STRATEGIES TO DETECT AND MITIGATE LISTERIA IN ARTISAN CHEESE FACILITIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0231072
• Project No.: VT-H01910
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017

Project Director
C Donnelly, IN, FO.

Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405

Performing Department
Nutritional Sciences

Non Technical Summary
Consumer interest in artisan and farmstead cheeses is driving explosive growth of on-farm cheese operations throughout the United States. As many of these enterprises are small to very small establishments, there is a need for focus on assuring the microbiological safety of cheeses produced on farm. During 2010, the U.S. Food and Drug Administration intensified its scrutiny of U.S. cheese makers. In particular, increased regulatory attention was focused on small scale artisan cheese makers and those producing cheeses from raw milk. Smaller operations are considered high risk due to lack of resources, capital and technical expertise to implement effective control programs. With 38 artisan cheese producers, Vermont boasts the highest number of artisan cheese makers per capita in the United States. In order to allow this industry to grow and prosper, it is essential that the safety of artisan cheeses be assured.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3430	1100	100%

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

Subject Of Investigation
3430 - Cheese;

Field Of Science
1100 - Bacteriology;

Keywords

listeria

artisan cheese

washed rind cheese

microbiological safety

Goals / Objectives
This project will provide continued research for small-scale cheese makers with the specific goal of assisting cheese makers producing high-risk, washed rind cheeses in managing microbiological risks through research to define best microbiological risk management practices which will be applied to develop written, preventive control plans for cheese making facilities. These written plans will help small scale artisan cheese makers meet requirements of the Food Safety Modernization Act. We will assess survival of Listeria monocytogenes during 60 days of aging of washed rind cheeses. Control of L. monocytogenes will be explored through use of bacteriopahges and competitive cultures, along with brush decontamination strategies. Our objectives are as follows: 1. Assess fate of L. monocytogenes in washed rind cheeses aged for 60 days. Assess the efficacy of anti-listerial products in mitigating Listeria contamination of washed rind cheeses. 2. Compare the efficacy of intervention procedures (sanitizers versus heat treatment) for decontamination of materials used for "smear" or "wash" application during cheese making. 3. Identify sources and niches of Listeria contamination in farmstead cheese facilities producing high risk, washed-rind cheeses. Compare use of novel and traditional testing systems which encompass microbiological monitoring of quality and safety of milk, cheese and environment. 4. Develop Risk Reduction Protocols to control Listeria contamination in farmstead cheese facilities producing high risk cheeses. Conduct a longitudinal study to assess efficacy of the risk reduction protocols.

Project Methods
Objective 1: We will obtain unripened raw milk wash rind cheeses post-brining from producers. Cheese type 1 will be a semi-soft washed rind cheese, while cheese type 2 will be a soft washed rind cheese. Cheeses will be inoculated with a six strain cocktail of L. monocytogenes and subjected to treatments which will include: inoculated soft cheese control; inoculated soft cheese with protective cultures; inoculated soft cheese with bacteriophage; inoculated soft cheese with both antilisterial products; inoculated semi-soft cheese control; inoculated semi-soft cheese cheese with protective cultures; inoculated semi soft-cheese with bacteriophage; and inoculated semi-soft cheese with both antilisterial products. At prescribed intervals (days 4,7,14,21,28,35,42,49,56,63 and 70) cheeses will be analyzed for presence of L. monocytogenes. We will also evaluate survival of Listeria in smear solutions with and without anti-listerial products. The general linear models procedure (GLM) will be used to determine the effect of cheese type and anti-listerial application on the rate of change in mean log cfu/g as well as the effect of time on changes in both pH and mean log cfu/g. Objective 2: Four commercially available smear application devices (small sponges, small plastic brushes, and two traditional cheese brushes will be inoculated with Listeria cultures. Tools will then be subjected to treatment with disinfectant treatments (sanitizers versus boiling)to evaluate efficacy as determined by measurement of Listeria survival/destruction. Objective 3: Environmental sampling for presence of Listeria will be conducted on nine on-farm artisan cheese processing facilities producing high-risk washed rind cheeses. Sites will be selected based on results obtained in previous sampling events. Samples will be analyzed for presence of Listeria using methods prescribed by the FDA, as well as modifications thereof. Isolates will be characterized using the automated Riboprinter Microbial Characterization System. Objective 4: Risk Reduction protocols will be developed for nine cheese producers. Using a HACCP approach, critical control points in the cheese making process will be identified. Environmental sampling of the facility will be conducted and samples subjected to analysis for presence of Listeria. Finding will be shared with cheese makers, along with recommendations for elimination of niches of contamination. Following implementation of recommendations, comprehensive microbiological analysis will be performed. Date will be compared to determine if changes have resulted in improved process control and improvements in cheese quality.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The target audience reached by this research consists of Vermont's artisan cheesemaking community comprised of approximately 50 producers.Cheesemakers are located throughout the State of Vermont. Most of these establishments are small to very small companies that lack technical and educational resources, so the work we have conducted is extremely significant. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?All personnel participating in this project have presented their findings at the Annual Meetings of the International Association for Food Protection over the duration of the project period. How have the results been disseminated to communities of interest?Project results have been shared with artusan cheesemakers attending the American Cheese Society Annual Meetings, the Innovation Center for U.S. Dairy and the Vermont Cheese Council, amnog others. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? we compared novel and traditional testing systems encompassing microbiological monitoring of the quality and safety of milk, cheese and the cheesemaking environment. We also evaluated fate of multi-drug resistant Salmonella in a Gouda style cheese, which was aged for 60 days. This work resulted in a publication listed below (see D'Amico et al. 2014). We included objectives focused on a comparison of detection procedures for L. monocytogenes, which evaluated a proprietary format developed by the 3M Corporation as well as evaluation of FTIR microspectroscopy for detection of Listeria that was subjected to acid and sanitizer treatments. These treatments mimic environmental conditions encountered by Listeria in a cheese due to acid development by the starter culture, as well as in the environment when sanitizers are applied. This work, conducted by my former Ph.D. student Esmond Nyarko, and resulted in 4 publications (Nyarko and Donnelly, 2014; Nyarko, Puzey and Donnelly, 2014; Nyarko et al. 2014 and Nyarko and Donnelly, 2015). In years 4 and 5, we utilized these novel detection methods to identify sources and niches of Listeria in Vermont artisan cheese facilities. Dr. Panos Lekkas, a recent Ph.D. graduate from my lab and current post-doc, explored farm sources of Listeria monocytogenes and impact on the microbiological quality of milk destined for artisan cheese manufacture. We studied a total of 5 Vermont farms. All farms tested were either producers of artisan cheese or they were supplying cheese makers with milk. The results show the presence of Listeria monocytogenes in all farms except Farm E (Table 1). Although pathogenic Listeria species were present on the farms, there was not an instance when cheese production was impacted during the duration of the study. Through a grant leveraged from my Hatch project, competitively funded by the George Walker Milk Fund entitled "Farm sources of Listeria monocytogenes and impact on the microbiological quality of milk destined for artisan cheese manufacture", we worked with select Vermont farms over a one-year period to understand practices that enhanced or decreased incidence of Listeria within the farm environment. We compared Listeria incidence on four farms; two (Farms A and D) fed dry hay or fed cows on pasture, while two others (Farms B and C) fed silage to animals. In both Farms B and C, the same ribotypes of L. monocytogenes found in silage were found in other areas of the farm environment, particularly in water sources. Advising artisan cheese producers making raw milk cheeses to eliminate silage feeding in favor of dry hay or pasture feeding is a strategy that shows promise to reduce the potential for presence of Listeria in milk used for cheese making. We wish to extend this work to analyze data from a three-year study period. An excellent article authored by Driehuis (2013) reviews other microbiological hazards that can be transmitted through silage feeding to milk used in artisan cheese production. Many PDO and AOC European cheese varieties prohibit silage feeding for certain varieties of cheese due to the known microbiological hazards associated with this practice. In our study, we could not detect the presence of Listeria in bulk tank milk from study farms. However, when milk filters were tested, presence of the pathogen was revealed. Encouraging cheesemakers to routinely test milk filters for presence of Listeria may be more effective in identifying potential presence of Listeria in milk. It is our experience that Listeria is rarely present in raw milk used for artisan cheese production. When we have found presence, it is present at levels below detection limits. Testing milk filters may increase the sensitivity of detection and provide confidence in negative results. We propose to expand our studies to determine the long term impact of feeding dry hay and pasture versus silage on the microbiological profile of milk intended for artisan cheese manufacture. If we can confirm the microbiological safety benefits associated with dry hay/pasture feeding, this will create an opportunity for dairy farmers to engage in value-added milk production for sale to cheese makers producing aged raw milk cheeses.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Trmcic , A., R. Ralyea , L. Meunier-Goddik , C. Donnelly, K. Glass, D. DAmico, E. Meredith, M. Kehler, N. Tranchina, C. McCue, M. Wiedmann. 2016. Consensus categorization of cheese based on water activity and pH  a rational approach to systemizing cheese diversity. Journal of Dairy Science 100:841847
• Type: Book Chapters Status: Published Year Published: 2017 Citation: D'Amico, D., and C.W. Donnelly. 2017. Growth and Survival of Microbial Pathogens in Cheese. In Cheese: Chemistry, Physics & Microbiology. 4th Edition. Academic Press.

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

Outputs
Target Audience:The target audience reached by our research efforts includes artisan cheesemakers, along with food scientists and regulatory officials working with artisan cheesemakers. Changes/Problems:No major changes have been made and no major problems encountered. What opportunities for training and professional development has the project provided?This work was presented at national meetings, including the International Association for Food Protection and the American Cheese Society Annual Conference. It was also presented internationally at the International Symposium on Problems of Listeriosis. In Vermont, this information has been shared with local cheesemakers and members of the Vermont Cheese Council. How have the results been disseminated to communities of interest?Our work has been published in refereed journals, presented in the form of posters at annual meetings of professional societies, and presented at annual meetings of the American Cheese Sociaty and the Vermont Cheese Council. What do you plan to do during the next reporting period to accomplish the goals?We will complete all project objectives, with a focus on farm visits and environmental surveillance to identify and mitigate sources of Listeria?in farm environments in order to reduce or eliminate this pathogen in milk intended for artisan cheese production.

Impacts
What was accomplished under these goals? We investigated the effects of L. monocytogenes on the composition of the surface microflora on washed rind soft cheese. Two trials with washed rind cheeses that were inoculated with 100cfu/cm-2 of a L. monocytogenes six strain cocktail were conducted. The first trial had to be terminated early (day 28) as contamination of Pseudomonas spp. from the initial brine did not produce the expected characteristics of the cheese during the aging period. For the second trial, cheese samples were aged in the lab for 60 days according to the cheesemakers specifications. Surface cheese rind samples were collected from both control and inoculated cheeses every 7 days. Cheese rind samples were analyzed through the standard BAM method for enumeration of L. monocytogenes and through amplification of the V4 region of 16S rRNA and ITS regions for identification of the surface rind bacterial and fungal communities, respectively. Our data showed that Pseudomonas spp. significantly changed the composition of the microorganisms found on the surface of the rind while L. monocytogenes had little effect. In addition, although the concentration of L. monocytogenes increased to levels of 106 cfu/cm-2 based on the enumeration data, the genetic data was not able to identify it in the flora due to the fact that other genera were found at much higher concentrations, which is a limitation of the molecular methods used for identification of pathogens in foods. The presence and incidence of L. monocytogenes was investigated on farms that either produce raw milk cheese or supply the milk for raw milk cheese production. Five farms were visited and in total 266 samples were collected from barn, environmental, and milk sites. L. monocytogenes prevalence was found to be at 6% from all the farms tested with 10 isolates found in the barn samples, 5 from environmental sites and 1 from milking equipment. Samples were identified to the genus level through a modified BAM method and speciated though multiplex PCR. Included in the pathogenic isolates was a DUP-1042B L. monocytogenes strain that has been implicated in major outbreaks, which emphasizes the adaptability and persistence of highly pathogenic stains in food manufacturing environments. Results from this study continue to support the fact that contaminated silage can be an important reservoir of the pathogen in a dairy farm setting. From our data and field observations we identified that drinking water sources for the animals is also an important reservoir of L. monocytogenes in farm environments. More importantly this study has shown the importance of continuous monitoring of environmental sites for the presence of the pathogen, particularly in silage.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Trm?i? , A., R. Ralyea , L. Meunier-Goddik , C. Donnelly, K. Glass, D. DAmico, E. Meredith, M. Kehler, N. Tranchina, C. McCue, M. Wiedmann. 2016. Consensus categorization of cheese based on water activity and pH  a rational approach to systemizing cheese diversity. Journal of Dairy Science 100:841847 Nyarko, E. B. and C.W. Donnelly. 2015. Listeria monocytogenes: strain heterogeneity, methods and challenges of subtyping.J. Food Sci. 80:M2868-M2878.
• Type: Books Status: Published Year Published: 2016 Citation: The Oxford Companion to Cheese, Catherine W. Donnelly, Editor
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Lekkas, Panagiotis, "The Microbial Ecology Of Listeria Monocytogenes As Impacted By Three Environments: A Cheese Microbial Community; A Farm Environment; And A Soil Microbial Community" (2016). Graduate College Dissertations and Theses. 463. http://scholarworks.uvm.edu/graddis/463
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lekkas, P. and C.W. Donnelly. 2016. Farm sources of Listeria monocytogenes and impact on the microbial safety of milk destined for artisan cheese production. P2-141 IAFP Annual Meeting, St. Louis, MO. August 2. Limoges, M. and C. Donnelly. 2016. FDAs compliance program guideline criteria for non-toxigenic Escherichia coli: Impacts on domestic and imported cheese. P2-142 IAFP Annual Meeting, St. Louis, MO. August 2. Gavell, J. and C. Donnelly. 2016. Validating the efficacy of cleaning procedures used to reduce microbial loads on wooden boards used for cheese aging. P2-143 IAFP Annual Meeting, St. Louis, MO. August 2. Donnelly,C. and P. Lekkas. 2016. Identification of farm sources of Listeria monocytogenes and incidence in milk destined for artisan cheese production. Problems of Listeriosis ISOPOL XIX, June 14-17, Institut Pasteur, Paris, France.

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

Outputs
Target Audience: The purpose of this project is to provide continued research for small-scale artisan cheese makers to help in the identification and management of microbiological risks. This is achieved by evaluating potential for survival of pathogens in cheeses during processing and aging, as well as validation of methods for detection and control of select pathogens of concern. In this project period,we evaluated a proprietary enrichment medium, mLRB, for recovery of acid-and nitrite-injured Listeria monocytogenes. We compared recovery in mLRB with enrichment media recommended by the USDA (UVM medium), FDA (buffered Listeria enrichment broth), and International Organization for Standardization (demi-Fraser broth). Listeria cell populations were significantly higher in mLRB versus UVM, BLEB and demi-Fraser. Delivery of selective agents to LRB via time-delayed release tablets maximized recovery of acid and nitrite-injured Listeria and saved time during food sample analysis. This format is suitable for utilization by cheese producers for analysis of cheeses and processing environments for Listeria. We evaluated Fourier Transform Infrared Spectroscopy (FT-IR) and Chemometrics as a means to accomplish differentiation of L. monocytogenes strains. Our research findings confirmed that the FT-IR method could be used as a differentiation/subtyping method for identification and source tracking of L. monocytogenes. We further conducted studies to compare the behavior of multi-drug resistant (MDR) and pansusceptible strains of Salmonella during the manufacture and aging of Gouda cheese. Our findings suggest that MDR Salmonella strains do not pose a greater threat to cheese safety than non-MDR Salmonella strains. These results have been published in scientific journals and will help inform cheese producers and regulators of improved methods for detection, control and risk evaluation of pathogens associated with cheese. Based on these outcomes, we will continue to identify and mitigate sources of bacterial pathogens in artisan cheese facilities to enhance the safety of these products. Publications: Nyarko, E. and C. Donnelly. 2014. Differentiation of mixed Listeria strains and also acid-injured, heat-injured, and repaired cells of Listeria monocytogenes using Fourier Transform Infrared spectroscopy. J. Food Prot. 78:540-548. Nyarko, E., K. Puzey and C. W. Donnelly. 2014. Rapid Differentiation of Listeria monocytogenes Epidemic Clones III and IV and Their Intact Compared with Heat-Killed Populations Using Fourier Transform Infrared Spectroscopy and Chemometrics. J. Food Sci.79:M1189-1196. DAmico, D.J., M. J. Druart and C. W. Donnelly. 2014. Comparing the Behavior of Multi-Drug Resistant and Pansusceptible Salmonella During the Production and Aging of a Gouda Cheese Manufactured from Raw Milk. J. Food Prot. 77:903-913. Nyarko, E., D. DAmico, P. Mach, W. Xia, and C. Donnelly. 2014. Delivery of selective agents via time-delayed release tablets improves recovery of Listeria monocytogenes injured by acid and nitrite. J. Food Prot. 77:772-780. Donnelly, C.W. and F. Diez-Gonzalez. 2013. Listeria monocytogenes, Chapter 3 In R.G. Labbe and S. Garcia (eds.) Guide to Foodborne Pathogens, Second Edition. Wiley Blackwell,West Sussex, UK, pages 45-74. Donnelly, C.W. 2013. From Pasteur to Probiotics: A Historical Overview of Cheese and Microbes. Microbiology Spectrum http://www.asmscience.org/content/journal/cm/10.1128/microbiolspec.CM-0001-12. Ryser, E.T., and C.W. Donnelly. 2014. Listeria. In Compendium of Method for the Microbiological Examination of Foods, 5th Edition. American Public Health Assoc., Washington, DC Relevancy Statement: UVM Researchers - develop methods for detection and evaluate potential for growth and survival of pathogens of concern to Vermont artisan cheese makers to improve cheese safety Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Information from our research studies is being utilized in development of Risk Reduction Plans for Artisan Cheesemakers in controlling microbial pathogens of concern. Our studies are also used to inform risk assessments regarding risks posed by bacterial pathogens to the safety of artisan cheeses. 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? During the next reporting period, we will compltet work which analyzes cheese microbial communities on washed rind cheese surfaces and their interactions with Listeria monocytogenes. We will also identify on-farm sources of Listeria contamination and strategies to mitigate these risks.

Impacts
What was accomplished under these goals? In this project period,we evaluated a proprietary enrichment medium, mLRB, for recovery of acid-and nitrite-injured Listeria monocytogenes. We compared recovery in mLRB with enrichment media recommended by the USDA (UVM medium), FDA (buffered Listeria enrichment broth), and International Organization for Standardization (demi-Fraser broth). Listeria cell populations were significantly higher in mLRB versus UVM, BLEB and demi-Fraser. Delivery of selective agents to LRB via time-delayed release tablets maximized recovery of acid and nitrite-injured Listeria and saved time during food sample analysis. This format is suitable for utilization by cheese producers for analysis of cheeses and processing environments for Listeria. We evaluated Fourier Transform Infrared Spectroscopy (FT-IR) and Chemometrics as a means to accomplish differentiation of L. monocytogenes strains. Our research findings confirmed that the FT-IR method could be used as a differentiation/subtyping method for identification and source tracking of L. monocytogenes. We further conducted studies to compare the behavior of multi-drug resistant (MDR) and pansusceptible strains of Salmonella during the manufacture and aging of Gouda cheese. Our findings suggest that MDR Salmonella strains do not pose a greater threat to cheese safety than non-MDR Salmonella strains. These results have been published in scientific journals and will help inform cheese producers and regulators of improved methods for detection, control and risk evaluation of pathogens associated with cheese. Based on these outcomes, we will continue to identify and mitigate sources of bacterial pathogens in artisan cheese facilities to enhance the safety of these products.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Nyarko, E. and C. Donnelly. 2015. Differentiation of mixed Listeria strains and also acid-injured, heat-injured, and repaired cells of Listeria monocytogenes using Fourier Transform Infrared spectroscopy. J. Food Prot. 78:540-548.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nyarko, E., K. Puzey and C. W. Donnelly. 2014. Rapid Differentiation of Listeria monocytogenes Epidemic Clones III and IV and Their Intact Compared with Heat-Killed Populations Using Fourier Transform Infrared Spectroscopy and Chemometrics. J. Food Sci.79:M1189-1196.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: DAmico, D.J., M. J. Druart and C. W. Donnelly. 2014. Comparing the Behavior of Multi-Drug Resistant and Pansusceptible Salmonella During the Production and Aging of a Gouda Cheese Manufactured from Raw Milk. J. Food Prot. 77:903-913.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nyarko, E., D. DAmico, P. Mach, W. Xia, and C. Donnelly. 2014. Delivery of selective agents via time-delayed release tablets improves recovery of Listeria monocytogenes injured by acid and nitrite. J. Food Prot. 77:772-780.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supported the training of one M.S. student and one P.h.D. student. A second M.S. student has joined the project. 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? Our continuing work will utilize genomic DNA extraction and pyrosequencing to identify microbial communities within the experimental and treatment cheeses. We will work with our experimental cheese making system to evaluate the impact of bacteriophages and competitive cultures in controlling Listeria growth on washed rind cheeses.

Impacts
What was accomplished under these goals? During the project period, we assessed survival of Listeria monocytogenes during 60 days of aging in washed rind artisan cheeses. Cheeses were produced at a commercial facility and washed once in a brine solution consisting of 3%NaCl and .5 DCU each of R2R and GEO 15 (Danisco, Denmark) per 1000ml of sterile deionized water. After the first wash the cheeses were transferred to lab scale cheese aging chambers where drying and ripening of the cheese was conducted. Cheeses were inoculated with L. monocytogenes at levels of 100 cfu/cm2 to mimic European Union limits. Cheeses were washed daily, with alternate surfaces being washed on alternate days, until surface mold began. Cheeses were turned daily until day 50 of ripening at which point the cheese were wrapped in parchment paper and placed back in the aging chamber with no added humidity following the procedure of the commercial manufacturer. Cheese samples were analyzed upon receipt, after pathogen inoculation (day 1) and during ripening. Samples (25g) were enumerated on CHROMagarTM Listeria plates. For the control cheeses (and instances where the above procedure did not produce detectable colonies of L. monocytogenes) cheese samples were enriched and screened for Listeria spp. using the DuPont Qualicon BAX. The pH of experimental cheeses was measured throughout aging, and the visual appearance of the cheese was recorded each day to monitor rind development. We also analyzed the growth interactions between L. monocytogenes and Pseudomonas spp. isolated from the surface of our experimental washed rind cheese. By the end of the 70-day study period the ripening conditions of the lab scale aging chambers conformed to the standards for washed rind cheese established by the commercial producer. During the first 2 weeks of the aging process the experimental cheeses experienced rind development that mimicked the rind development of the commercial cheese. However, in 2 of the 3 trials surface growth of Pseudomonas spp. was observed as florescent surface growth. Increased Pseudomonas spp. growth was associated with unfavorable rind development. All control cheeses and the cheeses from 1 of the 3 trials did not experience changes in rind development due to Pseudomonas spp. growth. The surface microflora developed during each of 3 independent trials did not exhibit sufficient anti-listerial activity to be used as a food safety tool. Pseudomonas spp. isolates from the surface of experimental cheese did not exhibit anti-listerial activity against any of the 6 individual L. monocytogenes strains used. All 6 of the L. monocytogenes strains tested did not exhibit anti-pseudomonas spp. potential (as measured by observable zones of clearing on Pseudomonas spp. bacterial lawns). These results were identical across 4 independent trials. Our continuing work will utilize genomic DNA extraction and pyrosequencing to identify microbial communities within the experimental and treatment cheeses. We will work with our experimental cheese making system to evaluate the impact of bacteriophages and competitive cultures in controlling Listeria growth on washed rind cheeses. During the project period, we assessed survival of Listeria monocytogenes during 60 days of aging in washed rind artisan cheeses. Cheeses were produced at a commercial facility and washed once in a brine solution consisting of 3%NaCl and .5 DCU each of R2R and GEO 15 (Danisco, Denmark) per 1000ml of sterile deionized water. After the first wash the cheeses were transferred to lab scale cheese aging chambers where drying and ripening of the cheese was conducted. Cheeses were inoculated with L. monocytogenes at levels of 100 cfu/cm2 to mimic European Union limits. Cheeses were washed daily, with alternate surfaces being washed on alternate days, until surface mold began. Cheeses were turned daily until day 50 of ripening at which point the cheese were wrapped in parchment paper and placed back in the aging chamber with no added humidity following the procedure of the commercial manufacturer. Cheese samples were analyzed upon receipt, after pathogen inoculation (day 1) and during ripening. Samples (25g) were enumerated on CHROMagarTM Listeria plates. For the control cheeses (and instances where the above procedure did not produce detectable colonies of L. monocytogenes) cheese samples were enriched and screened for Listeria spp. using the DuPont Qualicon BAX. The pH of experimental cheeses was measured throughout aging, and the visual appearance of the cheese was recorded each day to monitor rind development. We also analyzed the growth interactions between L. monocytogenes and Pseudomonas spp. isolated from the surface of our experimental washed rind cheese. By the end of the 70-day study period the ripening conditions of the lab scale aging chambers conformed to the standards for washed rind cheese established by the commercial producer. During the first 2 weeks of the aging process the experimental cheeses experienced rind development that mimicked the rind development of the commercial cheese. However, in 2 of the 3 trials surface growth of Pseudomonas spp. was observed as florescent surface growth. Increased Pseudomonas spp. growth was associated with unfavorable rind development. All control cheeses and the cheeses from 1 of the 3 trials did not experience changes in rind development due to Pseudomonas spp. growth. The surface microflora developed during each of 3 independent trials did not exhibit sufficient anti-listerial activity to be used as a food safety tool. Pseudomonas spp. isolates from the surface of experimental cheese did not exhibit anti-listerial activity against any of the 6 individual L. monocytogenes strains used. All 6 of the L. monocytogenes strains tested did not exhibit anti-pseudomonas spp. potential (as measured by observable zones of clearing on Pseudomonas spp. bacterial lawns). These results were identical across 4 independent trials. Our continuing work will utilize genomic DNA extraction and pyrosequencing to identify microbial communities within the experimental and treatment cheeses. We will work with our experimental cheese making system to evaluate the impact of bacteriophages and competitive cultures in controlling Listeria growth on washed rind cheeses.

Publications