREACH: Food Safety Research, Education and Extension at Food, Bioprocessing and Nutrition Sciences, NCSU. Fulbright Scholars Networking Event, November 5, 2013.
Type: Other Status: Published Year Published: 2017 Citation: Kathariou, S. 2017. EDUCATIONAL OUTREACH: 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. 2016. EDUCATIONAL OUTREACH: Food Safety: the academy, industry, regulation and public health nexus. Eisenhower School Agribusiness Study group site visit presentation
Type: Journal Articles Status: Other Year Published: 2013 Citation: Kathariou, S. 2013. Disinfectant and heavy metal resistance in Listeria: Genome signatures for environmental history. Invited seminar, FDA JFSAN, College Park, Maryland
Type: Websites 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: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Kathariou S, Evans P, Dutta V. 2017. Strain-Specific Virulence Differences In Listeria monocytogenes: Current Perspectives In Addressing An Old And Vexing Issue. In: M. Doyle, J. Kornacki and J. Gurtler (ed). Foodborne Pathogens: Virulence Factors and Host Susceptibility. Springer Publishing Co.
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: Book Chapters Status: Published Year Published: 2015 Citation: Marquis, H., D. A. Drevets, M. S. Bronze, S. Kathariou, T.G. Golos, and J. I. Iruretagoyena. 2015. Pathogenesis of Listeria monocytogenes in Humans. In: S. Singh (ed). Emerging and Re-Emerging Human Infections. John Wiley and Sons.
Type: Book Chapters Status: Published Year Published: 2015 Citation: Azizoglu RO, Dutta V, Breidt F Jr, Kathariou S. 2015. Resistance of Listeria monocytogenes Biofilms to Sanitizing Agents. In: Biofilms in the Food Environment, Second Edition. Pp. 51-83, Edited by Anthony L. Pometto and Ali Demirci. � 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.
Type: Book Chapters Status: Published Year Published: 2012 Citation: Lee, S, Siletzky RM, Kathariou S. 2012. Epidemiology, Pathogenesis, Ecology and Genetics of Listeria monocytogenes. In: S. M. Faruque (ed). Foodborne and Waterborne Bacterial Pathogens: Epidemiology, Evolution and Molecular Biology. Caister Academic Press.
Type: Journal Articles Status: Published Year Published: 2017 Citation: Dutta V, Lee S, Ward T, Orwig N, Altermann E, Jima D, 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: Parsons C, Lee S, Jayeola V, Kathariou S. 2017. Novel cadmium resistance determinant in Listeria monocytogenes. Appl Environ Microbiol 83(5). pii: e02580-16. doi: 10.1128/AEM.02580-16.
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: Pan P C, Palerme J S, Parsons C T, Kathariou S, Jacob ME. 2016. Isolation and Characterization of Listeria monocytogenes associated with a canine urinary tract infection. J Vet Diagn Invest. 28(5):604-7.
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: 2014 Citation: Gorski L, Walker S, Liang AS, Nguyen KM, Govoni J, Carychao D, Cooley MB, Mandrell RE. 2014. Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples with and without a selective secondary enrichment. Public Library of Science ONE 9:e92467.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Azizoglu, R. O., L. Gorski, and S. Kathariou. 2014. Listeria and produce: a troublesome liaison! New Foods. http://www.newfoodmagazine.com/15048/new-food-magazine/past-issues/issue-5-2014/listeria-produce-troublesome-liaison/
Type: Journal Articles Status: Published Year Published: 2014 Citation: Dutta V, Elhanafi D, Osborne J, Martinez MR, Kathariou S. 2014. Genetic characterization of plasmid-associated triphenylmethane reductase in Listeria monocytogenes. Appl Environ Microbiol. 80(17): 5379-5385.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Azizoglu RO, Elhanafi D, Kathariou S. 2014. Mutant construction and integration vector-mediated gene complementation in Listeria monocytogenes. 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: Azizoglu RO, Gorski L, Kathariou S. 2014. Isolation of Listeria monocytogenes from food and water: official and experimental protocols. Curr Protoc Microbiol. 2014 May 1;33:9B.5.1-19. doi: 10.1002/9780471729259.mc09b05s33.
Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Parsons C, Costolo B, Brown P, Kathariou S. Penicillin binding protein lmpo2229 mediates copper homeostasis in Listeria monocytogenes. (In Review, FEMS Lett. Microbiol)
Type: Journal Articles Status: Published Year Published: 2017 Citation: Wolfe B, Wiepz GJ, Schotzko M, Bondarenko GI, Durning M, Simmons HA, Mejia A, Faith NG, Sampene E, Suresh M, Kathariou S, Czuprynski CJ, Golos TG. 2017. Acute fetal demise with first trimester maternal infection resulting from Listeria monocytogenes in a nonhuman primate model. MBio. 2017 Feb 21;8(1). pii: e01938-16. doi: 10.1128/mBio.01938-16.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Lee S, Ward TJ, Graves LM, Tarr CL, Siletzky RM, Kathariou S.2014. Population structure of listeria monocytogenes serotype 4b isolates from sporadic human listeriosis in the United States, 2003-2008. Appl. Environ. Microbiol. 80(12): 3632-3644.
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: Journal Articles Status: Published Year Published: 2013 Citation: Wassinger A, Zhang L, Tracy E, Munson RS Jr, Kathariou S, Wang HH. 2013. Role of a GntR-family response regulator LbrA in Listeria monocytogenes biofilm formation. PLoS One. 2013 Jul 23;8(7):e70448. doi: 10.1371/journal.pone.0070448.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Dutta V, Elhanafi D, Kathariou S.2013. Conservation and distribution of the benzalkonium chloride resistance cassette bcrABC in Listeria monocytogenes. Appl. Environ. Microbiol. 79(19): 6067-6074.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Lee S, Rakic-Martinez M, Graves LM, Ward TJ, Siletzky RM, Kathariou S. 2013. Genetic determinants for cadmium and arsenic resistance among Listeria monocytogenes serotype 4b from sporadic human listeriosis. Appl. Environ. Microbiol. 79(7): 2471-2476.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Faith NG, Kim JW, Azizoglu R, Kathariou S, Czuprynski C. 2012. Purine biosynthesis mutants (purA and purB) of serotype 4b Listeria monocytogenes are severely attenuated for systemic infection in intragastrically inoculated A/J mice. Foodborne Pathog. Dis. 9(5):480-6. doi: 10.1089/fpd.2011.1013.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Katharios-Lanwermeyer S, Rakic-Martinez M, Elhanafi D, Ratani S, Tiedje JM, Kathariou S 2012. Coselection of cadmium and benzalkonium chloride resistance in conjugative transfers from nonpathogenic Listeria spp. to other listeriae. Appl. Environ. Microbiol. 78 (21): 7549-7556.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Kim JW, Dutta V, Elhanafi D, Lee S, Osborne JA, Kathariou S. 2012. A novel restriction-modification system is responsible for temperature-dependent phage resistance in Listeria monocytogenes ECII. Appl Environ Microbiol. 78(6): 1995-2004.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Ratani SS, Siletzky RM, Dutta V, Yildirim S, Osborne JA, Lin W, Hitchins AD, Ward TJ, Kathariou S. 2012. Heavy metal and disinfectant resistance of Listeria monocytogenes from foods and food processing plants. Appl Environ Microbiol. 78(19):6938-45.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Lee S, Ward TJ, Siletzky RM, Kathariou S. 2012. Two novel type II restriction-modification systems occupying genomically equivalent locations on the chromosomes of Listeria monocytogenes strains. Appl. Environ. Microbiol. 78(8): 2623-2630.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Lee S, Ward TJ, Graves LM, Wolf LA, Sperry K, Siletzky RM, Kathariou S. 2012. Characterization of atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette. Appl. Environ. Microbiol. 78(3): 660-667.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Brul S, Bassett J, Cook P, Kathariou S, McClure P, Jasti PR. 2012. Omics technologies in quantitative microbial risk assessment. Trends Food Sci. & Technol. 27 (1): 12-24.
Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Parsons, C T 2016. Functional Genomics of Heavy Metal Resistance in the Foodborne Pathogen Listeria monocytogenes. PhD Dissertation, North Carolina State University
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: 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. 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: 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: Theses/Dissertations Status: Published Year Published: 2014 Citation: Martinez, MR. 2014. Identification Of The Mechanisms Of Resistance And Potential Cross Resistance Of Listeria monocytogenes Upon Adaptation To Different Antimicrobials. Ph.D. Dissertation, University of Belgrade, Faculty of Veterinary Medicine
Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: Ratani, Shakir. 2012. Heavy Metal Resistance and Epidemic Clonal Characteristics among Listeria monocytogenes from a "Historical" Collection and Other Sources. MS Thesis, North Carolina State University
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: Parsons P, Brown P, Kathariou S. 2016. Metal homeostasis in Listeria monocytogenes: Which metals? Which genes? BioLunch, North Carolina State University
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: 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: 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, 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, Washington, DC June 15-16, 2015
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kathariou S. 2015. Genetic analysis of Listeria monocytogenes. FDA workshop on Listeria, Washington, DC June 15-16, 2015

Progress 02/15/15 to 02/14/16

Outputs
Target Audience:Target audiences included the produce industry, keen 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). 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. In addition to the heavy disease burden, Listeria-contaminated produce have caused numerous recalls with adverse economic repercussions. Discontinuation of production of certain types of produce due to implication in disease adversely impacts the economic vitality of this sector of US agriculture and compromises the wellbeing and sustainability of agricultural communities. 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. Visiting scientists and postdocs also benefited from further research experience with the molecular and microbiological components of interactions between Listeria and 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. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three undergraduate and eight graduate students acquired research experience working on the project during the past year. Experience included assays for adherence and growth on produce, genetic characterization and manipulation of LM, analysis of bioinformatics data, construction and screening of mutant libraries, biofilm assays 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. Four technical staff acquired further experience in microbiology, food safety and molecular biology. Technicians were trained on sequencer machines (Illumina Mi-Seq), biofilm assays, produce colonization assays, biofilm determinations 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. Postdoctoral scientists significantly furthered their experience in food safety, microbiology, molecular biology and bioinformatics. Graduate students, postoctoral scietists 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?Fresh Produce Industry: Findings were presented at the NC Governors Food Safety Task Force conference, which includes strong participation by the North Carolina fresh produce industry and the food industry in general. Research and Academic Community: Findings were presented via peer-reviewed manuscripts, posters and presentations at regional and international conferences, workshop participation and seminars at North Carolina State University and elsewhere. Findings from the project were regularly presented via case study formats and "teachable moments" in the Food Microbiology class that Kathariou taught during the past year 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?1. Objective 1. We will complete the assessments of the produce adherence and growth potential of clinical and produce-derived strains associated with recent produce outbreaks (stone fruit, caramel apple, leafy greens) and prepare the findings for publication, targeted for J. Food Prot. This analysis will include a panel of LM strains from non-produce-outbreaks but with genotypes similar to those of strains implicated in produce-related outbreaks. 2. Objective 2. Transposon mutants identified by the ampicillin selection process and found to be impaired for growth on produce will be characterized to determine insertion site and confirm the presence of a single transposon copy. For the most promising mutants genetic complementation will be pursued. Functional characterizations will include produce colonization, biofilm formation and virulence using the Galleria model. Pools of mutants obtained both from the ampicillin selection assay (enrichment for growth-impaired mutants) and from the repeat-rinse model (enrichment for adherence-impaired mutants)will be analyzed by next-generation sequencing. Selected non-adherent or growth-impaired mutants identified via the next-generation sequencing approach will be validated, genetically complemented and functionally characterized for produce colonization, biofilm formation and virulence. This Objective has proven more recalcitrant than expected due to the methodological challenges in the mutant enrichment protocols, but these challenges have now been resolved. The development and optimization of the enrichment protocols and the functional characterization of new genes involved in LM's ability to adhere and grow on fresh produce will be described in peer-reviewed publications, targeted for J. Food Prot., Applied & Environ. Microbiol., Foodborne Pathogens & Disease or similar journals. 3. Objective 3. Biofilm assessments will be completed for all LM strains, including on stainless steel coupons. Strains of fresh produce-derived bacteria will be used in mixed cultures with LM to assess LM fitness for biofilm formation on produce and on stainless steel. The confocal microscopy and biofilm findings will be prepared for peer-reviewed publications.

Impacts
What was accomplished under these goals? To further contribute to Objective 1, we have 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. We have further pursued Objective 2 by optimizing protocols utilizing ampicillin selection to enrich for mutants that are 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. A panel of mutants has been already identified, with four being especially promising. Collections of the recovered populations will be also analyzed at the Gorski and Miller laboratories (co-PIs) by the next-generation sequencing approach to identify insertions over-represented among the ampicillin-treated populations, in comparison with populations in the absence of ampicillin. 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 approx. 300 CFU/ml by the 4th successive rinse. Populations from the earlier successive rinses will be analyzed by co-PIs Gorski and Miller via next-generation sequencing, to identify mutants over-represented in the rinsate in comparison to their proportion in the mutant library employed as the original inoculum. To contribute to 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 had been 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 a panel of bacterial strains recovered from fresh cantaloupe rind. These will be utilized to assess LM colonization in mixed cultures with these other strains.

Publications

Type: Journal Articles Status: Published Year Published: 2016 Citation: Garner, D., and S. Kathariou. 2016. Fresh produce-associated listeriosis outbreaks, sources of concern, teachable moments and insights. J. Food Prot. 79(2):337-44.
Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Martinez, M. R., Osborne, J., Jayeola, V. O., 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. (in press; May 2016 issue)
Type: Journal Articles Status: Awaiting Publication 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. (in press)
Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Pan, P. C., Palerme, J. S., Parsons, Kathariou, S., Jacob, M.E. 2016. Isolation and Characterization of Listeria monocytogenes associated with a canine urinary tract infection. J.Vet. Diagn. Invest. (in press)
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: Submitted Year Published: 2016 Citation: Jahanafroozi, M., Fan, S., Jaeyola, V. O., Parsons, C., and Kathariou, S. 2016. Characterization of the genetic basis and stability of tetracycline resistance in a strain of Listeria monocytogenes from the 2011 cantaloupe outbreak. International Association for Food Protection Annual Meeting, St. Louis, MO.
Type: Conference Papers and Presentations Status: Submitted Year Published: 2016 Citation: Jayeola, V. O., Parsons, C., Miller, G. W., Gorski, and 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: Accepted Year Published: 2016 Citation: Parsons, C., Lee, S., and Kathariou, S. Novel locus mediating metal homeostasis in Listeria monocytogenes. American Society for Microbiology General Meeting, New Orleans.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Jayeola V. O., C. Parson, M. R. Martinez, S. Kathariou. Persistence of Listeria monocytogenes on Fresh Produce. Presentation at the NC Governors Food Safety Task Force Meeting. Salisbury, NC. March, 2016.
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Gorski, L., Liang, A., Romanolo, K. F., and 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 Annual Meeting. Portland, OR.

Progress 02/15/14 to 02/14/15

Outputs
Target Audience:Target audiences included the produce industry, keen on science-based strategies to control Listeria on fresh produce, especially since the 2011 cantaloupe outbreak. Relevant stakeholders in this target audience include farmers, workers, packing plant owners and operators, other individuals in the fresh produce distribution chain from farm to retail. In addition to the heavy disease burden, Listeria-contaminated produce have caused numerous recall with adverse, often catastrophic economic repercussions. Discontinuation of production of certain types of produce due to implication in disease adversely impacted the economic vitality of this sector of US agriculture and compromises the wellbeing and sustainability of farming communities. 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. Visiting scientists also benefited from further research experience with the molecular and microbiological components of interactions between Listeria and 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. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five undergraduate and seven graduate students acquired research experience working on the project during the past year. Experience included assays for adherence and growth on produce, genetic characterization and manipulation of LM, analysis of bioinformatics data, construction and screening of mutant libraries, biofilm assays 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. Technical staff acquired further experience in microbiology, food safety and molecular biology. Technicians were trained on sequencer machines, confocal microscopy, 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. Visiting scientists significantly furthered their experience in food safety, microbiology, molecular biology and bioinformatics. 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? Fresh Produce Industry: Findings were presented at industry-attended conferences and an FDA workshop that included strong participation by the fresh produce industry and the food industry in general. Kathariou informally shared findings from the project during the 1015 Center for Produce Safety (CPS) Research Symposium (Atlanta, GA). Project-related information was disseminated also via an article in New Food, a journal with wide reach to the industry, and with a blog in Food Safety News. Research and Academic Community: Findings were presented via posters and presentations at regional and international conferences, workshop participation and seminars at North Carolina State University and elsewhere. Findings from the project were regularly presented via case study formats and "teachable moments" in the Food Microbiology class that Kathariou taught during the past year 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. The Food Safety News blog (http://www.foodsafetynews.com/2015/04/listeriosis-and-produce-whats-the-connection/#.VcPguSpVikq) reached numerous individuals in the community. What do you plan to do during the next reporting period to accomplish the goals? Objective 2: Deep sequencing will be completed with a panel of strains to identify sequences required for adherence to fresh produce (cantaloupe and romaine lettuce leaves). Objective 2: The ampicillin selection assay will be employed using mutant library inoculums, to identify mutants unable to grow on cantaloupe and romaine lettuce leaves. Objectives 2 and 3: Genes identified by the deep sequencing approach will be targeted by deletion. Deletion mutants with impaired adherence on produce will be genetically complemented and also characterized for biofilm formation on PVC and stainless steel. Objectives 2 and 3: Mutants identified by the ampicillin selection approach will be confirmed, and promising mutants will be genetically complemented and also characterized for biofilm formation on PVC and stainless steel. Objective 3. Listeria (wildtype and mutant) biofilm formation on produce and on stainless steel will be examined using monocultures as well as mixed cultures with produce-derive microbes. A total of nine manuscripts for peer review will be prepared and submitted. In addition, material will be prepared for online or trade publications. Three manuscripts will focus on the roles of specific targeted genes (listeriolysin, prfA, RNA helicase) on produce colonization. Five additional manuscripts will concern: adherence and growth of outbreak strains on cantaloupe rind, flesh and extract at different temperatures; adherence of 24 LM strains, their growth on romaine lettuce leaves at different temperatures, and their biofilm formation potential; virulence of produce outbreak strains in the Galleria model; novel genes required for adherence, and identified via deep sequencing; genes required for growth on produce, identified by the ampicillin selection. One review article on Listeria from fresh produce outbreaks is being prepared for submission.

Impacts
What was accomplished under these goals? To contribute to Objective 1, we continued the characterization of adherence and growth of Listeria monocytogenes (LM) strains derived from produce outbreaks, specifically cantaloupe, celery and caramel apple. All strains were able to adhere to and grow on cantaloupe fragments at 4, 8 and 25oC, even though strain-specific differences in adherence were noted. Log increases on rind were significantly greater than on flesh or in cantaloupe extract for certain temperature-time combinations. Significant differences in growth among strains were not noted. Furthermore, the propensity for more growth on rind than on flesh or in extract was also observed for strains from unrelated outbreaks (involving deli meats and cheese). Numerous efforts were made to establish a lettuce seedling model to assess ability of different LM strains to adhere and grow, and also for subsequent testing of mutant libraries. However, the lettuce seedling model continue to be challenging (little or no colonization, inconsistent data). Therefore, for lettuce colonization assays we switched to romaine lettuce leaves available at retail. Pieces of lettuce were exposed to LM by immersing in a suspension of the pathogen for 90 seconds, and then allowing adherence for 90 minutes before vigorous washing to enumerate cells attached to the plant tissue. We also tested growth on lettuce by incubating the washed lettuce pieces for 24 hours. 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 oubtreaks. 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 pursued with two of the cantaloupe outbreak strains, but was unsuccessful, in spite of repeated efforts. Mutant libraries with the mariner-based transposon have been 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. Non-hemolytic mutants with single insertions in the gene for listeriolysin (hly) or prfA were not impaired in ability to adhere to and grow on cantaloupe at either 25 or 37oC. This was surprising, as prfA has been recently implicated in control of genes impacting biofilm formation in LM, and we had hypothesized that biofilm formation was involved in the observed growth on the rind. In fact, the mutants were impaired in biofilm on PVC microtiter plates. Findings suggest that the virulence genes hly and prfA were not required for adherence and growth of LM on cantaloupe, even though they did impact biofilm formation on abiotic surfaces. Two motility-impaired mutants were characterized for adherence and growth on cantaloupe. Both 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). To contribute to Objective 2, Mutant libraries constructed and validated at the Kathariou lab were made available to the Miller and Gorski labs so that deep sequencing will be employed to identify the location of the transposon in mutant sub-populations enriched for those failing to adhere onto cantaloupe. At the same time, at the Kathariou lab assays were optimized to use ampicillin for isolation of mutants that fail to grow on the cantaloupe. The hypothesis is that ampicillin will only inhibit growing cells, thus enriching for mutants that fail to grow. Several strain pairs were identified to optimize the method, with promising results. To contribute to 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.

Publications

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: V. Dutta, P. Evans, S. Kathariou. 2014. Comparative analysis of gene synteny for adaptive molecular determinants utilizing sequenced genomes of Listeria spp. Proceedings of 113th General Meeting of American Society for Microbiology, May 17-20, 2014, Boston, MA.
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: L. Gorski. 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: L. Gorski, A. Liang, K. Romanolo, and S. Walker. 2015. Examination of Environmental Isolates of Listeria monocytogenes Indicates that Their inlA Genotypes Are Intact and the Strains Potentially Virulent. IAFP, July 2015, Portland OR
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: V. Dutta, R. Price, C. Parsons, V Jayeola, G. Ryan, P. Evans, S. Kathariou. 2015. Development of Restriction-Modification Isogenic Mutants to Study Global Methylation Patterns in Listeria monocytogenes authors: Proceedings of 114th General Meeting of American Society for Microbiology, New Orleans, LA
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: V. Dutta, T. J. Ward, S. Lee, C. Parsons, S. Kathariou. 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, LA
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: M. Rakic-Martinez, C. Parsons, V. Jayeola, R. Price, L. Gorski and S. Kathariou. 2015. Molecular Mechanisms for Interactions Between Listeria monocytogenes and produce. NIFA Investigators Annual Meeting, Portland, OR.
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Kathariou. 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: S. Kathariou. 2015. Listeria Virulence. FDA workshop on Listeria, Washington, DC June 15-16, 2015
Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Kathariou. 2015. Listeria Ecology. FDA workshop on Listeria, Washington, DC June 15-16, 2015
Type: Other Status: Published Year Published: 2015 Citation: D. Garner. 2015 Listeriosis and Produce: Whats the Connection? Food Safety News (April 01, 2015). http://www.foodsafetynews.com/2015/04/listeriosis-and-produceKwhats-the-connection/#.VcPguSpVikq (D. Garner was graduate student at kathariou's lab)
Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: R. O. Azizoglu, V. Dutta, F. Breidt, Jr. and s. Kathariou. 2015. Resistance of Listeria monocytogenes Biofilms to Sanitizing Agents. In: Biofilms in the Food Environment, Second Edition. Pp. 51-83, Edited by Anthony L. Pometto and Ali Demirci. � 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

Progress 02/15/13 to 02/14/14

Outputs
Target Audience: Graduate and undergraduate students were reached by efforts centered on exposure to research methodologies and tools relevant to project, including sample processing, data collection, analysis and presentation. Additional efforts included project-derived case studies discussed during formal classroom activities supporting the courses Food Safety and Public Health (FS 540) and Food Microbiology (FS405), a guest lecture for the Microbiology class MB360 (Scientific Inquiry in Microbiology: At the Bench), a seminar and a presentation at the Fulbright Scholars Networking Event at North Carolina State University, directed to Fulbright scholars from pre-doctoral programs throughout the United States. Pre-college students were reached through participation in the CALS 3D program sponsored by the Diversity program at North Carolina State University and directed to High School students from under-represented minorities. Program involved research immersion of students in a project assessing Listeria adherence on cantaloupe. The food Safety community was reached through presentations at the American Society for Microbiology General Meeting, the International Association for Food Protection Annual Meeting, the North Carolina Food Safety and Defense Task Force monthly meeting, an invited presentation at the Western Food Safety Summit, Hartnell College, Salinas, CA, participation and an invited presentation at the North Carolina State-University of Adelaide Food Security and Health Workshop, and an invited presentation at the FDA. The fresh produce grower community was reached by participation in outreach directed to regional cantaloupe production and packing facilities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Project director and co-project directors enhanced their academic and professional development and growth through leadership in project design, data analysis, data dissemination (conferences, invited seminars, workshops, invited book chapters, manuscripts) and outreach. Graduate and undergraduate students obtained research experience in data collection, analysis and presentation through presentations and manuscripts. Their professional development was furthered through presentations at conferences and authorship in manuscripts. Technical staff enhanced their research skills through adoption of novel methodology. Their professional development was furthered through presentations at conferences and authorship in manuscripts. Co-authors from other institutions expanded their academic and professional portfolios through presentations at conferences and co-authorship in manuscripts. Pre-college students from under-represented minorities obtained research experience though an experimental immersion program at the project director’s lab with focus on produce safety and the molecular analysis of produce-associated Listeria. How have the results been disseminated to communities of interest? Project results were disseminated to the scientific community and produce safety community through presentations in conferences, invited seminars, a book chapter and manuscripts in the peer-reviewed literature. Project results were disseminated to the produce safety community (scientists, regulatory staff, commercial sector stakeholders) via a presentation at the North Carolina Food Safety and Defense Task Force meeting (Kathariou) and a presentation at the Western Food Safety Summit, Hartnell College, Salinas, CA (Gorski). Project results were disseminated to the college and university student community through case studies discussed in Food Microbiology and Food Safety classes, a guest lecture and through participation in specific events and workshops (e.g. the Fulbright Scholars workshop on Food security at North Carolina State University). Project results were disseminated to pre-college students from under-represented minority groups through presentations and laboratory immersion experiences. Project results were disseminated to the produce grower community through outreach to cantaloupe farmers in conjunction with produce safety team site visits. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period we anticipate submission of at least four manuscripts to the peer review literature; two in the trade magazine New Food; and one book chapter. We also anticipate several presentations in conferences, guest lectures and invited seminar. At the NCSU lab we anticipate continuation of the pre-college student research immersion program, and continued exposure of undergraduate and graduate students through case studies. Graduate and undergraduate students, visiting scientists and technical staff will continue to participate in the project under guidance of project and director and co-project directors. Research tasks to be pursued and reported upon are as follows: Objective 1. Additional strains will be examined at the NCSU lab for adherence, survival and growth on cantaloupe and celery. These additional trains will include those recently isolated from produce and environmental samples of produce production facilities. At USDA-ARS, lettuce and spinach from retail will be used in dip and spot inoculations to assess the role of leaf age on Listeria adherence, survival and growth on leafy greens. Objective 2. Previous efforts to obtain mutant sub-libraries enriched for adherence-impaired mutants were compromised by overgrowth of other bacteria from the cantaloupe rind. To address this, Listeria from the rinsates will be grown on selective media (MOX agar) and the colonies (representing the adherence-impaired mutant population) swabbed, suspended in sterile water, and used as inoculum (with the process repeated 10 times). If this continues to be challenging, indigenous bacterial populations will be reduced by rinsing the cantaloupe with hydrogen peroxide, which was found to markedly reduce aerobic plate counts. Treated produce will be rinsed with sterile water and then inoculated with the Listeria mutant libraries. If data warrant, gamma-irradiated produce will be obtained from the irradiation facility at NCSU and utilized for the inoculations. The original mutant library and the adherent mutant-enriched sub-libraries will be made available to co-project director William Miller (USDA-ARS) for genome sequencing-based determinations of differences in insertion site distributions between original mutant library and adherence-impaired enriched populations. Data on insertions over-represented in the non-adherent pool will be used to guide the construction of deletion mutants in the relevant genes, and mutants will be characterized for adherence and growth on cantaloupe and celery (NCSU) and with leafy greens (USDA-ARS). Promising mutants already identified (see Accomplishments in Objective 2) will be examined with other produce types, including celery and leafy greens. Cold-sensitive mutants and additional motility-impaired mutants will be characterized for adherence and growth on cantaloupe and celery (NCSU) and with leafy greens (USDA-ARS). Mutants of special interest will be characterized for virulence in the Galleria model. Objective 3. Mutants of special interest to be identified at the Miller laboratory (USDA-ARS) and those identified in the Kathariou laboratory through specific mutant screens (see Objective 2 accomplishments) will be characterized at NCSU for their ability to form biofilms on stainless steel, and on cantaloupe rind; GFP-tagged parental strains will be constructed together with deletion derivatives and utilized for cantaloupe biofilm assessments. The GFP-tagged strains and mutants will be characterized at the Gorski laboratory, USDA-ARS, for their ability to form biofilms on the surface of leafy greens.

Impacts
What was accomplished under these goals? IMPACT In the United States, the bacterium Listeria monocytogenes remains a leading cause of severe disease (listeriosis), hospitalizations and deaths due to foodborne infections. In 2011, a listeriosis outbreak with unprecedented mortality involved fresh cantaloupe the rind of which had become contaminated with Listeria. Current trends suggest increasing implication of fresh produce in human listeriosis in the United States. However, mechanisms involved in adherence and growth of Listeria on fresh produce remain poorly understood, inhibiting development of novel science-based interventions. In this project, we characterized strains from outbreaks of listeriosis for their ability to survive and grow on rind, flesh and extract from fresh cantaloupe, as well as on lettuce of different varieties, and we examined the role of specific genes in survival and growth on cantaloupe rind. At North Carolina State University under guidance by S. Kathariou graduate student Mira Rakic-Martinez discovered that all tested Listeria strains were able to grow on cantaloupe, with growth depending on time and temperature. She also discovered that under certain time-temperature combinations Listeria grew significantly more on rind than on flesh or in extract from fresh cantaloupe. Furthermore, she found that washing of the fruit surface with water or hydrogen peroxide resulted in temporary but significant reductions of the Listeria populations on the rind, suggesting that if the cantaloupe is utilized promptly after washing it will have a greater safety margin. She and graduate student Robert Price characterized mutants constructed and characterized by graduate student Cameron Parsons and undergraduate student Ben Costolo. A gene essential for motility was found to be critical for the ability of Listeria to colonize cantaloupe rind. At the co-director’s laboratory at USDA-ARS laboratory in California, Lisa Gorski and her technician assessed growth of Listeria on lettuce seedlings from six commercial varieties and found limited or no growth on the seedlings; whole leaves from lettuce available at retail are being examined. At the co-director’s laboratory at USDA-ARS William Miller has designed an improved protocol that utilizes genome sequencing tools to identify genes essential for adherence and growth on cantaloupe. This protocol will be employed utilizing strain constructs developed at the NCSU lab. The findings suggest that fresh produce colonization by Listeria is a complex process that depends critically on multiple factors including specific bacterial attributes (e.g. certain genes), produce type and location of contamination on the produce, storage temperature and treatment of the produce. Such complexity creates challenges but also opportunities, as multiple factors critical for colonization also suggest the availability of multiple targets for intervention strategies. ACCOMPLISHMENTS Objective 1: To assess relative produce colonization ability of a panel of strains of Listeria monocytogenes, including representatives of different lineages and sources. At NCSU the Kathariou laboratory examined a panel of strains representing all major serotypes and lineages implicated in human listeriosis (serotype 1/2a [lineage II] and serotypes 1/2b and 4b [lineage I]). Following spot inoculation on the rind, all tested strains survived and grew on the cantaloupe fragments at 4 and 8ºC (monitored for up to 21 days) and at 25ºC, monitored for up to 7 days. At 4ºC populations on the rind increased by an average of 1.0 to 1.2 logs during the 21-day incubation while following 7 days at 8ºC and 72 h at 25ºC increases averaged at 1 and 1.8 logs, respectively. No significant differences in growth were noted among different strains. After 7 days at 4°C and 72 h at 25°C all strains grew more on the rind than on the flesh or in the extract (p=0.02 and p<.0001, respectively). Following washing with sterile water for 2 minutes, Listeria populations on the cantaloupe rind were reduced by approx. 1.5-2 logs for all tested strains. Even though populations increased thereafter at 25ºC, for the first 48 h they remained consistently lower (by at least 1 log) than those on the untreated fragments. We also confirmed previously reported effectiveness of rinsing with hydrogen peroxide (1.5%, 2 minutes). CFUs of Listeria on the rind decreased (at least 1 log) immediately upon peroxide treatment and were at least 2 logs lower than those on untreated fragments after 24 h at 25°C. At USDA-ARS the Gorski laboratory examined ability of produce-derived Listeria strains to colonize lettuce seedlings of 6 different varieties. Black Seeded Simpson and Waldmann's Dark Green varieties supported Listeria survival best but none of the lettuce cultivars supported Listeria growth. Levels of Listeria on the lettuce leaves immediately after dip inoculation of 25-day old seedlings were 2-3 log CFU/g, but levels on day 3 varied from not detectable to nearly 4 log CFU/g, even on leaves from the same plant. Findings suggest that some leaves may be more capable of supporting Listeria growth and survival than others. Objective 2: To identify and characterize genes of Listeria monocytogenes required for produce colonization (adherence, survival, growth). At NCSU, the Kathariou laboratory generated mariner-based transposon libraries of three produce-derived strains, including one each from the 2011 cantaloupe and 2010 celery outbreaks. The libraries were utilized to produce sub-libraries enriched for adherence-impaired mutants, by inoculating cantaloupe rind with the mutant library, rinsing the cantaloupe to obtain populations failing to adhere, growing the rinsates overnight in medium with erythromycin (erythromycin resistance is conferred by the transposon) and then utilizing these cultures as inoculum, with the process repeated 10 times. However, bacteria from the microbial community indigenous on the rind became predominant, resulting in limited recovery of Listeria. The mutant libraries from two strains, including one from the 2011 cantaloupe outbreak, were also individually established in 96-well plates, preserved at -80ºC and screened for specific attributes, specifically hemolysin (a key virulence determinant for Listeria), cold growth ability and motility. Mutants of interest were characterized to confirm that they harbored single transposon insertions, and insertion location was determined by PCR and sequencing. Non-hemolytic mutants with insertions in hly (listeriolysin O gene) and prfA (virulence regulator) were isolated; non-motile mutants harbored insertions in an RNA (DEAD-box) helicase gene and in 6 different genes implicated in motility and chemotaxis. Selected mutants were characterized for adherence and growth on cantaloupe rind, alone as well as (for relative fitness assessments) in co-cultures with their parental wildtype counterpart. It was found that hly or prfA were not required for adherence and growth on the cantaloupe, while the RNA helicase gene was critical for adherence, growth and prolonged survival. At the Kathariou laboratory, parental strains and mutants of interest have been characterized for virulence using larvae of the greater waxworm, Galleria mellonella, as model system. Non-hemolytic mutants were markedly impaired in virulence, confirming relevance of the model. Differences in virulence were found in this model system among the different produce-derived strains, a finding which will be explored in future work with Galleria and with collaborators employing murine and cell culture models. Objective 3: To assess role of the genes on biofilm formation by Listeria monocytogenes on stainless steel and produce surfaces. This objective will be addressed during the remaining period of the project.

Publications

Type: Journal Articles Status: Accepted Year Published: 2014 Citation: R. Azizoglu, L.Gorski and S. Kathariou. 2014. Isolation of Listeria monocytogenes from food and water. Current Protocols in Microbiology.
Type: Book Chapters Status: Accepted Year Published: 2014 Citation: R. O. Azizoglu, D. Elhanafi and S. Kathariou. 2014. Mutant construction and integration vector-mediated gene complementation in Listeria monocytogenes. Listeria monocytogenes: Methods and Protocols. Methods in Molecular Biology, Humana Press, Springer Science, New York, NY.
Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Gorski, L., Walker, S. Liang, A. S., Nguyen, K. M., Govoni, J. Carychao, D., Cooley, M. B., and Mandrell, R. E. 2014 Comparison of Subtypes of Listeria monocytogenes Isolates from Naturally Contaminated Watershed Samples with and without a Selective Secondary Enrichment. PLoS ONE: submitted
Type: Conference Papers and Presentations Status: Submitted Year Published: 2014 Citation: Cameron Parsons, Mira Rakic-Martinez, Driss Elhanafi and Sophia Kathariou. 2014. Genetic evidence for confirmation of the role of a novel gene (cadA4) in cadmium resistance of Listeria monocytogenes, and possible impact of the gene in virulence in an insect model. 113th American Society for Microbiology General Meeting.
Type: Conference Papers and Presentations Status: Submitted Year Published: 2014 Citation: Robert Price, Sophia Kathariou, Cameron Parsons and Ben 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: Submitted Year Published: 2014 Citation: Sangmi Lee and Sophia 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: Submitted Year Published: 2014 Citation: Vikrant Dutta, Peter Evans and Sophia Kathariou. 2014. Comparative analysis of gene synteny for adaptive molecular determinants utilizing sequenced genomes of Listeria spp. 113th American Society for Microbiology General Meeting.
Type: Book Chapters Status: Accepted Year Published: 2014 Citation: Gorski, L. 2014. Serotype Assignment by Sero-Agglutination, ELISA, and PCR. In K. Jordan (ed). Listeria monocytogenes: Methods and Protocols. Humana Press, Springer Science, New York, NY., in press (Book Chapter)
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Gorski, L., Liang, A. S., Walker, S., Nguyen, K. M., Carychao, D., Cooley, M. B., Govoni, J., and Mandrell, R. E. 2013. Comparison of methods for Salmonella enterica and Listeria monocytogenes isolation to measure incidence in a central california watershed. 112th American Society for Microbiology General Meeting.
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S.Lee, S. Kathariou. 2013. Distribution of a Genomic Island Harboring Arsenic Resistance Genes in Listeria monocytogenes and Other Listeria spp. 112th General Meeting of American Society for Microbiology
Type: Theses/Dissertations Status: Accepted Year Published: 2014 Citation: Identification Of The Mechanisms Of Resistance And Potential Cross Resistance Of Listeria monocytogenes Upon Adaptation To Different Antimicrobials. Ph.D. Thesis, M. Rakic-Martinez.

Progress 02/15/12 to 02/14/13

Outputs
OUTPUTS: We characterized the ability of a panel of Listeria monocytogenes strains, representing different serotypes (1/2a, 1/2b and 4b) to grow and persist on fresh produce stored at different temperatures (4, 8 and 25 degrees C) for up to 21 days. The panel included three different strains from the 2011 cantaloupe outbreak, a strain from the 2010 celery outbreak, and a strain isolated from celery and previously found to excel in produce colonization ability. Bacteria were inoculated on the rind, on freshly exposed flesh, in freshly extracted cantaloupe juice and freshly extracted celery juice. The impact of surface washing of the rind on removal and subsequent growth of the inoculated bacteria was tested. We pursued the construction of mariner-based transposon mutant libraries in four different strains of L. monocytogenes (two of serotype 1/2a and one each of serotype 1/2b and 4b) from produce (one strain) and produce-related outbreaks (three strains). Several transposon mutants and their wildtype parental counterparts were characterized for their ability to colonize produce. Mutants earlier identified based on their reduced susceptibility to disinfectant and other antimicrobials were also characterized for adherence on produce. We examined the ability of the cantaloupe outbreak-derived strains to serve as recipients of disinfectant resistance genes in conjugations with non-pathogenic listeriae that may serve as reservoirs for such genes. In consultation with co-PIs Gorski and Miller we developed a protocol to pilot-test the mutant library screening on produce. PARTICIPANTS: Project involved participation of PI Kathariou and co-PIs Gorski, Mandrell and Miller. Kathariou had oversight of the overall project design and implementation including construction and characterization of transposon mutant libraries and assessing Listeria'a ability to grow on produce (cantaloupe rind, flesh and extract, celery extract). Gorski was responsible for implementing the lettuce seeding model to assess Listeria survival and test mutant libraries. Mandrell and Miller were responsible for pilot trials of the novel, sequencing-based protocol for identification of putative mutants. R. Siletzky participated as lab manager and research technician in project. S. Ratani (Masters student), Rakic-Martinez (predoctoral student) and C. Parsons (predoctoral student) participated as graduate students. K. Berger and R. Poe participated as undergraduate research assistants. TARGET AUDIENCES: Target audiences include the produce industry which is interested in pre-harvest reductions of Listeria, especially since the 2011 cantaloupe outbreak. Listeria-contaminated produce is subject to recall with adverse economic repercussions. Discontinuation of production of certain types of produce due to implication in disease adversely impacts the economic vitality of this sector of US agriculture and compromises the well being and sustainability of farming communities. The food safety and public health sector are relevant target audiences, since their mission includes reductions in prevalence of human listeriosis, for which contaminated produce has emerged as a major vehicle. Graduate 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. The scientific community is served by acquisition of further knowledge on contamination of fresh produce by Listeria. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
All tested Listeria monocytogenes strains were able to grow on cantaloupe inoculated on the rind, freshly exposed flesh, and in juice from cantaloupe or celery. Data were statistically analyzed with SAS using the linear mixed effects model. L. monocytogenes populations increased by approximately 10fold following 21 days incubation at 4 or 8C, and by approximately 100fold following 7 days incubation at 25C. Increases were higher on the rind than on the flesh or in the juice, with statistically significant differences after 7 days of incubation at 4C and 72 hr at 25C. No significant differences were noted among the three different strains from the cantaloupe outbreak, but certain other strains exhibited enhanced potential to grow on the inoculated produce. Rinsing of the inoculated rind resulted in temporary reductions of the L. monocytogenes population but the remaining bacteria resumed growth more rapidly than those on inoculated rind in the absence of rinsing. Mariner-based transposon mutant libraries were constructed in four strains, including the celery-derived strain F8027 previously reported to excel in produce colonization potential and three strains from recent (2010, 2011) listeriosis outbreaks involving cantaloupe and celery. A panel of mutants with selected phenotypes (cold sensitive; catalase-negative; non-hemolytic; deficient in purine biosynthesis ; reduced tolerance to cadmium; altered phage susceptibility) were tested for growth on inoculated rind, flesh and extract together with their wild type parental counterparts and found to exhibit intact ability to grow and persist on inoculated cantaloupe. Mutants earlier identified based on their reduced tolerance to disinfectants and other antimicrobials were found to be intact in their produce colonization potential. The cantaloupe outbreak-derived strains were found to serve as recipients of disinfectant resistance genes in conjugations with non-pathogenic listeriae that may serve as reservoirs for such genes. An optimized scheme for screening the entire mutant library for produce colonization was developed in consultation with co-PIs Gorski and Miller. This scheme involves the use of cantaloupe rind inoculations with the mutant library to serially enrich for transposon mutants unable to colonize the produce. Non-adherent mutants in the rinsate will be utilized as inoculum in successive inoculations. The mutant library screening in this cantaloupe rind model will allow pilot testing of the method prior to testing by co-PIs Gorski and Miller on the more demanding lettuce seedling model.

Publications

Katharios-Lanwermeyer S, Rakic-Martinez M, Elhanafi D, Ratani S, Tiedje JM, Kathariou S. 2012. Coselection of cadmium and benzalkonium chloride resistance in conjugative transfers from nonpathogenic Listeria spp. to other Listeriae. Appl Environ Microbiol. 2012 Nov;78(21):7549-56.
Martinez, M. R., Siletzky, R. M., and Kathariou, S. 2013. Survival and growth of outbreak strains of Listeria monocytogenes on cantaloupe. Intern Assoc. Food Protection (IAFP) Conference, July 2013.
Parsons, C., Lee, S., and Kathariou, S. 2013. A novel cadmium resistance determinant in Listeria monocytogenes. American Society for Microbiology (ASM) General meeting, May 2013.