Control of Food-Borne Pathogens in Pre- and Post-Harvest Environments

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1430	1100	20%
712	3260	1100	20%
712	3320	1100	20%
712	4010	1030	10%
712	4010	1040	10%
712	4010	1090	10%
712	4010	1100	10%

Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: Food composition is an important factor for the initial attachment of pathogenic bacteria; however the persistent presence of pathogens such as Listeria may also be affected by background bacteria remaining on the surfaces after the cleaning and sanitizing process. It is critical to assess the level of microbial diversity on the food-contact surfaces. We assessed the extent and distribution of microbial diversity of deli slicers by identification of pathogenic & background bacteria. We completed overall goals for controlling Listeria in these systems as follows:a)Compared Listeria spp.for their sensitivity to antimicrobial plant essential oils. The antimicrobial mechanism of action was explored by determining if synergism with cell wall/membrane affecting antibiotics & bacteriocins existed. b)Determined the optimal formulation of food grade sanitizer system containing antimicrobial plant essential oils for dispersal & inactivation of surface attached L. monocytogenes. c)Used denatured gradient gel electrophoresis(DGGE)to profile microbial populations in deli slicers to determine patterns of microbial populations associated with microbial contamination.d)Initiated studies on ecology of norovirus surrogates & transfer potential in retail food environments with different cleaning cloths. Samples from six deli slicers that were currently being used in the food service industry to slice all cheese, vegetables & RTE deli meat in the same slicers were collected from restaurants in Fayetteville,AR and Houston,TX. Ten different surface areas in each slicer were selected & included; the cover for blade sharpener, back plate, blade guard, blade, carriage tray, side wall of carriage tray, collection area, side area of collection area, underneath the slicer, & the back of blade. The sample collection for microbial analysis was performed when the slicers were not in use & after their regular cleaning & sanitization process, which was between 3 to 5 times a day. After DNA extraction, DNA amplification & DGGE were performed. Sequence analysis was performed by excising the bands of interest from the DGGE gel. Bacteria may survive & attach to food-contact surfaces by residual food components and/or background bacteria which may subsequently transfer to other food products. Pseudomonads were identified as the dominant bacteria following with Enterobacteriaceae family & lactic acid bacteria(Lactococcus lactis). Streptococcus thermophilus were also found. Bacterial distribution was similar for all surface areas while the blade guard exhibited the greatest diversity. This study provided a profile of the microbial ecology of slicers using DGGE & to develop more specific sanitation practices to reduce cross-contamination during slicing. PARTICIPANTS: S.C. Ricke (Professor and Project Director), P.G. Crandall and M.G. Johnson, (Professors, Co-PDs and collaborators), A. Muthaiyan (Postdoc), J. Lingbeck (Research Assistant Professor), S. Milillo (Postdoc), K. Gibson (Postdoc), O. Koo (Postdoc), R. Story (Technician), E. van Loo (M.S. student), E. Shannon (M.S. student). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This study was the first to demonstrate the application of DGGE methodology to investigate the microbial diversity of food-contact surfaces such as the deli slicer. These results can further enhance the understanding of the microbial ecology of food-contact surfaces and aid in developing effective cleaning and sanitization methods. L. monocytogenes is a gram positive, facultative anaerobic, rod bacterium that the food industry regards as an important foodborne pathogen The organism is responsible for a life-threatening disease "listeriosis" that is able to cause different ailments including maternofetal- or neonatal -listeriosis, bloodstream infection, and meningoencephalitis. L. monocytogenes is not only a public health burden because of high mortality and high hospitalization associated with listeriosis cases but also an economic problem for the food industry due to costly food recalls when an outbreak occurs. It is a significant bacterial foodborne pathogen associated with RTE meat products, such as deli meat. The most recent CDC/FDA/FSIS risk assessment found that consumption of deli meats results in the highest risk of contracting listeriosis. Moreover, it has been estimated that turkey deli meat alone contributes significantly to the total number of listeriosis cases. Because L. monocytogenes contamination of deli meats is primarily due to post-processing contamination, in order to successfully combat potential listeriosis cases effective methods for preventing its survival and persistence in deli meat processing/handling environments must be developed.

Publications

Jiang, W., R.S. Pinder, J.A. Patterson, and S.C. Ricke. 2012. Sugar phosphorylation activity in ruminal acetogens. J. Environ. Health Sci., Part A 47: 843-846.
Millilo, S.R., C.A. OBryan, Shannon, E.M., M.G. Johnson, P.G. Crandall, and S.C. Ricke. 2012. Enhanced inhibition of Listeria monocytogenes by a combination of cold pressed terpeneless Valencia orange and antibiotics. Foodborne Pathogens Dis. 9: 370-372.
Crandall, P.G., C.A. OBryan, E.M. Martin, H.M. Kuefner, S. Pendleton, E.M. Shannon, J.A. Marcy and S.C. Ricke. 2012. Efficacy of cleaning and sanitizing agents against surface attached Listeria monocytogenes on meat slicer components. Food Prot. Trends. 32: 68-72.
Gibson, K.E., P.G. Crandall, and S.C. Ricke. 2012. Removal and transfer of viruses on food contact surfaces by cleaning cloths. Appl. Environ. Microbiol. 78: 3037-3044.
Masuku, S.M., D. Babu, E.M. Martin, O.K. Koo, C.A. OBryan, P.G. Crandall, and S.C. Ricke 2012. Cleaning and decontamination efficacy of wiping cloths and silver dihydrogen citrate on food contact surfaces. J Appl. Microbiol. 113: 89-95.
Ndahetuye, J.B., O.K. Koo, C.A. OBryan, S.C. Ricke, and P.G. Crandall. 2012. Role of lactic acid bacteria as a bio-sanitizer to prevent attachment of Listeria monocytogenes on deli slicer contact surfaces. J. Food Prot. 75: 1429-1436.
Milillo, S.R., J.C. Stout, I.B. Hanning, A. Clement, E.D. Fortes, H.C. den Bakker, M. Wiedemann, and S.C. Ricke. 2012. Characteristics of Listeria from pasture-reared poultry reveals L. monocytogenes and hemolytic L. innocua in poultry ceca and the pasture environment. Poultry Sci. 91: 2158-2163.
Koo, O.K., E.M. Martin, R. Story, D. Lindsay, S. C. Ricke, and P.G. Crandall 2012. Comparison of cleaning fabrics for bacterial removal from food contact surfaces. Food Control 30: 292-297.
Koo, O.K., E.M. Martin, R. Story, D. Lindsay, S. C. Ricke, and P.G. Crandall 2012. Comparison of cleaning fabrics for bacterial removal from food contact surfaces. Food Control 30: 292-297.
Perumalla, A.V.S., N.S. Hettiarachchy, K.F. Over, S.C. Ricke, E.D. Gbur, J. Zhang, and B. Davis. 2012. Effect of potassium lactate and sodium diacetate combination to inhibit Listeria monocytogenes in low and high fat chicken and turkey hotdog model systems. The Open Food Science J. 6: 16-23.
Gibson, K.E., O.K. Koo, C. A. OBryan, J.A. Neal, S. C. Ricke, and P. G. Crandall. 2013. Observational and relative quantification of cross-contamination within a mock retail delicatessen environment. Food Control 31: 116-124.
Milillo, S.R., R.S. Story, D. Pak, and S.C. Ricke. 2013. Antimicrobial properties of three lactic acid bacterial cultures and their cell free supernatants against Listeria monocytogenes. J. Environ. Health Sci., Part B 48: 63-68.
Milillo, S.R., E.C. Friedly, J.C. Saldivar, A. Muthaiyan, C.A. OBryan, P.G. Crandall, M.G. Johnson, and S.C. Ricke. 2012. A review of the ecology, genomics and stress response of Listeria innocua and Listeria monocytogenes. Crit. Reviews in Food Sci. and Nutr. 52: 712-725.
Gibson, K.E. and S.C. Ricke. 2012. Human noroviruses and food safety. Agric., Food, Anal. Bacteriol. 2: 25-34.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We have overall goals for controlling Listeria in food systems as follows: A) Characterize the mechanism(s) of action of antimicrobial plant essential oils and oil components against Listeria: The six Listeria spp. will be compared for their sensitivity to antimicrobial plant essential oils/oil components. These experiments will help elucidate what cellular components contribute to resistance or susceptibility as the Listeria spp. are highly homologous and at least five of the six species have been sequenced. Additionally, the antimicrobial mechanism of action of will be explored by determining if synergism with cell wall/membrane affecting antibiotics and bacteriocins exists as well as screening for spontaneous resistance and characterizing the source of the mutation/resistance. B) Determine the optimal formulation of food grade sanitizer system containing antimicrobial plant essential oils for dispersal and inactivation of surface attached L. monocytogenes: Methods for complete sanitization of food processing/handling equipment are urgently required. Antimicrobial plant essential oils formulated in food grade sanitizers represent a promising avenue for sanitization due to their potential for both dispersal and inactivation of attached bacteria. Using coupons of materials representative of those found in ready to eat (RTE) food processing and handling environments, L. monocytogenes serotype 1/2a and 4b strains as well as L. innocua will be examined for attachment. Subsequently, attached Listeria will be subjected to plant essential oil sanitizer treatments and the treatments evaluated for their effectiveness at dispersing and inactivating the attached cells. PARTICIPANTS: S.C. Ricke,Professor and Project Director, A. Muthaiyan, Postdoc, J. Lingbeck, Research Assistant Professor, S. Milillo, Postdoc, K. Gibson Postdoc, R. Story, Technician, E. Van Loo, M.S. student, E. Shannon, M.S. student. TARGET AUDIENCES: Food safety remains an ongoing concern from a public health perspective. Consequently, there is a need to develop a variety of control measures at all levels of food production that have different mechanisms for inhibiting pathogen growth and survival. Successful development of these interventions will give the food industry more choices to meet specific needs at different steps during processing. PROJECT MODIFICATIONS: All natural, plant essential oils will be screened for antimicrobial activity against the Listeria species as well as against important listeriosis-associated L. monocytogenes serotypes using minimum inhibitory concentration agar disc diffusion and well diffusion technique approaches. Plant essential oils derived from herbs and spices (e.g. carvacrol from thyme and oregano and eugenol from clove) as well as those from waste products of the fruit juice industry will be screened. For candidate essential oils that represent mixtures of compounds, the essential oils themselves will be characterized to determine the active component(s). Furthermore, a variety of environmental conditions will be tested, such as low temperature, nutrient deprivation, and/or oxygen restriction, since these are the likely conditions in which L. monocytogenes would encounter the plant essential oils/components in food systems. In the second phase the mechanism of action of the most effective plant essential oil/oil component will be investigated by initially testing L. monocytogenes with and without exposure to plant essential oils for susceptibility to a range of cell wall/membrane active antibiotics (e.g. penicillin and ampicillin) and bacteriocins (e.g. nisin).

Impacts
Listeria monocytogenes is a significant bacterial foodborne pathogen associated with RTE poultry products, such as deli meat. The most recent CDC/FDA/FSIS risk assessment found that consumption of deli meats results in the highest risk of contracting listeriosis. Moreover, it has been estimated that turkey deli meat alone contributes significantly to the total number of listeriosis cases. Because L. monocytogenes contamination of deli meats is primarily due to post-processing contamination, in order to successfully combat potential listeriosis cases effective methods for preventing its survival and persistence in deli meat processing/handling environments must be developed.

Publications

Crandall, P.G., E.C., Friedly, M. Patton, C.A. OBryan, A. Gurubaramurugeshan, S. Seideman, S.C. Ricke, and R. Rainey. 2011. Consumer awareness of and concerns about food safety at three Arkansas Farmers markets. Food Prot. Trends 31: 156-165.
Limayen, A., I. B. Hanning, A. Muthaiyan, K. Illeghems, J.-W. Kim, P.G. Crandall, C.A. OBryan, and S.C. Ricke. 2011. Alternative antimicrobial compounds to control potential Lactobacillus contaminants that occur in bioethanol fermentations. J. Environ. Health Sci. 46B: 709-714.
Muthaiyan, A., A. Limayen, and S.C. Ricke. 2011. Antimicrobial strategies for limiting bacterial contaminants in fuel bioethanol fermentations. Prog. Energy Comb. Sci. 37: 351-370.
Van Loo, E., V. V. Caputo, R.M. Nayga, Jr., J.-F. Meullenet, and S.C. Ricke. 2011. Consumers willingness to pay for organic chicken breast: Evidence from choice experiment. Food Qual. Preference. 22: 603-613.
Njongmeta, N.L.A., H. Benli, K.D. Dunkley, C.S. Dunkley, D.R. Miller, R.C. Anderson, C.A. OBryan, J.T. Keeton, P.G. Crandall, and S.C. Ricke. 2011. Application of acidic calcium sulfate and ε-polylysine to pre-rigor beef rounds for reduction of pathogens. J. Food Safety 31: 395-400.
Shannon, E., S.R. Milillo, M.G. Johnson, and S.C. Ricke. 2011. Inhibition of Listeria monocytogenes by exposure to a combination of nisin and cold-pressed terpeneless valencia oil. J. Food Sci. 76: 600-604.
Shannon, E.M., S.R. Milillo, M.G. Johnson, and S.C. Ricke. 2011. Efficacy of cold-pressed terpeneless Valencia oil and its primary components on inhibition of Listeria species by direct contact and exposure to vapors. J. Food Sci. 76: M500-M503.
Ravichandran, M., N.S. Hettiarachchy, V. Ganseh, S.C. Ricke and S. Singh. 2011. The enhancement of antimicrobial activities of naturally occurring phenolic compounds by nanaoparticle mediated delivery against Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium in broth and chicken meat system. J. Food Safety 31: 462-471.
Lungu, B., C.A. OBryan, A, Muthaiyan, S. R. Milillo, M.G. Johnson, P.G. Crandall, and S.C. Ricke. 2011. Listeria monocytogenes: Antibiotic resistance in food production. Foodborne Path. Dis. 8: 569-578.
Crandall, P.G., J.A. Neal Jr., C.A. OBryan, C.A. Murphy, B.P. Marks, and S.C. Ricke. 2011. Minimizing the risk of Listeria monocytogenes in retail delis by developing employee focused, cost effective training. Agric., Food, Anal. Bacteriol. 1:159-174.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We have overall goals for controlling Listeria in food systems as follows: A) Characterize the mechanism(s) of action of antimicrobial plant essential oils and oil components against Listeria: The six Listeria spp. will be compared for their sensitivity to antimicrobial plant essential oils/oil components. These experiments will help elucidate what cellular components contribute to resistance or susceptibility as the Listeria spp. are highly homologous and at least five of the six species have been sequenced. Additionally, the antimicrobial mechanism of action of will be explored by determining if synergism with cell wall/membrane affecting antibiotics and bacteriocins exists as well as screening for spontaneous resistance and characterizing the source of the mutation/resistance. B) Determine the optimal formulation of food grade sanitizer system containing antimicrobial plant essential oils for dispersal and inactivation of surface attached L. monocytogenes: Methods for complete sanitization of food processing/handling equipment are urgently required. Antimicrobial plant essential oils formulated in food grade sanitizers represent a promising avenue for sanitization due to their potential for both dispersal and inactivation of attached bacteria. Using coupons of materials representative of those found in ready to eat (RTE) food processing and handling environments, L. monocytogenes serotype 1/2a and 4b strains as well as L. innocua will be examined for attachment. Subsequently, attached Listeria will be subjected to plant essential oil sanitizer treatments and the treatments evaluated for their effectiveness at dispersing and inactivating the attached cells. PARTICIPANTS: S.C. Ricke (Professor and Project Director, A. Muthaiyan (Postdoc), J. Lingbeck (Research Assistant Professor), S. Milillo (Technician/Postdoc), K. Gibson (Postdoc), E. van Loo (M.S. student), E. Shannon (M.S. student). TARGET AUDIENCES: Food safety remains an ongoing concern from a public health perspective. Consequently, there is a need to develop a variety of control measures at all levels of food production that have different mechanisms for inhibiting pathogen growth and survival. Successful development of these interventions will give the food industry more choices to meet specific needs at different steps during processing. PROJECT MODIFICATIONS: All natural, plant essential oils will be screened for antimicrobial activity against the Listeria species as well as against important listeriosis-associated L. monocytogenes serotypes using minimum inhibitory concentration agar disc diffusion and well diffusion technique approaches. Plant essential oils derived from herbs and spices (e.g. carvacrol from thyme and oregano and eugenol from clove) as well as those from waste products of the fruit juice industry will be screened. For candidate essential oils that represent mixtures of compounds, the essential oils themselves will be characterized to determine the active component(s). Furthermore, a variety of environmental conditions will be tested, such as low temperature, nutrient deprivation, and/or oxygen restriction, since these are the likely conditions in which L. monocytogenes would encounter the plant essential oils/components in food systems. In the second phase the mechanism of action of the most effective plant essential oil/oil component will be investigated by initially testing L. monocytogenes with and without exposure to plant essential oils for susceptibility to a range of cell wall/membrane active antibiotics (e.g. penicillin and ampicillin) and bacteriocins (e.g. nisin).

Impacts
Listeria monocytogenes is a significant bacterial foodborne pathogen associated with RTE poultry products, such as deli meat. The most recent CDC/FDA/FSIS risk assessment found that consumption of deli meats results in the highest risk of contracting listeriosis. Moreover, it has been estimated that turkey deli meat alone contributes significantly to the total number of listeriosis cases. Because L. monocytogenes contamination of deli meats is primarily due to post-processing contamination, in order to successfully combat potential listeriosis cases effective methods for preventing its survival and persistence in deli meat processing/handling environments must be developed.

Publications

Muthaiyan, A. and S.C. Ricke. 2010. Current perspectives on detection of microbial contamination in bioethanol fermentors. Bioresource Technol. 101: 5033-5042.
Chalova, V.I., C.A. Froelich, Jr.*, and S.C. Ricke. 2010. Potential for development of an Escherichia coli based biosensor for assessing bioavailable methionine: A review. Sensors 10: 3562-3584.
OBryan, C.A., D.M. Johnson, K. Shores-Ellis, P.G. Crandall, J. A. Marcy, S.C. Seideman, and S.C. Ricke. 2010. Designing an affordable usability test for E-Learning modules. J. Food Sci. Educ. 9: 6-10.
Lungu, B., J.C. Saldivar, S.C. Ricke, and M.G. Johnson. 2010. The combination of energy-dependent internal adaptation mechanisms and external factors enable Listeria monocytogenes to express a strong starvation survival response during multiple-nutrient starvation. Foodborne Pathogens Dis. 7: 499-505.
Martin, E.M., C.A. OBryan, R.Y. Lary Jr., C.L. Griffis, K.L.S. Vaughn, J.A. Marcy, P.G. Crandall, and S.C. Ricke. 2010. Spray application of liquid smoke to reduce or eliminate Listeria monocytogenes surface inoculated on frankfurters. Meat Sci. 85: 640-544.
Van Loo, E.*, S.C. Ricke, S.R. Milillo, S. Seideman, and P.G. Crandall. 2010. Consumer food safety perceptions of ready-to-eat deli foods in Northwest Arkansas. Food Prot. Trends 30: 635-643.
Crandall, P.G., E.C., Friedly, E.C., M. Patton, C.A. OBryan, A. Gurubaramurugeshan, S. Seideman, S.C. Ricke, and R. Rainey. 2010. Estimating the demand for organic foods by consumers at farmers markets in Northwest Arkansas. J. Agric. and Food Info. 11: 185-208.
Crandall, P.G., C.A. OBryan, E.M. Martin, S. Pendleton, E. Shannon*, J. Marcy, and S.C. Ricke. 2010. Dry thermal inactivation of Listeria innocua on deli slicer components. Food Prot. Trends 30: 588-592.
Bozic, A.A. , R.C. Anderson, T.R. Callaway, D.J. Nisbet, S.C. Ricke, P.G. Crandall, and C.A. OBryan. 2010. In vitro comparison of nitroethane, 2-nitro-1-propanol, lauric acid, lauricidin(registered trademark), and the Hawaiian marine algae, Chaetoceros, for potential broad spectrum control of anaerobically grown Staphylococcus aureus. J. Appl. Res. Vet. Med. 8: 180-184.
OBryan, C.A, R.S. Dittmar, V.I. Chalova, M.M. Kundinger, P.G. Crandall, and S.C. Ricke. 2010. Assessment of a food microbiology senior undergraduate course as a potential food safety distance education course for poultry science majors. Poultry Science 89: 2542-2545.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: In other collaborative work, it was shown that there was a low prevalence of L. monocytogenes present in older swine sows at the time of culling. Post harvest studies: A wild type (wt) strain of L. monocytogenes, 10403S and a delta sigB mutant, FSLA1-245, were stressed by starvation in phosphate buffered saline and by exposure to chemicals with or without oxygen. In control tests, the mutant survived starvation about as well as the wt, suggesting that the starvation survival response (SSR) in this organism was sigBindependent. In the presence of chemical stresses, the SSR results differed depending on the chemical used. Thus for sodium chloride, both strains were able to express a SSR under aerobic conditions but not under anaerobic conditions. Conversely, for sodium propionate (SP) or sodium lactate (SL), the mutant respectively produced 2 and 3 log lower counts than the wt under anaerobic conditions. The latter results appeared to suggest that the L. monocytogenes cells had a sigB- dependent SSR response after exposure to SP or SL under anaerobic conditions. Additionally, the better survival of stressed cells in presence of SL under aerobic conditions may indicate the better ability of this organism to grow on food products after the anaerobic packaging film is first opened by the consumer. Interestingly, stressed cells exposed to sodium diacetate (SD) were unable to express a SSR, with no detectable cells observed after 14-21 days under aerobic or anaerobic conditions. Additionally, while a recent review of the literature indicates that this pathogen has not been documented to be associated with any foodborne illness outbreaks involving pre-cut, prepackaged lettuce, this food is still a possible theoretical vector as this food is currently processed and marketed in the USA. PARTICIPANTS: The major participants on this project were Dr. Steve Ricke and his Post Doc associates, Drs Arun Muthaiyan, Irene Hanning, and Sara Milillo. Other team members were Mr. Robert Story, Program Associate and Erin Shannon, graduate student. TARGET AUDIENCES: Researchers and food processors interested in understanding some of the mechanisms by which this pathogen can persist in pre-harvest and raw product processing environments and persist in post-harvest, post thermal processing environments by resisting approved GRAS status chemical preservatives during cold storage and distribution of cooked RTE foods. As persistence and resistance mechanisms are better understood, more comprehensive processing methods can be developed to more reliably control and eliminate this pathogen in our food systems. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Post - harvest: The above results with sodium lactate (SL) and sodium diacetate (SD) differ dramatically. Collectively, the results showing the better survival of stressed cells in presence of SL under aerobic conditions than under anaerobic conditions with no oxygen present may indicate the better ability of this organism to grow on food products after the anaerobic packaging film is first opened by the consumer. Conversely, the fact that stressed cells exposed to SD were unable to express a SSR, showing no detectable viable cells after 14-21 days under aerobic or anaerobic conditions is very encouraging from the standpoint of using the hurdle theory to inhibit pathogen growth in foods during storage. The latter result suggests that the latter chemical, unlike SL, is able to prevent growth of L. monocytogenes cells in such food packages after oxygen is introduced by opening the food product package.

Publications

Lungu, B., S.C. Ricke, and M.G. Johnson. 2009. Growth, survival, proliferation and pathogenesis of L. monocytogenes under low oxygen or anaerobic conditions: a review. Anaerobe 15: 7-17.
Anderson, R.C., S.C. Ricke, B. Lungu, M.G. Johnson C. Oliver, S.M. Horrocks and D.J. Nisbet. 2009. Food safety issues and the microbiology of beef. pp. 115-145 In: Microbiologically Safe Foods, N. Heredia, I. Wesley and S. Garcia (eds.) John Wiley & Sons, Inc. Hoboken, NJ.
Shannon, E., S. Milillo, S.C. Ricke, and M.G. Johnson. 2009. Effect of cell membrane disrupting antimicrobial agents on Listeria monocytogenes during cold growth. Retail Food Safety, A Catalyst for Change, Arkansas Association for Food Protection, Tyson Food Headquarters, Springdale, AR, Oct. 8-9, 2009.
Crandall, P.G., S. Seideman, S.C. Ricke, C.A. OBryan, A.F. Fanatico, and R. Rainey. 2009. Organic poultry consumer perceptions, opportunities and regulatory issues. J. Appl. Poultry Res. 18: 795-802.
Sirsat, S.A., A. Muthaiyan, and S.C. Ricke. 2009. Antimicrobials for pathogen reduction in organic and natural poultry production. J. Appl. Poultry Res. 379-388.
Kim, W.K., L.J. Weeks, R.C. Anderson, D.J. Nisbet, K. Dunkley, and S.C. Ricke. 2009. Effects of nitrocompounds on uric acid utilizing microorgansisms, nitrogen retention, and microbial community in laying hen manure. J. Environ. Sci. Health, Part B 44: 403-406.
OBryan, C.A., P.G. Crandall, K. Shores-Ellis, D.M. Johnson, S.C. Ricke, and J. Marcy. 2009. Optimizing web based instruction, a case study using poultry processing unit operations. J. Food Sci. Educ. 8: 93-100.
Park, S. H., H.J. Kim, W.H. Cho, J.H. Kim, M.H. Oh, S.H. Kim, B.K. Lee, S.C. Ricke, and H.Y. Kim. 2009. Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR. FEMS Microbiol. Letts. 301: 137-146.
Ganesh, V., N.S. Hettiarachchy, M.G. Johnson, J.-F. Meullenet, and S.C. Ricke. 2009. Electrostatic spray treatments with organic acids and plant extracts to decontaminate Salmonella Typhimurium in spinach. Institute of Food Technologists Annual Meeting and Food Expo, Albany, CA.
Martin, E.M., S.J. Pendleton, E.M. Shannon, S.R. Milillo, P.G. Crandall, S.C. Ricke, M.G. Johnson, C.A. OBryan and J.A. Marcy. 2009. Thermal inactivation of Listeria innocua as a surrogate for Listeria monocytogenes on deli slicer components. Amer. Soc. Microbiol. General 109th Annual Meeting, Philadelphia, PA.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Pre-harvest studies: The persistence of Listeria monocytogenes in tissues of turkey poults exposed to L. monocytogenes on days 4 to 12 when exposed to challenges by an aerosol or oral dosing with one of three treatments, a pathogenic to turkeys strain of E. coli, L. monocytogenes Scott A, serotype 4b or a combination of both, E.c. and L.m was examined. Further stresses were cold stress and immunosuppressive doses of dexamethasone (Dex) given at 7 weeks of age. There were no significant differences in cumulative mortality, body and organ weights or airsacculitis scores. At 1 week post-challenge, L. m. was isolated from up to 50 percent of liver or knee synovial tissues by pre enrichment and culturing and was detected in up to 67 percent of knee synovial tissues using pre enrichment followed by real time PCR. Interestingly, by 2 weeks post-challenge the previously positive birds appeared to give negative results, suggesting this pathogen at levels used could be cleared by the turkey's immune system. In other collaborative work, it was shown that there was a low prevalence of L. monocytogenes present in older swine sows at the time of culling. Post harvest studies: A wild type (wt) strain of L. monocytogenes, 10403S and a delta sigB mutant, FSLA1-245, were stressed by starvation in phosphate buffered saline and by exposure to chemicals with or without oxygen. In control tests, the mutant survived starvation about as well as the wt, suggesting that the starvation survival response (SSR) in this organism was sigB- independent. In the presence of chemical stresses, the SSR results differed depending on the chemical used. Thus for sodium chloride, both strains were able to express a SSR under aerobic conditions but not under anaerobic conditions. Conversely, for sodium propionate (SP) or sodium lactate (SL), the mutant respectively produced 2 and 3 log lower counts than the wt under anaerobic conditions. The latter results appeared to suggest that the L. monocytogenes cells had a sigB- dependent SSR response after exposure to SP or SL under anaerobic conditions. Additionally, the better survival of stressed cells in presence of SL under aerobic conditions may indicate the better ability of this organism to grow on food products after the anaerobic packaging film is first opened by the consumer. Interestingly, stressed cells exposed to sodium diacetate (SD) were unable to express a SSR, with no detectable cells observed after 14-21 days under aerobic or anaerobic conditions. Additionally, while a recent review of the literature indicates that this pathogen has not been documented to be associated with any foodborne illness outbreaks involving pre-cut, prepackaged lettuce, this food is still a possible theoretical vector as this food is currently processed and marketed in the USA. PARTICIPANTS: The major pre- harvest cooperator teams on this project were Dr. Gerry Huff of the USDA-ARS Fayetteville, AR Poultry Production and Product Safety Unit along with her M.S. student in Poultry Science, Mr. Vik Dutta who graduated in Dec 2007 and in Jan 2008 moved to North Carolina St U to begin his Ph.D. degree and Dr. Irene Wesley and her team members at NADC, USDA-ARS in Ames, IA . Post- harvest cooperators included Dr. Steve Ricke and his Post - Doc associates, Drs Arun Muthaiyan and Irene Hanning, and by Dr Johnson's team of Mr. Robert Story and two graduate students, Ph.D. student, Dr. Bwalya Lungu who moved in January 2008 to start a Post -Doc appointment with Dr. Joe Frank at the U of Georgia and Mr. Josh Saldivar, M.S. student, who moved to Ohio State U in 2008 to pursue his Ph.D. degree. TARGET AUDIENCES: Researchers and food processors interested in understanding some of the mechanisms by which this pathogen can persist in pre- harvest and raw product processing environments and persist in post -harvest, post thermal processing environments by resisting approved GRAS status chemical preservatives during cold storage and distribution of cooked RTE foods. As persistence and resistance mechanisms are better understood, more comprehensive processing methods can be developed to more reliably control and eliminate this pathogen in our food systems. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Pre- harvest: Earlier collaborative work showed that this pathogen could be detected in knee joints of turkeys. The possible role and persistence of latent infections in joint tissues of meat animal as a potential source of cross contamination to further processed products by L. monocytogenes in poultry slaughter plants has not been thoroughly investigated. The results from the model turkey poult system used suggest that environmentally acquired L. monocytogenes cells can transiently colonize the liver and synovial tissues of stressed turkey and may be a sporadic source of contamination of further processed poultry in processing plants. Post - harvest: The above results with sodium lactate (SL) and sodium diacetate (SD) differ dramatically. Collectively, the results showing the better survival of stressed cells in presence of SL under aerobic conditions than under anaerobic conditions with no oxygen present may indicate the better ability of this organism to grow on food products after the anaerobic packaging film is first opened by the consumer. Conversely, the fact that stressed cells exposed to SD were unable to express a SSR, showing no detectable viable cells after 14-21 days under aerobic or anaerobic conditions is very encouraging from the standpoint of using the hurdle theory to inhibit pathogen growth in foods during storage. The latter result suggests that the latter chemical, unlike SL, is able to prevent growth L. monocytogenes cells in such food packages after oxygen is introduced by opening the food product package.

Publications

Dutta, V., Huff, G. R., W.E. Huff, M.G. Johnson, R. Nannapaneni, R.J Saylor. 2008. The effects of stress on respiratory disease and transient colonization of turkeys with Listeria monocytogenes Scott A. Avian Dis.:52:581-589 .
Lungu, B., S.E. Dowd, A. Muthaiyan, S.C. Ricke, M.G. Johnson. 2008. Transcriptional analysis of the growth of L. monocytogenes 10403S and as Sig B mutant strain following exposure to dinitrophenol or sodium arsenite. Abstract Amer. Soc. for Microbiology Annual General Mtg. Boston, MA. June 1-5, 2008, Abstract P-005, p 486.
Lungu, B., J.C. Saldivar, R.P. Story, S.C. Ricke and M.G. Johnson. 2008. Protein synthesis and metabolic energy strategies in Listeria monocytogenes 10403S and a Sig B mutant during multiple nutrient starvation. Abstract Amer. Soc. for Microbiology Annual General Mtg. Boston, MA. June 1-5, 2008, Abstract P-018, pp 488-9.
Wesley, I.V., S. Larsen, H. S. Hurd, J. D. McKean , R. Grifffith, F. Rivera, R. Nannapaneni, M.Cox, M. G. Johnson , D. Wagner & M. DeMartino . 2008. Low prevalence of Listeria monocytaogenes in cull sows. J. Food Prot. 71(No.3):545-549.
Hanning, I, M.G. Johnson, and S.C. Ricke. 2008. Pre-cut pre-packaged lettuce: A risk for listeriosis Foodborne Pathogens & Dis. Vol 5(No. 6):731-746. (Manuscript FPD 2008-0142, R-2, accepted July 9, 2008 , available on line Dec 1, 2008).
Huff. G.R., W.E. Huff, V. Dutta, M.G. Johnson, and R. Nannapaneni. 2008. Pathogenicity of Listeria monocytogenes Scott A after oral and oculonasal challenges of day-old turkey poults. Avian Dis. 52:444-450.
Lungu, B., S. C. Ricke, and M.G. Johnson. 2008. Growth, survival, proliferation and pathogenesis of Listeria monocytogenes under low oxygen or anaerobic conditions: A review. Anaerobe XX: (Manuscript ANAE 07-59R1, accepted Sept 1, 2008).
Lungu, B., S. C. Ricke and M.G. Johnson. 2008. Resistance of a nutrient deprived Listeria monocytogenes 10403S and a delta sig B mutant to chemical stresses in the presence or absence of oxygen. J. Food Sci. 73(No. 7):pp M 339 - M 345.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: The starvation survival response (SSR) of L. monocytogenes wild type strain 10403 and a sig B mutant were characterized under complete exogenous nutrient deprivation. The effects on cell viability and SSR of adding a protein synthesis inhibitor, chloramphenicol (Chlo) and of inhibitors of substrate - level phosphorylation (SLP) and oxidative level phosphorylation (OLP) on the two strains over 28 days was measured. Survival of buth the wt and mutant were affected by addition of Chlo during the first 4 h of starvation but cells were able to mount a SSR if Chlo were added at 6 h but the mutant was affected more. Neither strain was able to elicit a SSR when exposed to the SLP inhibitors 0.1 M sodium fluoride (SF) or 0.01 M sodium arsenite (SAs), suggesting that reaction downstream from the enolase and pyruvate dehydrogenase enzyme reactions are important in retaining cell viability, possibly through the production of energy needed for cell maintenance and protein synthesis during exogenous nutrient starvation. Conversely, both strains were able to mount a SSR when exposed to two OLP inhibitors, 0.1 M sodium arsenate (SA) or 0.02 M dinitriophenol (DNP). Two possibilities suggested by the latter results are that this organism in using oxygen as a terminal proton and electron acceptor: 1) may not use the classic cytochrome metabolic chain of intermediate enzyme steps but instead may us a direct NADH oxidase system as is reported for a related Gram positive, Enterococcus faecalis; or 2) is not sensitive to these two inhibitors at the levels used. In related work 21 cold sensitive mutants were constructed from above strain 10403 using the plasmid pLTV3 which contains the transposon Tn917. These 21 strains were grouped into 2 categories based on the reduction in growth rates compared to the wild-type strain at 4 degree C: slightly reduced (10-15% reduction) and significantly reduced (20-30% reduction). When examined for survivability at -20 degree C and -80 degree C, both groups showed equal survival at sub-freezing temperatures compared to the wild-type. These findings suggest that bacterial genes involved in low temperature growth of L. monocytogenes may not be important in aiding the adaptation and survival of L. monocytogenes at sub-freezing temperatures. PARTICIPANTS: Major cooperators in this project were Dr. Gerry Huff of the USDA-ARS Fayetteville Poultry Production and Product Safety Unit along with her M.S. student in Poultry Science, Mr. Vik Dutta who graduated in Dec 2007 and in Jan 2008 moved to North Carolina St U to begin his Ph.D. degree. Two graduate students of MGJ completed their M.S. (Mr. Josh Saldivar, who plans to go on to work for the Ph.D. degree with Dr Ricke) and Ph.D. (Dr. Bwalya Lungu who graduated in Dec 2007 and effective Jan 2008 moved to the U of Georgia to work with Dr. Joe Frank in a post doc position) degrees in Food Science Dept at UAF. Collaboration with Drs Steve Ricke and Arun Muthiyan were fruitful in employing some mutant techniques to look for cold sensitive mutants. TARGET AUDIENCES: Researchers interested in understanding some of the mechanisms that this pathogen can employ to resist loss of nutrients and cold temperatures. PROJECT MODIFICATIONS: The use of plasmids to modify the cold tolerance of Listeria monocytogenes was employed using standard procedures.

Impacts
Above studies indicate that surviving portion of a L. monocytogenes cell population can cope with loss of outside nutrients by living on waste products sloughed off by that portion of the cell population that dies. This mechanism would explain how such surviving cells remain quite viable for long periods of time, up to 1 year or more, without receiving any exogenously added carbohydrate or nitrogen sources.

Publications

Lungu, B. R. Story, S.C. Ricke and M.G. Johnson. 2007. The role of protein synthesis in the starvation survival responses of L. monocytogenes 10403S and sig B mutant. Abstract Amer. Soc. for Microbiology Annual General Mtg. Toronto, Ontario, CANADA, May 21-25, 2007.
Lungu, Bwalya. 2007. Ph.D. dissertation . Physiological and molecular characterization of the response of Listeria monocytogenes 10403S and Sig B mutant strain following exposure to various external stresses. U of Arkansas. Fayetteville, AR .
Saldivar, J.C., B. Lungu, V.I. Chalova, S.C. Ricke, and M.G. Johnson. 2007. Isolation and characterization of L. monocytogenes 10403S mutant strains showing impaired growth at 4 C compared to wild type strain 10403S. Abstract Amer. Soc. for Microbiology Annual General Mtg. Toronto, Ontario, CANADA. May 21-25, 2007.
Dutta, V., G.R. Huff, R.J. Sayler, W.E. Huff, N.C. Rath, M.G. Johnson, and R. Nannapaneni. 2007. The effects of stress and concurrent E coli infection on the isolation of L. monocytogenes from synovial tissue of turkey poults. Abstract Amer. Soc. for Microbiology Annual General Mtg. Toronto, Ontario, CANADA. May 21-25, 2007.