Source: PENNSYLVANIA STATE UNIVERSITY submitted to
REDUCING INCIDENCES OF FOODBORNE DISEASE THROUGH PREHARVEST INTERVENTION, POSTHARVEST CONTROL, MOLECULAR TRACKING AND KNOWLEDGE OF THE BIOLOGICAL BASIS OF PATHOGEN FUNCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0233376
Grant No.
(N/A)
Project No.
PEN04522
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 2013
Project End Date
Mar 31, 2018
Grant Year
(N/A)
Project Director
Dudley, ED, .
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Food Science
Non Technical Summary
Infectious diseases are one of the leading causes of death worldwide. In the U.S., foodborne pathogens are the greatest threat to food safety and are a growing problem due to the growing population of at-risk individuals and the emergence of new pathogens, many of which are also now resistant to multiple antibiotics. As a result of this emerging antibiotic resistance, prevention of infectious diseases increasing relies on preventing contamination. Much remains to be done to reach the national health objectives for foodborne- and healthcare-related illnesses. Enhanced measures are needed to reduce or prevent contamination in the food and healthcare system and to educate stakeholders more effectively about risks and prevention measures. Such measures can be better focused when the sources of human infections (i.e., animal reservoir species and transmission route) are known. Understanding of the routes of pathogen transmission requires the development of tracking methods that are capable of identifying organisms to the strain level. Once the ecology and spread within the food system is understood, principles of Hazard Analysis and Critical Control Points (HACCP) can be applied to identify pre-harvest and post-harvest control points, and to devise mechanisms for inactivating pathogens at these steps. Additionally, research directed at further understanding the biological basis of pathogen persistence along transmission routes is needed to devise evidence-based methods of decreasing the prominence of pathogens from farm-to-fork. Consumers are demanding safe foods, but at the same time they are demanding foods that are minimally processed with few or no food additives. This situation has created a major dilemma for the food industry, and highlights the need for practices and technologies that produce foods that are fresh and nutritious, but also free of pathogens. The project describes several key areas where faculty at Penn State's Department of Food Science will work collaboratively to develop improved methods of tracking and reducing pathogen prevalence throughout the food supply. Such an outcome would have tremendous benefits to food safety and public health in the U.S. and throughout the world.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5013260110012%
5011470110012%
5013450110012%
5013320110012%
7123260110013%
7121470110013%
7123450110013%
7123320110013%
Goals / Objectives
The objectives of this proposal include: 1) Develop pre-harvest interventions for the produce and mushroom industry 2) Develop novel intervention methods to reduce pathogen prevalence in the food system 3) Develop novel molecular tracking systems for identifying pathogen transmission routes in the food system 4) Enhance understanding of the biological mechanisms promoting the survival and virulence of foodborne pathogens Milestones and goals include: Objective 1: Collecting data on sanitizer trials that will assess the safety of produce irrigation water; quantification of the survival of human pathogens during mushroom composting and synthesis of recommendations for improving cleaning and sanitation protocols for facilities. Objective 2: Data collection to support recommendations for new packaging materials and non-thermal processing treatments to reduce the prevalence of pathogens on fresh meat and produce. Objective 3: Detection of novel epidemic clones of L. monocytogenes and E. coli through development of improved molecular subtyping methodologies; identify the routes of transmission of both pathogens within processing environments, especially those manufacturing ready-to-eat mushrooms; identification of new persistent strains of pathogens. Objective 4: Identification of the biological mechanism(s) promoting L. monocytogenes persistence within food processing environments; understand the evolutionary forces driving diversification of Shiga toxin-encoding bacteriophage and the impact this has on the ecology of Shiga toxin-producing E. coli from farm-to-fork.
Project Methods
Objective 1: A split-block design experiment will be used at the Horticultural Research Farm at Rock Springs, PA. Various disinfectants will be applied and we will analyze yield and coliform counts. Additionally, at the Penn State Univerisity Mushroom Research Center we will culture mushrooms using various composts and growing conditions. Standard microbiological methods will be followed to quantify pathogen prevalence. Objective 2: Antimicrobial films containing nicin, colicin, and/or lysozyme will be prepared and observed for their ability to inhibit pathogen growth on meat surfaces using standard microbiological methods. Similar methods will be used to determine the effect of various antimicrobial dips on pathogen reduction on meat surfaces, and the impact of food processing conditions such as high pressure processing and pulsed light. Lastly, standard methods will be used to determine whether ultrasound can reduce pathogen prevalence on inoculated alfalfa seeds. Objective 3: DNA sequencing and other molecular methods will be used to identify genomic regions of Listeria monocytogenes and Shiga toxin-producing Escherichia coli that are useful for separating unrelated isolates. Objective 4: Standard methods will be used to evaluate the survival of Listeria monocytogenes during extended incubation and ability to persist within meat plant environments. DNA sequencing will be used to analyze the diversity within Shiga toxin-converting bacteriophage carried by E. coli O157:H7 strains.

Progress 04/01/13 to 03/31/18

Outputs
Target Audience:Research scientists in academia, government, and industry who are interested in methods of improving food safety. Employees of the Pennsylvania and great United States food industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained undergraduate, graduate, and postdoctoral students in food safety, and a large number of industry personel through extension and short courses (see "Other products"). How have the results been disseminated to communities of interest?Short courses, websites, extension, and research publications. 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: Results from the completed longitudinal survey of Listeria spp. in a commercial mushroom packing and fresh-cut processing facility were published in the Journal of Food Protection. A new project was initiated to determine the occurrence of Listeria monocytogenes in tree fruit packing houses in Pennsylvania. Preliminary data show between 0 and 50% of non-food-contact surfaces samples taken between October 2015 and May 2016 were positive for the pathogen. A one year longitudinal study at 3 packing houses over a one year period was initiated in September of 2016. Because commercial mushroom slicers are difficult to disassemble for regular and frequent deep cleaning and sanitization, there is a need to validate the effectiveness of a rapid thermal sanitization treatment. Heat tolerance studies in the laboratory determined D-values of 11.53, 1.90, and 0.99 minutes at 50, 60, and 70oC, respectively. Kinetic data for destruction of a selected strain of Listeria innocua showed that it would be an adequate non-pathogenic surrogate for a pilot plant challenge study. The surrogate was inoculated at the pre-determined cold-spot within the disassembled slicer head. Upon re-assembly, the slicer head was subjected to temperatures between 55 and 75oC for up to 90 minutes. The results showed complete destruction of the inoculated Listeria innocua from which we can infer the same for L. monocytogenes. Objective 2: There is an increased need for regulatory compliance of non-heat treated, shelf-stable products. Though a heat treatment is effective in reducing pathogens of interest, it may impart undesirable sensory characteristics in traditional, fermented, semidry sausages. We determined that traditional processing (fermentation and drying; no heat step) could result in a >3 log10CFU/g reduction of Escherichia coli O157:H7 (EC), Listeria monocytogenes (LM) and Salmonella Typhimurium (ST) in experimentally-inoculated landjäger. This study was the first to demonstrate that traditional processing of Landjäger may result in a safe product. The information from this research may be of interest to the meat processing industry, regulatory agencies, and researchers. Several antimicrobials of interest (ex. nanoparticles, nisin, lauric arginate) were incorporated alone or in combination into edible films made from pullulan. Films were found to inhibit foodborne bacteria in plate overlay assays. Films exhibiting the greatest inhibitory activity against the select pathogens were then evaluated in situ (ex. meat, poultry, eggs). Food surfaces were experimentally inoculated with test organisms (ex. E. coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, or Brochothrix thermosphacta) and inhibition determined over long term storage. Remaining bacterial populations were determined by standard microbiological culture methods, analyzed, and log reductions determined. These experiments demonstrated that antimicrobial films made with pullulan inhibited pathogens significantly. The results from this study suggest that incorporation of antimicrobials into pullulan films has the potential to improve the quality and safety of foods by inhibiting the growth of foodborne microorganisms. High pressure processing (HPP) has previously been shown to be effective at reducing Escherichia coli O157:H7 in meat products. However, few studies have determined whether HPP may be effective at reducing non-O157:H7 Shiga toxin producing E. coli (STEC) in ground beef. This study investigated the efficacy of short and repeated HPP treatments to reduce non-O157:H7 STEC inoculated into ground beef. HPP treatments produced >2.0 log10 CFU/g reductions of each E. coli serogroup, and reductions ranged from 2.35-3.88 and 2.26-4.31 log10 CFU/g in 80:20 and 90:10 samples, respectively. These results suggest that HPP could be an effective, post-processing intervention to reduce the risk of non-O157:H7 STEC contamination in ground beef. We performed sensory tests with consumers of ground beef, using commercially-processed ground beef patties subjected to high pressure processing using short, repeated cycles (as described above). HHP-treated patties received significantly lower ratings for overall liking, texture, flavor and juiciness when compared to control patties. Analyses also indicated the HHP patties were drier and less flavorful than the control patties. The results of these studies indicate that high pressure processing of ground beef, while effective for reducing foodborne pathogens, can affect the eating quality of the beef. As such, it may be helpful to determine the degree to which consumers are willing to balance a loss of sensory quality against food safety. Objective 3: We have shown that CRISPR-MVLST is a comparable method to PFGE, the current 'gold standard' in Salmonella subtyping. Given the rapidity and tractability of CRISPR-MVLST, we extended our studies to analyze Salmonella isolates from outbreaks (S. Newport and S. Typhimurium). We also participated in a collaboration initiated by the Food and Drug Administration that involves twelve different methods of Salmonella typing analysis. In this study, CRISPR-MVLST did extremely well, producing subtyping results that were discriminatory and epidemiologically concordant as well as being the least expensive method employed. We conclusively demonstrated that CRISPR can be developed into an effective subtyping marker for tracking foodborne outbreaks of Salmonella enterica serovars Enteritidis, Typhimurium, Heidelberg, and Newport. We worked with several government agencies including the Food and Drug Administration, Centers for Disease Control, and Pennsylvania Department of Health to develop this method using patient isolates. The Department of Food Science has also taken the lead for the state of Pennsylvania in the FDA's GenomeTrakr program. In this capacity, we are sequencing food and environmental isolates of E. coli and S. enterica for public databases, and working with the PA Department of Health to characterize pathogen isolates from retail meats purchased within the state. Objective 4: Through DNA sequencing, we determined that the stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well. We also determined that non-pathogenic E. coli isolates are capable of enhancing Shiga toxin production of E. coli O157:H7 strain, using a pathogenic isolate designated PA2 as a model. During co-culture with the non-pathogenic strain E. coli C600, PA2 produced Stx2a levels that were two to 12-fold higher than those observed during co-culture with Sakai and EDL933, respectively. Germ-free mice co-colonized by PA2 and C600 showed greater kidney damage, increased Stx2a accumulation in feces, and more visible signs of disease than mice given PA2 or C600 alone. These data suggest one mechanism by which microorganisms associated with the colonic microbiota could enhance the virulence of E. coli O157:H7, particularly a subset of clade 8 strains.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Scheinberg JA, Dudley EG, Campbell J, Roberts B, DiMarzio M, DebRoy C, Cutter CN. 2017 Prevalence and Phylogenetic Characterization of Escherichia coli and Hygiene Indicator Bacteria Isolated from Leafy Green Produce, Beef, and Pork Obtained from Farmers' Markets in Pennsylvania. J. Food Prot. 80:237.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Uhlich GA, Paoli GC, Zhang X, Dudley EG, Figler HM, Cottrell BJ, Andreozzi E. 2017. Whole-Genome Sequence of Escherichia coli Serotype O157:H7 Strain PA20. Genome Announc. 2017 Jan 12;5(2)
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Zhang H, Dudley EG, Davidson PM, Harte F. 2017. Critical Concentration of Lecithin Enhances the Antimicrobial Activity of Eugenol against Escherichia coli. Appl Environ Microbiol. 2017 Mar 31;83(8).
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Zhang H, Dudley EG, Harte F. 2017. Critical Synergistic Concentration of Lecithin Phospholipids Improves the Antimicrobial Activity of Eugenol against Escherichia coli. Appl Environ Microbiol. 2017 Oct 17;83(21).
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Gerst MM, Dudley EG, Xiaoli L, Yousef AE. 2017. Draft Genome Sequence of Bacillus velezensis GF610, a Producer of Potent Anti-Listeria Agents. Genome Announc. 2017 Oct 12;5(41).


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

Outputs
Target Audience:Our target audiences are small and very small food processors throughout Pennsylvania and beyond. We have reached these audiences via Extension programs, including HACCP, Sanitation Shortcourse, Food Microbiology Shortcourse, Food Mycology Shortcourse, GAP's, etc. and via presentations at national and international conferences. Also, the dairy food processing industry, including small-scale processors, academic and government researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We have shared the information from our research projects with researchers via scientific publications and books, as well as to small and very small food processors via Extension programs, outreach activities, and presentations at local, international, and national food safety/food science meetings. What do you plan to do during the next reporting period to accomplish the goals?We propose to incorporate a crude avocado seed extract (acetogenin), alone or in combination with lauric arginate into pullulan films to improve the safety and shelf life and quality of fresh and further processed meat and poultry products. We will evaluate the effect of drying and lowered water activity on the survival of Listeria monocytogenes, Salmonella spp., and Campylobacter jejuni in duck prosciutto.

Impacts
What was accomplished under these goals? We evaluated the differences between E. coli O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) O26, O45, O103, O121, and O145 subjected to stressful conditions relevant to the processing of dried, fermented sausages, both in vitro and in situ. Results from experiments conducted using in vitro models (modified TSB and dry filter paper) suggest that E. coli O157:H7 is better able to survive than the non-O157:H7, regardless of acid-adaptation. Furthermore, prior acid-adaptation did not confer STEC protection from subsequent stressors. In ground beef slurries with adjusted aw, no significant differences were observed between E. coli O157:H7 and the non-O157:H7 STEC. These results suggest non-O157:H7 STEC serogroups are equally or more susceptible than E. coli O157:H7 when exposed to the stressors tested in this research. Finally, the survival of acid-adapted E. coli O157:H7 and the "big six" non-O157:H7 STEC were evaluated during the production of a dry fermented sausage (DFS). Results indicated that all STEC were able to survive during processing of a dry, fermented sausage. Although cells were recovered by the last sampling day, the inoculua used were artificially high and are unlikely to represent a real-world processing scenario. E. coli O157:H7 obtained higher reductions than the non-O157:H7 by the end of the experiment; however, it was not significantly different from the reductions obtained by the non-O157:H7 STEC. These results suggest that the non-O157:H7 STEC behave similarly to E. coli O157:H7 when exposed to conditions of lowered pH and aw. Therefore, interventions used to control E. coli O157:H7 may be equally effective in controlling non-O157:H7 STEC during processing conditions similar to those used in this experiment. To determine whether or not trehalose protects Bifidobacterium animalis, and if so, at what concentration best preserves cell viability over a 35-day period.Cultures of Bifidobacterium animalis subsp. lactis BB-12 were suspended in prepared solutions of trehalose (0%, 1%, 5%, 10%, & 20%). After 30 minutes in suspension with the trehalose solutions, the bacteria were incorporated into a pullulan film solution (10% pullulan, 0.5% glycerol) and left to dry for 48 hours in a biological safety hood. On sampling days (0, 1, 2, 7, 14, 28, and 35), films were aseptically cut, dissolved, serially diluted, and plated on modified MRS agar. Morphologic and genetic confirmation of Bifidobacterium animalis subsp. lactis BB-12 was performed using standard culture methods and polymerase chain reaction (PCR), respectively.The results from these experiments demonstrated that 20% and 10% trehalose treatments allowed for more viable B. animalis after 28 days of storage than the 0, 1%, or 5% trehalose treatments. Treatment of B. animalis cells with trehalose prior to casting of pullulan films may preserve cell viability over time and allow for a better delivery of this probiotic to the intestinal tract of human. Resources for small raw milk cheesemakers to conduct science-based risk assessments, funded by NE SARE began October 1, 2016. We conducted initial visits with 5 cheesemakers to take survey of their food safety knowledge and attitudes, conducted 2 visits at each of the 5 cheesemakers collaborators to collect environmental microbial samples and invited stakeholders to participate in food safety attitudes & practices survey, and for hazard analysis workshop sent out via Extension Dairy Foods Newletter and via email to local cheese guilds

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Rivera-Reyes, M., J. A. Campbell, and C. N. Cutter. 2017. Pathogen Reductions Associated with Traditional Processing of Landj�ger. Food Control 73:768-774.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fasoyiro, S.B., Gourama, H. and Cutter, C.N. 2017. Stability and safety of maizelegume-fortified flours stored in various packaging materials. Eur. Food Res. Technol. 243:1861.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pastor, T. and C. N. Cutter. 2017. Enhancement of Bifidobacterium animalis survivability in pullulan films by treatments with trehalose. Penn State University, Gamma Sigma Delta Research Expo, March 28, 2017.


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

Outputs
Target Audience:This year's accomplishments included academic and regulatory research targeting food and food processors in Pennsylvania and beyond. We have reached these audiences via Extension programs, including HACCP, Sanitation Shortcourse, Food Microbiology Shortcourse, Food Mycology Shortcourse, Good Agricultural Practices, etc. and via presentations at national and international conferences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. K.Kaylegian is now certified as a Lead Instructor for teaching Preventive Controls for Human Food. The approved FDA curriculum will be augmented with information specific to dairy processors and at least 2 courses will be held. Food safety information will be incorporated into the Dairy Foods newsletters and websites as appropriate. A research project titled, "Development of resources for small raw milk cheesemakers to conduct science-based risk assessments," was awarded by the NE SARE organization and begins October 1, 2016. How have the results been disseminated to communities of interest?We have shared the information from our research projects with researchers via scientific publications and books, as well as to small and very small food processors via Extension programs, outreach activities, and presentations at local, international, and national food safety/food science meetings. What do you plan to do during the next reporting period to accomplish the goals?We propose to incorporate a probiotic organism (ex. Bifidobacteria spp.) into edible, pullulan films and improve the stability of the bacterium through treatments with trehalose. We also propose to incorporate food grade antimicrobial compounds into squid-like films and evaluate the activity of these films against undesirable spoilage and pathogenic bacteria. And finally, we will evaluate the effect of exposure of fermentation processes, varying salt concentrations, drying and lowered water activity, and pH treatments on the adaptation of Shiga-toxin producing E. coli in vitro (broths and meat slurries) and in situ (fermented sausages).

Impacts
What was accomplished under these goals? Milk can be contaminated with pathogens on the farm directly from the animals or during milking, and in the processing plant before, during or after product manufacture. Milk is an ideal matrix to support the growth of pathogens. Proper sanitation and handling of dairy products is critical, and continued training of the dairy industry personnel is essential to minimizing foodborne illness from dairy products. Training on sanitation was provided to 61 dairy processors at the Dairy Basics: Fundamentals of Food Safety workshops. The Sanitation Controls section added to the Penn State Extension Dairy Foods website last year received 6803 unique views on the Writing Sanitation Standard Operating Procedures page, the Key Concepts in Sanitation Controls had 3745 unique views, and the Sanitizing Wood Boards for Cheese making page had 301 unique views from 10/1/15 to 9/30/16. The pages on regulations were added in July and have received less than 100 unique views during this time, and advertising of the availability of this information is ongoing. Information on the new requirements of the Food Safety Modernization Act, and tools to meet these requirements for dairy processors was provided to 250 direct contacts through workshops and training sessions, and to 600 indirect contacts through Dairy Foods Newsletter and Extension Dairy Foods website. Pigeon pea seeds were pre-treated by blanching and dehulled by shredding at different times and varying seed to water ratio (seed:water), using a simple blender. Degree of dehulling and dehulling efficiency increased with a decrease in seed:water and increased shredding times. The information from this research may be of interest to individuals who are looking for low-cost ways to improve dehulling efficiency of pigeon pea for the manufacture of flour or other uses. In an attempt to control mold growth in maize-legume fortified flours, fermented maize, blanched pigeon pea, and soybean seeds were dried milled, and stored in four packaging materials for up to 24 weeks. The stored products were assessed periodically for water activity (aw) and fungal counts. Dominant mold isolates from the flours were further characterized and identified. Results demonstrated that the aw increased slightly after 24 weeks. Fungal counts ranged from 1.69 to 2.31 log10 CFU/g initially to 2.6 to 3.79 log10 CFU/g by the end of storage. Seven fungal species were identified from the flours, with Cladosporium cladosoporioides the dominant mold species in all the flours after 24 weeks of storage. Subsequent aflatoxin analyses indicated low levels (<20 ppb) throughout the storage period, regardless of packaging materials. This study demonstrates that aw, fungal counts, and aflatoxin levels of the flours were within tolerable limits. The information from this study may be useful to processors, regulators, or researchers who are interested in identifying suitable packaging materials for these types of flours. Pickling has been used for centuries to preserve and extend the shelf life of different foods, ranging from fruits and vegetables, to eggs and meat. The pickling process inhibits microorganism growth by reducing pH, water activity (aw) and atmospheric oxygen. Since very little is known about the safety of cold fill pickling processes, a series of separate experiments was performed using a brine solution (5% acetic acid and 5% salt at ~22-23°C) to pickle pre-cooked, RTE beef and pork sausages inoculated with various spoilage and pathogenic microorganisms. In two separate experiments, spoilage organisms consisting of lactic acid bacteria (LAB) (Lactobacillus curvatus, L. sakei, Debaryomyces hansenii, Candida zeylanoides, Penicillium nalgiovense, and Cladosporium cladosporiodies) and pathogenic bacteria (Salmonella Typhimurium, S. Senftenberg and S. Montevideo, Listeria monocytogenes, and Staphylococcus aureus) were inoculated onto RTE sausages prior to pickling. Microbial populations were evaluated initially (day 0) and up to 28 days after pickling and storage at room temperature. Other parameters, such as pH of the sausage, pH of the brine, aw of the sausages, and percent salt of both the sausage and the brine, were measured over time. Results demonstrated that the cold fill pickling process is capable of inhibiting and destroying contamination from both spoilage and pathogenic microorganisms within 24 hours after initial pickling. Results of these studies may provide scientific validation to manufacturers that cold fill pickled sausages in order to meet requirements for their HACCP plans and for regulatory compliance with the USDA-FSIS. While a heat treatment can reduce foodborne pathogens significantly, it may impart undesirable sensory characteristics in traditional, fermented, semi-dry sausages. Through a series of experiments, we demonstrated that traditional processing (fermentation and drying; no heat step), along with vacuum packaging resulted in a 5 log10CFU/g reduction of Escherichia coli O157:H7 (EC), Listeria monocytogenes (LM) and Salmonella spp. (S) in experimentally-inoculated landjäger. The information from this study may be of interest to processors, regulators, and researchers who are validating processes (ex. fermentation, drying, and packaging) for pathogen reduction in fermented and dried sausages. A chapter was written for a book to address the use of pullulan as a suitable biopolymer for antimicrobial food packaging applications. The increasing demands for alternatives to petroleum-based polymers, biodegradability, cost-effective solutions and moreover, high performance properties from alternative food packaging materials provide the opportunity for scientists to explore new combinations and applications in the area of biopolymers. We reviewed recent trends, developments, and applications of the biobased material pullulan, produced by Aureobasidium pullulans, in foods.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Murugesan, L., K. Zuzana, S. J. Knabel and L. F. LaBorde. 2015. Predominance and distribution of a persistent Listeria monocytogenes clone in a commercial fresh mushroom processing environment. J. Food Prot.78:88-98.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Fasoyiro, S., R. Hovingh, H. Gourama, and C. N. Cutter. 2016. Change in water activity and fungal counts of maize-pigeon pea flour during storage utilizing various packaging materials. Humanitarian Technology: Science, Systems and Global Impact 2016, HumTech2016, 7-9 June 2016, Massachusetts, USA (poster presentation).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fasoyiro, S., R. Hovingh, H. Gourama, and C. N. Cutter. 2016. Change in water activity and fungal counts of maize-pigeon pea flour during storage utilizing various packaging materials. Procedia Engineering 159:7276.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Knabel, S. J. 2015. Persister cells of Listeria monocytogenes. Invited paper presented at the joint JIFSAN-GMA Workshop on Evaluation of Risk Factors for Foodborne Listeriosis in Washington, DC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Knabel, S. J. 2015. Tracking and controlling Listeria monocytogenes in food processing plants. 2015. Presented at the Annual Meeting of the Society for Industrial Microbiology and Biotechnology in Philadelphia, PA.
  • Type: Websites Status: Published Year Published: 2016 Citation: Penn State Extension Dairy Foods Website, http://extension.psu.edu/food/dairy
  • Type: Websites Status: Published Year Published: 2015 Citation: Sanitation Controls, http://extension.psu.edu/food/dairy/sanitation-controls
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Gaydos, N. J., C. N. Cutter, and J. A. Campbell. 2016. Survival of Pathogenic Bacteria Associated with the Production of Pickled Sausage Using a Cold Fill Process. Journal of Food Protection 79:1693-1699.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Trinetta, V. A., and C. N. Cutter. 2016. Pullulan: A suitable biopolymer for food packaging applications. In Antimicrobial Food Packaging. Elsevier Publishing. Chapter 30, pp. 385-397.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Developing a Contemporary Dairy Foods Extension Program: A Training and Technical Resource Needs Assessment of Pennsylvania Dairy Foods Processors. 2015. Syrko, J., and K. E. Kaylegian. Journal of Extension. 53(5):http://www.joe.org/joe/2015october/rb1.php
  • Type: Other Status: Published Year Published: 2016 Citation: Dairy Foods Newsletter, sent by mail to 480 contacts " February 2016 issue " July 2016 issue
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Knabel, S. J. 2015. What the Industry has learned from the recent Listeria outbreaks and recalls. Invited paper presented at the Mid-Atlantic Fruit and Vegetable Conference.
  • Type: Websites Status: Published Year Published: 2016 Citation: Dairy Food Regulations, http://extension.psu.edu/food/dairy/dairy-food-regulations
  • Type: Websites Status: Published Year Published: 2016 Citation: Dairy Products, http://extension.psu.edu/food/dairy/dairy-products


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

Outputs
Target Audience:The accomplishments of the project this year reached the academic and regulatory research communities and the food processing industry. Many of the Extension education programs are targeted at small and very small food processors throughout Pennsylvania and the U.S. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Kaylegian. Milk can be contaminated with pathogens on the farm directly from the animals or during milking, and in the processing plant before, during or after product manufacture. Milk is an ideal matrix to support the growth of pathogens. Proper sanitation and handling of dairy products is critical, and continued training of the dairy industry personnel is essential to minimizing foodborne illness from dairy products. Training on sanitation was provided to 165 dairy processors at the Dairy Basics: Fundamentals of Food Safety workshop and Pennsylvania Association for Milk, Food, and Environmental Sanitarians annual conference. Twenty people from the dairy industry were trained at the Dairy HACCP workshop. A Sanitation Controls section was added to the Penn State Extension Dairy Foods website and received 408 visits from 8/1/15 to 9/30/15. Training materials on Key Concepts in Cleaning and Sanitizing and Writing Sanitation Standard Operating Procedures were developed and handed out during the training workshops and are available on the website and through the Ag Publications office. How have the results been disseminated to communities of interest?Research papers and book chapters were published to reach the scientific research communities and food industry. Research results were presented at national food safety and food science meetings. Training materials were delivered directly to the food industry through workshops. Food safety training materials were made available as website content and through the Extension Publications office. What do you plan to do during the next reporting period to accomplish the goals?Cutter. We propose to identify newly identified generally recognized as safe (GRAS) antimicrobials or probiotic organisms (ex. Bifidobacteria spp.) that can be incorporated into edible films and use this approach to reduce or inhibit undesirable microflora in vitro and in situ. We would like to determine the impact of pickling processes on the spoilage and/or pathogenic microflora of sausages. We will be evaluating rapid and/or molecular methods to quantitate bacterial populations associated with challenge/validation studies. Doores. We are examining the range of amplitude, cycling and pulsing that would retain the integrity of the seed and allow germination while reducing the level of salmonella in inoculated seed. Dudley. Continue the research project as described in the proposal. Learn new bioinfomatic techniques for studying data sets. Knabel. Continue the research project as described in the proposal. Kaylegian. The Penn State Extension Dairy Foods website and dairy industry workshops will be updated to include information to help dairy processors meet the requirements of the FSMA Preventive Controls final rule. Additional workshops will be held in Pennsylvania to assist dairy manufacturers understand and comply with the FSMA requirements.

Impacts
What was accomplished under these goals? Cutter. Several antimicrobials of interest (ex. nanoparticles, nisin, lauric arginate) were incorporated alone or in combination into edible films made from pullulan. Films were found to inhibit foodborne bacteria in plate overlay assays. Films exhibiting the greatest inhibitory activity against the select pathogens were then evaluated in situ (ex. meat, poultry, eggs). Food surfaces were experimentally inoculated with test organisms (ex. E. coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, or Brochothrix thermosphacta) and inhibition determined over long term storage. Remaining bacterial populations were determined by standard microbiological culture methods, analyzed, and log reductions determined. There is an increased need for regulatory compliance of non-heat treated, shelf-stable products. Though a heat treatment is effective in reducing pathogens of interest, it may impart undesirable sensory characteristics in traditional, fermented, semi-dry sausages. We determined that traditional processing (fermentation and drying; no heat step) could result in a >3 log10CFU/g reduction of Escherichia coli O157:H7 (EC), Listeria monocytogenes (LM) and Salmonella Typhimurium (ST) in experimentally-inoculated landjäger. This study is the first to demonstrate that traditional processing of Landjäger, without a heat step, may result in a safe product. High pressure processing (HPP) has previously been shown to be effective at reducing Escherichia coli O157:H7 in meat products. However, few studies have determined whether HPP may be effective at reducing non-O157:H7 Shiga toxin-producing E. coli (STEC) in ground beef. This study investigated the efficacy of short and repeated HPP treatments to reduce non-O157:H7 STEC inoculated into ground beef. HPP treatments produced >2.0 log10 CFU/g reductions of each E. coli serogroup, and reductions ranged from 2.35-3.88 and 2.26-4.31 log10 CFU/g in 80:20 and 90:10 samples, respectively. These results suggest that HPP could be an effective, post-processing intervention to reduce the risk of non-O157:H7 STEC contamination in ground beef. Doores. The use of ultrasound technology may be effective in reducing or eliminating naturally occurring pathogens as well as other microorganisms on alfalfa seeds. The seed coat can harbor such organisms in the cracks and crevices, thus making it difficult to thoroughly eliminate microbes. We have found that subjecting seeds directly to ultrasound causes a "popcorn" effect, which destroys the seed, thus preventing germination. However, placing the seeds in varying amounts of liquid can be more effective in reducing numbers. Furthermore, exposure to ultrasound using the appropriate amplitude, cycling and pulsing did not affect germination or grow out of the sprout. Dudley. Escherichia coli O157:H7 is a notorious foodborne pathogen due to its low infectious dose and the disease symptoms it causes, which include bloody diarrhea and severe abdominal cramps. In some cases, the disease progresses to hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), due to the expression of one or more Shiga toxins (Stx). Isoforms of Stx, including Stx2a, are encoded within temperate prophages. In the presence of certain antibiotics, phage induction occurs, which also in- creases the expression of toxin genes. Additionally, increased Stx2 accumulation has been reported when O157:H7 was cocultured with phage-susceptible nonpathogenic E. coli. We characterized an E. coli O157:H7 strain, designated PA2, that belongs to the hypervirulent clade 8 cluster. Stx2a levels after ciprofloxacin induction were lower for PA2 than for the prototypical outbreak strains Sakai and EDL933. However, during coculture with the nonpathogenic strain E. coli C600, PA2 produced Stx2a levels that were 2- to 12-fold higher than those observed during coculture with EDL933 and Sakai, respectively. Germfree mice cocolonized by PA2 and C600 showed greater kidney damage, increased Stx2a accumulation in feces, and more visible signs of disease than mice given PA2 or C600 alone. These data suggest one mechanism by which microorganisms associated with the colonic microbiota could enhance the virulence of E. coli O157:H7, particularly a subset of clade 8 strains.We also isolated and sequenced the genomes of 22 stx2a-converting phage, identifying three distinct phage types (PST). E. coli O157:H7 that showed the greatest amplification of Shiga toxin in the above assay were lysogenized with phage from PST2. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well. Knabel. Multi-Virulence-Locus Sequence Typing (MVLST) revealed that most clinical isolates in India were Epidemic Clone I. MVLST was also used to detect a persistent and predominant clone in a Pennsylvania mushroom processing facility.Research demonstrated that the frequency of Listeria monocytogenes persister cells increased rapidly following the log phase until they reached 100% in the long-term-survival (LTS) phase. Tryptone, but not Soytone, in Trypticase Soy Yeast Extract broth was required for the formation and long-term survival of persister cells.Persister cells were tolerant to penicillin, but not to antibiotics that targeted protein synthesis.Persister cells germinated rapidly in the presence of excess nutrients and again become sensitive to penicillin.

Publications

  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Doores, S. 2014. The Genus Sporolactobacillus. Chapter 30 in Lactic Acid Bacteria Biodiversity and Taxonomy, W. H. Holzapfel and B. J. Wood, Editors. John Wiley and Sons Ltd. Pages 533-543.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Doores, S. 2015. Lipolytic Organisms. Chapter 15 in Compendium of Methods for the Microbiological Examination of Foods. 5th Edition. Y. Salfinger and M. L. Tortorello. American Public Health Association. Pages 201-208.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Goswami, K., Chen, C., Xiaoli, L., Eaton, K.A., Dudley, E.G. 2015. Co-culturing Escherichia coli O157:H7 with a non-pathogenic E. coli strain increases toxin production and virulence in a germ-free mouse model. Infect Immun. 83:4185.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Jiang, Y., Scheinberg, J.A., and Cutter, C.N. 2015. The efficacy of short and repeated high-pressure processing treatments on the reduction of non-O157:H7 Shiga-toxin producing Escherichia coli in ground beef patties. Meat Science 102:22-26.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Knabel, S. J. 2014. Multilocus Sequence Typing: An Adaptable Tool for Understanding the Global Epidemiology of Bacterial Pathogens in DNA Methods in Food Safety: Molecular Typing of Foodborne and Waterborne Bacterial Pathogens, O. Oyarzabal and S. Kathariou (Eds), Wiley.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Morsy, M.K., Khalaf, H.H., Sharoba, Am.M., H.El-Tanahy, H., and Cutter, C.N. 2015. Efficacy of Pullulan-based Coating to Improve Internal Quality and Shelf-life of Chicken Eggs During Storage. Journal of Food Science 80:1066-1074.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Murugesan, L., Kucerova, Z., Knabel, S.J., and LaBorde, L.. 2015. Predominance, distribution, and persistence of Listeria monocytogenes in a commercial mushroom processing facility. J. Food Prot. (in press)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Pattanayaiying, R., H-Kittikun, A., and Cutter, C.N. 2015. Optimization of Formulations for Pullulan Films Containing Lauric Arginate and Nisin Z. LWT - Food Science and Technology. 63:1110-1120.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Rivera, M., J. A. Campbell, and C. N. Cutter. 2015. Pathogen reductions associated with traditional processing of landj�ger: a pilot study. Annual Meeting of International Association of Food Protection, July 2015; Portland, OR
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Yin, S., Rusconi, B., Sanjar, F., Goswami, K., Xiaoli, L., Eppinger, M., and Dudley, E.G. 2015. Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages. BMC Genomics, 16:733.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The main target audiences reached during this reporting period were agricultural andfood industries facing microbiologicalfood safety challenges, as well as consumers, especially those most at-risk for foodborne disease. Another target audience was government agencies responsible for disease prevention and control, which were involved in research, regulation and disease tracking and prevention. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Numerous graduate students and undergraduate students received training on various aspects of microbial food safety as a result of this project. The project also resulted in extensive training for post-doctoral scholars and visiting scientists. The project also resulted in the training of cooperative extension agents as well as agricultural and food industry personnel on various ways to control foodborne pathogens and thus foodborne disease. How have the results been disseminated to communities of interest? The results of research associated with this project were shared with participants in our many short-courses and workshops, as well as with extension agents during our Annual Extension In-Service Conference. Many of the PIs also shared the results of this project with governmental agencies, such as USDA and FDA. What do you plan to do during the next reporting period to accomplish the goals? We will use of multivariate analysis to compare biofilm formation by various strains and use this information to describe the ecological niches of L. monocytogenes lineages. Nutrient availability, temperature, pH and water activity (aw) at three different levels will be used to determine biofilm formation by 41 strains of L. monocytogenes. We will investigate the efficacy of short and repeated HPP treatments to reduce non-O157:H7 STEC experimentally inoculated into ground beef. We will continue research to reveal how persister cells are formed, and how this makes them tolerant to various stresses. We will use this information to propose more effective strategies for eliminating foodborne pathogens in food processing facilities to reduce the incidence of contamination, especially on ready-to-eat foods. We will develop novel molecular methods for rapidly detecting serotypes of Listeria monocytogenes, so that those serotypes that cause foodborne illness can be more rapidly detected and controlled.

Impacts
What was accomplished under these goals? Based on our research project, which involved a comprehensive survey for Listeria monocytogenes in a mushroom processing facility we were able to detect the location of Listeria monocytogenes contamination within this facility.Multi-Virulence-Sequence Typingof the isoaltes revealed that a specific virulence type (Virulence Type 11) was responsible for most of the contamination and was located mostly in the floor within the slicing room in the processing plant, where food and moisture were available for growth. Based on our results, the company took action to remove harborage sites within the floors and instituted a rigorous cleaning and sanitatizing program, which dramatically reduced the incidence of Listeria monocytogenes within the processing plant. The results of this research project are in the process of being publsihed and shared with scientific community and the food industry to help them reduce the incidence of Listeria monocytogenes in raw, ready-to-eat produce, including mushrooms. A critical review on the development of the biofilm concept by Listeria monocytogenes, focused on the practical implications, strengths, and weaknesses of the current definitions was addressed. This approach, with the help of new and improved molecular characterization and typing techniques, may assist researchers to better understand bacterial ecology and determine reservoirs, as well as transmission pathways of L. monocytogenes. Collectively, this information may provide researchers with a better understanding of the impact of environmental conditions on L. monocytogenes growth or biofilm formation. We performed sensory tests with consumers of ground beef, using commercially-processed ground beef patties subjected to high pressure processing using short, repeated cycles. HHP-treated patties received significantly lower ratings for overall liking, texture, flavor and juiciness when compared to control patties. Analyses also indicated the HHP patties were more dry and less flavorful than the control patties. This research may be helpful in determining the degree to which consumers are willing to balance a loss of sensory quality against their food safety Novel molecular tracking systems were developed and applied to new problems during the period. MVLST was applied to subtype Listeria monocytogenes isolates in Italy and India.InIndia it was found that Virulence Type I (EpidemicClone I) was the major clone associated with animals and foodborne disease, while in Italy Virulence Type 59 (Epidemic Clone V) appears to be increasing in incidence. CRISPR arrays were used to subtype Salmonella from various sources in PA and in the U.S. See the list of publications for descriptions of these advances.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Valderrama, W. B., and C. N. Cutter. 2013. An ecological perspective of Listeria monocytogenes biofilms in food processing facilities. Current Reviews in Food Science and Nutrition 53:801817.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hayes, J. E., C. R. Raines, D. A. DePasquale, and C. N. Cutter. 2014. Consumer acceptability of high hydrostatic pressure (HHP)-treated ground beef patties. LWT - Food Science and Technology. 56:207-210. Accessed at: http://www.sciencedirect.com/science/article/pii/S0023643813004179
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 35. NACMCF. 2013. Expedited response to the questions posed by the United States Department of Agriculture Marketing Service to support ground beef purchanse for the Federal Food and Nutrition Assistance Programs. J. Food Prot. 70:523-537.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Viswanath P, Murugesan L, Knabel SJ, Verghese B, Chikthimmah N, LaBorde LF. 2013. Incidence of Listeria monocytogenes and Listeria spp. in a small-scale mushroom production facility. Journal of Food Protection. 76(4):608-615.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Rocha, P. R., S. Lomonaco, M. T. Botero, A. Dalmasso, A. Dondo, C. Grattanola, F. Zuccon, B. Julini, S. J. Knabel, M. T., Caupucchio and C. Casalone. 2013. Listeria monocytogenes strains from ruminant rhombencephalitis constitute a genetically homogeneous group related to human outbreak strains. Appl. Environ. Microbiol. 79:3059-3066.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Wen, J., S. Karthikeyan, R. C. Anantheswaran, and S. J. Knabel. 2013. Listeria monocytogenes responds to cell density as it transitions to the long-term-survival phase. Int. J. Food Microbiol. 165:326-331.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Wood, T. K., S. J. Knabel and B. W. Kwan. 2013. Bacterial persister cell formation and dormancy. Appl. Environ. Microbiol. 79:7116-7121.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Doijad, S., S. Lomonaco, K. Poharkar, S. Garg, S. Knabel, S. Barbuddhe and B. Jayarao. 2014. Multi-Virulence-Locus Sequence typing of 4b Listeria monocytogenes isolates obtained from different sources in India over a 10-year period. Appl. Environ. Microbiol. 7:511-516.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Knabel, S. J. 2014. Multilocus Sequence Typing: An Adaptable Tool for Understanding the Global Epidemiology of Bacterial Pathogens in DNA Methods in Food Safety: Molecular Typing of Foodborne and Waterborne Bacterial Pathogens, O. Oyarzabal and S. Kathariou (Eds), Wiley.


Progress 04/01/13 to 09/30/13

Outputs
Target Audience: Academic, government, and industry personel who are interested in farm-to-fork reduction of pathogens within the food system. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will continue to pursue the stated goals of the project.

Impacts
What was accomplished under these goals? This project was recently approved, however initial progress on stated objectives include: Objective 1: This project is just starting. A drip and spray irrigation system has been set up at the Horticulture Research Farm in Rock Springs, PA. Studies are underway to determine the feasibility of injecting select sanitizers at various doses into water lines for reducing bacterial populations. Objective 2: For 2013-2014, we plan to develop and validate antimicrobial delivery systems with edible packaging films (ex. pullulan) to inhibit or reduce spoilage and/or pathogenic bacteria on fresh or processed muscle foods (poultry, beef, pork, lamb, seafood, etc.). We plan to identify and validate novel antimicrobial compounds (ex. nanoparticles, esters, bacteriocins, etc.) to inhibit or reduce spoilage and/or pathogenic bacteria on fresh or processed muscle foods. And finally, we plan to identify or validate novel non-thermal intervention strategies (ex. pickling, high pressure processing, pulsed light, UV light, HPP) to inhibit or reduce spoilage and/or pathogenic bacteria on fresh or processed meat and poultry products. Objective 3: Repeated sampling of a commercial mushroom processing facility determined that a MVLST Virulence Type 11 serotype 1/2c strain of Listeria monocytogenes was the most predominant, persistent and widely distributed strain in the facility. Most isolates of this strain were obtained from floor and drain samples, but isolates of this strain were also obtained from non-food-contact surfaces located between the floor and the food-contact surfaces. Isolates of this persistent strain were only isolated from samples taken in the wet areas of the processing plant. Research is ongoing to determine why this particular strain appears to predominate, persist and spread widely in the wet areas of this processing plant. Objective 4: Preliminary data indicate that tolerance to various antibiotics increases dramatically as cells of Listeria monocytogenes transition from log phase to stationary phase to long-term-survival (LTS) phase. Research to determine the dormant nature of these cells is ongoing using fluorescence activated cell sorting (FACS), along with SEM and TEM analysis of cells in the LTS phase. Research is also being conducted to determine if LTS phase cells rapidly lose tolerance to antibiotics upon germination in fresh media

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Weil JD, Cutter CN, Beelman RB, LaBorde LF. 2013. Inactivation of human pathogens during phase II composting of manure-based mushroom growth substrate. J Food Prot. 76:1393-1400