Annual/Termination Reports:

[09/10/2007] [08/11/2008] [09/30/2009] [09/02/2010] [09/13/2011]

Report Information

Participants

Barefoot, Susan, Clemson University; Brashears, Mindy, Texas Tech; Chen, Haiqiang, University of Delaware; Dawson, Paul, Clemson University; Goodridge, Larry, Colorado State University; Harris, Linda, UC Davis; Hung, Yen-Con, University of Georgia; Janes, Marlene, LSU; Karunasena, Enusha, Texas Tech; Kotara, Lane, Texas Tech; LeJeune, Jeff, Ohio State University; Marshall, Doug, Mississippi State University; Phister, Trevor, NCSU; Price, Stuart, Auburn University; Ryser, Elliott, Michigan State University; Siebert, Karl, Cornell University; Sofos, John, Colorado State University; Wang, Hua H., Ohio State University; Williams, Rob, Virginia Tech; Worobo, Randy, Cornell University

Brief Summary of Minutes

Accomplishments

Price, Toro, McKee and Hoerr (Auburn U., AB) explored whether Salmonella-targeted bacteriophages treatment can be integrated with competitive inhibition and vaccination to further reduce colonization of chickens. Tap water caused major reductions in titers of three of five bacteriophage cocktails after 48 h exposure; addition of powdered milk to tap water prevented reductions. Four of five cocktail bacteriophages survived transit through chicks; however, findings indicate need for more work on treatment timing and experimental endpoint. In an experimental treatment/challenge model, the combination of bacteriophage treatment plus competitive exclusion, and the bacteriophage treatment alone, significantly reduced cecal counts of Salmonella compared to untreated controls. Quantitative PCR methods for enumerating Salmonella in cecal samples showed good correlation with traditional colony-counting enumeration methods.<br /> <br /> Oyarzabal (Auburn U., AB) directly enumerated Campylobacter species from poultry feces on mCCDA, Campy-Cefex (mCC) agar, Campy-Line (CL) and Campy BAP (CB) agar plates. Both mCCDA and mCC are suitable plates for the isolation of Campylobacter from fecal or cecal samples from broiler. CL is too selective and yields a statistically lower number of Campylobacter spp. The 96-microwell plate dilution method with mCC appears to be similar to the counts determine by serial dilution and spread plating. However, these results require a further validation with a larger number of samples. Isolates from different plate media have similar PFGE patterns, suggesting that any differences found in the PFGE profiles may be due to the colonization of the flock with multiple isolates and is not a consequence of the antimicrobial composition of the media. <br /> <br /> Johnson (University of Arkansas, AR) addressed starvation survival response of L. monocytogenes by detailing viable counts in absence of added carbon and nitrogen sources. <br /> <br /> Sofos, Belk, and Scanga (Colorado State University, CO) assessed effectiveness of single and multiple preharvest intervention strategies on prevalence of Escherichia coli O157 on/in cattle before transport to harvest. Treatments included Control (CT; No treatment), Bovamine (Bov; a Lactobacillus acidophilus NPC-747 dietary product), NEOMIX (Neo; feeding of neomycin sulfate), E. coli O157:H7 bacterin vaccine (Vac), and all combinations of single treatments. Preharvest mitigation strategies used singly or in combination may be effective in reducing prevalence of E. coli O157 in market-ready feedlot cattle. In a second study they evaluated the behavior of Escherichia coli O157:H7 on beef during aerobic storage, following storage in vacuum packages. The practice of storing fresh beef in vacuum packages for subsequent storage in aerobic retail packing does not promote growth of E. coli O157:H7. The residual antimicrobial effect of hot water followed by lactic acid on both E. coli O157:H7 and natural flora was confirmed. Combined use of temperature control and decontamination with hot water and lactic acid may increase the shelf-life of fresh beef. <br /> <br /> Sofos et al. characterized growth rates and heat and acid death rates for 25 Listeria monocytogenes strains of various serotypes and sources, including clinical and food isolates associated with outbreaks. Extensive variation was observed among growth and stress resistance characteristics of L. monocytogenes strains. Serotype appeared to play a significant role only with respect to the heat resistance of the organism, with 4b isolates as a group exhibiting lower thermal resistance than isolates representing all other serotypes combined. Outbreak-related isolates of serotype 4b demonstrated higher acid resistance compared to the rest of the strains belonging to this serotype. Selecting Scott A as the sole strain to be used in low temperature or acid challenge studies, due to its clinical origin or the strong epidemiologic association of serotype 4b with human listeriosis, may not assure the assessment of the worst case scenario from a food safety perspective.<br /> <br /> The antilisterial effect of post-processing antimicrobial treatments on commercially manufactured frankfurters formulated with and without 1.5% potassium lactate-0.05% sodium diacetate was evaluated by Sofos et al. Results indicated that dipping frankfurters in post-processing antimicrobial treatments including Nisaplin resulted in 2.4 to >3.8 log CFU/cm2 reductions of initial L. monocytogenes populations. Furthermore, by applying post-processing antimicrobial dipping treatments to frankfurters formulated with potassium lactate-sodium diacetate, inhibitory and listericidal effects were obtained during storage at 10°C. This approach may be refined and validated for commercial application.<br /> <br /> Sofos et al evaluated post-processing chemical dipping solutions for their antilisterial effects on commercial smoked sausage formulated with or without 1.5% potassium lactate plus 0.05% sodium diacetate, and contaminated (approximately 3-4 log cfu/cm2) with 10-strain composite Listeria monocytogenes inocula prepared under various conditions. Dipping of sausage formulated with potassium lactate-sodium diacetate in Nisaplin followed by AA or LA would satisfy Alternative 1 of the interim final rule, due to substantial reductions of initial contamination levels and subsequent inhibition of growth during storage. Processors, however, need to develop and validate their own formulations to fit their product specifications and expectations.<br /> <br /> Sofos et al modeled the effect of drying temperature (52, 57 and 63°C) and pre-drying treatments on inactivation of Listeria monocytogenes on beef jerky. Results of this study confirmed that the acidified pre-drying treatment increased pathogen inactivation during drying, regardless of drying temperature and the models developed appeared to be a promising means of predicting the responses of this bacterium during dehydration of beef jerky. The use of the mathematical models to predict the drying time or survival of L. monocytogenes can be useful in beef jerky processing. <br /> <br /> Janes (Louisiana State University, LA) developed a direct colony immunoblot (DCI) assay for enumeration of Vibrio vulnificus. Vibrio alginolyticus (Va), V. cholerae (Vc), V. parahaemolyticus (Vp), V. damsela (Vd), V. mimicus (Vm), V. fluvialis (Vf) and several Vv strains, were plated onto Vv agar (VVA), Thiosulfate Citrate Bile Salts sucrose (TCBS) agar, and modified Cellobiose Polymyxin Colistin (mCPC) agar and incubated. Colonies were transferred to polyvinylidene fluoride membranes and treated with anti-H Vv antibodies. Membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG and color was developed. Our results showed that the DCI from the inoculated VVA plates had less non-specific color development compared to TCBS and m-CPC agar plates for Va and Vm. Vc, Vp. Vf and Vd were not detected by the DCI. At 9 log CFU/ml all Vv strains were detected by the DCI whereas Vp was not detected. In a Vv and Vp mixed culture containing 7.9 log CFU/ml of each bacteria the DCI only detected Vv. Total time duration for enumeration of Vv by the DCI was 31/2 h. <br /> <br /> Janes conducted a second study to determine the concentration of Cetylpyridinium Chloride (CPC) that effectively reduces Listeria monocytogenes Lm on the surface of white Gulf shrimp. Treatment of shrimp with 1.0% CPC (no water rinse) reduced L. monocytogenes counts by >2 log CFU/g on day 0. The largest reduction (3 logs) occurred for shell-on cook shrimp,. The shelled raw, shelled cooked and peeled raw shrimps treated with 1.0% CPC and a water rinse on day 0 had reductions of 1.05, 2.19 and 0.8 log CFU/g, respectively. Shell-on raw shrimp treated with 0.05, 0.4 and 1.0 % CPC (no water rinse) had reductions on day 3 of 1.26, 1.89, and 2.06 log CFU/g, respectively. By day 9 the shell-on cooked shrimp at 1.0% CPC with or without a water rinse had reduced Lm counts by 3 log CFU/g.<br /> <br /> Ryser (Michigan State University, MI) is addressing : risk and communication regarding Listeria monocytogenes contamination of deli meats and slicers. Accomplishments include: (1) acquisition of four model 2612 Hobart slicers (including two types of blades), as well as two Berkel slicers that was redesigned for enhanced cleanability; (2) set-up of the three Hobart slicers and one Berkel slicer at a local delicatessen for one year of use and monthly sampling; (3) identification of the product contact areas on the slicers for sampling; (4) optimization of the blade inoculation method using surface-inoculated ham; and (5) collection of initial data on the potential and extent for sequential transfer of L. monocytogenes from the slicer blade to ham and other areas of the slicer during slicing of the product at two different temperatures. They are now focusing on transfer of Listeria from the artificially contaminated G+B German blade to ham with different moisture and fat contents stored at 4 and 22Ú C; work with the remaining two blades for the Hobart slicer and the Berkel slicer will follow. 56 environmental samples per month from the retail deli will be examined for Listeria spp. They will continue to work on the development of mathematical models and risk communication. For the mathematical model, a compartmental model of L. monocytogenes cross-contamination among deli products, deli contact surfaces (e.g., different parts of a slicer), employees hands covered (gloved and ungloved) , and the environment with which hands may come into contact, defined as the immediate environment, will be developed. This model will be used to consider which parameters have the most profound impact upon the prevalence and level of L. monocytogenes contamination occurring during delicatessen slicing.<br /> <br /> Ryser is also investigating a risk-based approach to determine best consume by dates to control exposure of Listeria monocytogenes in deli meats. Eight L. monocytogenes strains of different pulsed-field gel electrophoresis (PFGE) types from RTE meat products are being used. Growth of L. monocytogenes is being assessed in five different brands each of uncured turkey, cured turkey, ham, and roast beef. These products were selected based on differences in pH, moisture, aw, salt, fat content, as well as presence/absence of nitrite, Listeria growth inhibitors, lactate and diacetate. Whole chubs of cured turkey, uncured turkey, ham and roast beef are purchased from retail delis in major supermarkets on the day of delivery. One chub is being inoculated on the day of delivery. The remaining chubs are held at 4Ú C for inoculation at the midpoint and at the last allowable date for sale. All experiments are being conducted in triplicate using three different lot numbers of the same product from the same manufacturer.<br /> <br /> Marshall (Mississippi State University, MS) investigated a rapid chill intervention process to control Vibrio vulnificus in raw Gulf Coast oysters. Oysters were harvested during the warmer months (April-September) that were expected to have high numbers of naturally occurring V. vulnificus were subjected to a rapid chill process (10°C) for two weeks using a recirculating water tank containing 12, 16, and 20 ppt salt seawater. Control oysters were placed in a similar tank at 23°C. After two weeks, aerobic plate counts, V. vulnificus counts, and MIDI GC-FAME bacterial profiles were determined. In addition, oysters were removed from the tanks and stored at 4°C for two weeks to simulate retail shelf life. After this storage period, bacterial profiles were again performed. Results demonstrated that the chill process reduced V. vulnificus counts by 2-3 logs but counts were not below FDA target levels of less than 30 MPN/g. Salinity had little effect on counts. Bacterial profiles predominantly shifted from vibrios to psychrotrophic spoilage bacteria such as Pseudomonas and related genera. The oral hygiene antimicrobial cetylpyridinium chloride was tested for its activity against Salmonella Typhimurium attached to ready-to-eat shrimp. Levels up to 1% CPC reduced Salmonella counts by less than 1 log.<br /> <br /> LeJeune (Ohio State University, OH) reported the need to validate probiotic preparations individually and identified birds as a potential vector for E. coli O257 dissemination among dairy farms. E. coli O157 prevalence in cattle was benchmarked against cattle in a country where E. coli O157 disease in humans is rare. Prevalence rate in cattle may impact human disease incidence. The sensitivity of IMS methods for isolation of E. coli O157 from bovine fecal samples containing less than 1000 CFU/ml was determined to be poor (<30%). Removal of oxytetracyline from conventional swine production has miminal impact on the prevalence of oxytetracycline resistant fecal E. coli in pigs.<br /> <br /> Dawson and Jiang (CLEMSON UNIVERSITY, SC) are assessing the prevalence of vancomycin resistant enterococci in rendered animal byproducts and during composting. About 200 suspected enterococci isolates were first confirmed as enterococci through biochemical tests and real-time PCR. Enterococci were examined for resistance to vancomycin and for minimum inhibitory concentration (MIC). 76 % of the isolates belong to enterococci genus based on PYR results and approximately 92% based on the other tests. Sixteen enterococci were resistant to vancomycin (VRE). Seventeen showed additional resistance to at least five other antibiotics. Rendered animal by-products contain multiple drug resistant VRE. The fate of VRE during active composting was assessed and antibiotic resistance of VRE isolates was further characterized. Duplicate dairy compost heaps were constructed on an outdoor, fenced site. Enterococci populations inside the composting heaps decreased rapidly from ca.7.0 to 5.0 log cfu/g within 3 days of active composting, followed by a relative slow reduction of cell numbers during a 120-day composting. Except at bottom of the compost heap, neither enterococci nor VRE could be detected inside composting heaps at 60 days. Vancomycin-resistant enterococci accounted for ca. 2-18% of total enterococci populations in initial dairy compost, and were inactivated inside the compost heaps at similar rates as enterococci. However, on the surface, like enterococci, VRE survived very well with ca. 2 ~ 3.5 logs reduction in cell populations at the end of experiment. Isolates of those VRE were further analyzed for the resistance to vancomycin and other antibiotics. Most VRE are resistant to multiple antibiotics, and over 93% of VRE had minimal inhibitory concentration (MIC) e128 µg vancomycin/ml. Our results revealed that a large number of vancomycin-resistant enterococci cells survived active composting on the surface or inside the heaps when temperature was low, and the existence of these VRE could serve as the potential source for disseminating vancomycin-resistance among microbes on farm.<br /> <br /> Dawson and Jiang studied antimicrobial activity of lysozyme absorbed on immunonanoparticles against L. monocytogenes Scott A. Polystyrene nanoparticles with active carboxyl groups were conjugated with anti-L. monocytogenes through covalent bounding. Immunonanoparticles were then coated with lysozyme by direct adsorption. The antimicrobial activity of lysozyme adsorbed on immunonanoparticles was compared to that of free lysozyme in phosphate buffered saline. Amount of anti-L. monocytogenes and lysozyme and adsorption times were optimized for most efficient inhibition. Nanoparticles were conjugated with 0.04 µg anti-L. monocytogenes per ml, and at that concentration, immunonanoparticles demonstrated enhanced antimicrobial activities of lysozyme. Lysozyme (35 µg/ml) adsorbed on immunonanoparticles reduced the L. monocytogenes Scott A population from 5.2 log CFU/ml to below the detection limit (<1 log CFU/ml) in 3 h. However, when free lysozyme (500 µg/ml) was used, ca. 2.2 log CFU/ml of the L. monocytogenes Scott A remained culturable after 5 h treatment. Our study revealed that the use of immunonanoparticles coated with lysozyme is a more efficient method than direct addition of lysozyme in inhibiting L. monocytogenes.<br /> <br /> Dawson and Jiang assessed the effects of thickness on in-package pasteurization against Listeria monocytogenes on ready-to-eat meats. The surface heating rate (³) and final surface temperature (±) during in-package pasteurization were determined for different thickness levels of two types of bologna having different (13% and 18%) fat content. Three different thickness levels (4, 12, and 20 mm) corresponding to 1, 3, and 5 slices of bologna were each vacuum-packaged separately in clear polymer pouches after placing thermocouples on the surface. Refrigerated samples were immersed into a water bath set to one of four pre-determined temperatures (60, 70, 80, and 90° C) and time and temperature data were recorded for 10 min. Surface- heating rate was fastest in the thinnest (4mm) and slowest in the thickest (20mm) samples for all four pasteurization temperatures. Surface- heating rate was slower in bologna with higher fat content compared to lower fat bologna. Final surface temperature attained after 3 min was lower with increased thickness levels for all pasteurization temperatures. Thus meat sample thickness and fat content significantly affect surface heating rate and final surface temperature during in-package pasteurization of bologna.<br /> <br /> Dawson and Jiang investigated efficacy of in-package pasteurization combined with pre-surface application of nisin and/or lysozyme to reduce and prevent the growth of Listeria monocytogenes on bologna. Sterile bologna slices were treated with 0.1 ml (500 AU) of nisin (2 mg/ml = 5000 AU/ml), 0.1 ml (8 AU) of lysozyme (10 mg/ml = 80 AU/ml), or 0.1 ml of nisin + lysozyme (250 AU of nisin + 4 AU of lysozyme) solutions. These samples were inoculated with 0.1 ml of a 9 log10 cfu/ml culture then packaged and subjected to 65 C for 32 seconds (after a 60 s come-up time). This heat treatment was calculated to attain a 3-log reduction. Bologna was sampled immediately and weekly through 12 weeks of refrigerated storage for L. monocytogenes population. The combination of heat with nisin + lysozyme or nisin alone treatments significantly reduced the time required to reduce L. monocytogenes populations compared to heat combined with no antimicrobial or lysozyme. The rate of log reduction at each of the three temperatures did not fit a linear model thus a Weibull model was used to fit data at 65 and 62.5OC while a log-logistic model was used to best describe the log reduction vs. time relationship.<br /> <br /> Grants Received:<br /> <br /> Control of Salmonella in Infected Chickens by Combining Application of Bacteriophages, Competitive Exclusion, and Maternal Immunity. USDA NRICGP. PI: Price; Co-I: Toro, McKee, Hoerr. 9/15/06  9/14/09. $298,271.<br /> <br /> Novel Approaches for Reducing Shedding of Salmonella from Chickens. Animal Health and Disease Research Program, College of Veterinary Medicine, Auburn University. PI: Price; Co-I: Toro. 10/1/05-9/30/07. $36,319.<br /> <br /> Monoclonal antibody probes for C. jejuni and differential enumeration of Campy from poultry Nannapaneni, R. et al:. CSREES/USDA/Food Safety Consortium. M. G. Johnson.<br /> <br /> Persistence, and virulence of starved L. monocytogenes. CSREES/USDA/Food Safety Consortium Nannapaneni, R. et al:. M. G. Johnson.<br /> <br /> Ciprofloxacin resistance and virulence of Campy from poultry. Nannapaneni, R. et al: CSREES/USDA/Food Safety Consortium. M. G. Johnson.<br /> <br /> USDA/CSREES/NIFSI. AGREEMENT 2004-51110-02160 9/15/2004-9/14/2007. John N. Sofos, PI. .<br /> <br /> USDA/CSREES/NIFSI.AGREEMENT 2005-51110-03278; 9/15/2005-9/14/2009. John N. Sofos, P.I.<br /> <br /> USDA/CSREES/NIFSI. AGREEMENT 2006; 8/15/2006-8/14/2009. John N. Sofos, PI.<br /> <br /> USDA/CSREES/NIFSI. AGREEMENT 2004-51110-02160 9/15/2004-9/14/2007. John N. Sofos, PI. .<br /> <br /> USDA/CSREES/NIFSI.AGREEMENT 2005-51110-03278; 9/15/2005-9/14/2009. John N. Sofos, P.I.<br /> <br /> USDA/CSREES/NIFSI. AGREEMENT 2006; 8/15/2006-8/14/2009. John N. Sofos, PI.<br /> <br /> Microbial Drug Resistance in Vibrio spp. from Oysters. USDA Aquaculture Special Grant, September 2006/October 2007, Funded for $24,000. Lead PI  Beilei Ge, Co  PIs Marlene Janes, Witoon Prinyawiwatkul.<br /> <br /> Determining the Total Aerobic counts, coliform and E. coli counts, and Yeast and Mold counts for mash samples flooded by Hurricane Rita Cooperative Research Agreement, McIlhenny, October 2005, Funded for $10,000. Lead PI  Marlene Janes.<br /> <br /> Control of Listeria monocytogenes in crawfish and shrimp processing facilities using copper, copper-based alloys or coatings containing copper ions,USDA Aquaculture Special Grants, September 2005/September 2006, Funded for $25,460. Lead PI Marlene Janes, Co  PIs Jon Bell.<br /> <br /> Detection of Vibrio vulnificus by direct colony immunoblot,Louisiana Sea Grant College Program, , June 2005/July 2007, Funded for $94,537. Lead PI  Marlene Janes, Co  PIs Janet Simonson and Jon Bell.<br /> <br /> Ecology and control of pathogenic strains of Vibrio vulnificus and Vibrio parahaemolyticus in U.S. Gulf Coast oysters USDA CSREES NRI CGP, October 2004/September 2007, Funded for $415,504. Lead PI  Lee-Ann Jaykus, Co  PIs Andy De Paola, Marlene Janes, Jon Bell, and John Supan.<br /> <br /> A risk-based approach to determine best consume by dates to control exposure to L. monocytogenes in deli meats. CSREES/USDA. Todd, E.C.D., E.T. Ryser, L.A. Jaykus. $599,999.<br /> <br /> . L. monocytogenes contamination of deli meat slicers  risk and communication. NAFSS/USDA Todd, E.C.D., and E.T. Ryser. $225,000.<br /> <br /> Screening for potential non-pathogenic surrogates for Salmonella enteritidis PT-30 during moist-air convection heating of almonds. Almond Board of California. Marks, B.P., A. Orta-Ramirez, E.T. Ryser, K.D. Dolan. $32,478.<br /> <br /> Mechanisms of E. coli O157:H7 attachment to and transfer between meat and equipment surfaces. National Cattlemens Beef Association. Ryser, E.T., A. Orta-Ramirez, B.P. Marks, and A.M. Booren. $105,999.<br /> <br /> Determining the likelihood that Salmonella develops heat resistance during processing of commercial whole muscle products. American Meat Institute. Marks, B.P., A. Orta-Ramirez, A.M. Booren, and E. T. Ryser. $44,290.<br /> <br /> Efficacy of two commercial sanitizers against L. monocytogenes E. coli O157:H7, and Salmonella on conveyor belts during normal operation. Ecolab, Inc. Ryser, E.T., and B.P. Marks. $34,500.<br /> <br /> Efficacy of two commercial sanitizers against L. monocytogenes E. coli O157:H7, and Salmonella on conveyor belts during normal operation. Midwest Advanced Food manufacturing Allience. Ryser, E.T., and B.P. Marks. $50,000.<br /> <br /> Risk and protection factors for foodborne illnesses in perinatal and infant populations. CSREES #2006-51110-03663. Medeiros, L(PI), LeJeune, Jeffery T; Sofos, J Kendall, P. $1,500,000<br /> <br /> Phage encoded transfer of antibiotic resistance genes in Salmonella. Nat Cattlemen's Beef Assn. <br /> LeJeune, Jeffery T. $59,000<br /> <br /> Biophysical and ecological processes impacting the growth and survival of E. coli O157 on and in vegetables. CSREES. Lejeune, Jeffery T; Lee, K, Miller, S. $537,816<br /> <br /> The role of European starlings in the epidemiology of E. coli O157 of dairy cattle. CSREES. Lejeune, Jeffery T. $1,146,635 <br /> <br /> Genetic profiles of bovine-origin salmonellae. Nat Cattlemen's Beef Assn. Lejeune, Jeffery T. $47,163<br /> <br /> Incidence, significance, and control of Listeria monocytogenes in the home environment. USDA/ARS. Medeiros, L (PI) Lejeune, Jeffery T; Scharf, R Sofos, J and Kendall, P. $540,000 <br /> <br /> Communicating and advancing prudent antibiotic use among dairy farmers. USDA/FSIS. Lejeune, Jeffery T; Doohan, D, Hooker, N and Tucker, M. $28,862<br /> <br /> Evaluation of a bacteriophage cocktail to reduce Escherichia coli 0157:H7 shedding in beef cattle. Univ of Wyoming. NCBA Subcontract. Lejeune, Jeffery T. $37,073 <br /> <br /> Development of Pre-harvest Nutritional Management Strategies to Reduce E. coli O157:H7 and Salmonella spp. Gastrointestinal Survival and Shedding. Nat Cattlemen's Beef Assn. Henery Zerby (PI), LeJeune et al. $84,346.00 <br /> <br /> Phage encoded dissemination of Shiga toxin genes among bovine E. coli. Nat Cattlemen's Beef Assn. Lejeune, Jeffery T. $89,787.00<br />

Publications

Impact Statements

1. Bacteriophages can be delivered to chicks via drinking water, using powdered milk to prevent loss of titer. Bacteriophage treatment alone is as effective as when combined with competitive exclusion; further work with optimizing intervention strategies is needed. Quantitative PCR may displace traditional bacteriological methods for enumerating Salmonella in cecal samples.
2. E. coli O157:H7 may be transmitted through consumption of undercooked ground beef. Contamination of RTE meat and poultry products with L. monocytogenes after exposure to a lethal treatment (heat) during processing remains of concern. This project evaluates potential human health risks as affected by potential for pathogen growth, and develops antimicrobial alternatives for control of the pathogen at the production, distribution and consumption level of RTE meat and poultry products.
3. The newly developed direct colony immunoblot for detection of V. vulnificus could be used as a rapid enumeration method by regulatory agencies or the seafood industry. The CPC washing solution showed potential for reducing L. monocytogenes on the surfaces of shrimp. Results indicated that the effectiveness of oyster lysozyme was enhanced when combined with nisin incorporated in the calcium alginate coating, which could be used to preserve ready-to-eat smoked salmon at refrigerated temperatures.
4. Lab-based studies, modeling, and communication/education is used to fill specific data gaps regarding the likelihood of L. monocytogenes contamination occurring via the deli slicing process. This knowledge will be used to develop risk communication and education strategies. L. monocytogenes is seven times more likely to be found in deli-sliced as pre-packaged luncheon meats. Adoption of best consumed by times for deli meats will help reduce human exposure to high levels of this organism.
5. Harvesters and processors of Gulf Coast oysters destined for the raw consumption market are under pressure to develop interventions to control pathogenic vibrios. Although the rapid chill process was effective in significantly reducing V. vulnificus counts, the residual counts remain above satisfactory levels (>30/g). Additional interventions coupled with rapid chilling may be useful. CPC did not appear to be an effective control substance to inactivate Salmonella on ready-to-eat shrimp.
6. Determination of both antibiotic resistant bacteria and heat resistant bacteria in animal co-products will help the rendering industry to develop processing parameters to control these in rendered animal meal and thus reduce their prevalence in the food animal production cycle.
7. Quantitating the synergism between pasteurization and antimicrobials in ready-to-eat meat will assist the meat industry in reducing the incidence of L. monocytogenes in these products. Also, determination of the heat-inactivation models will allow a clearer understanding the patterns of how these bacteria are destroyed by heat will result in better treatments to ensure consumer safety.

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Report Information

Participants

Barefoot, Susan sbrft@clemson.edu - Clemson University; Boyer,Renee boyer@vt.edu - Virginia Tech; Chen, Haiqiang haiqiang@udel.edu - University of Delaware; Dickson, James jdickson@iastate.edu - Iowa State University; Goodridge, Lawrence Lawrence.Goodridge@colostate.edu - Colorado State University; Harris, Linda ljharris@ucdavis.edu - University of California-Davis; Hung Yen-Con yhung@uga.edu - University of Georgia; Janes, Marlene mjanes@agctr.lsu.edu - Louisiana State University; Lejeune, Jeff lejeune.3@osu.edu - Ohio State University; Rajashekara, Gireesh rajashekara.2@osu.edu - Ohio State University; Rao, Ram rrao@csrees.usda.gov - USDA CSREES; Ryser, Elliot ryser@msu.edu - Michigan State University; Sofos, John john.sofos@colostate.edu - Colorado State University; Wang, Hua wang.707@osu.edu- Ohio State University.

Brief Summary of Minutes

Accomplishments

Objective 1: Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments including meat, poultry, seafood, fruits and vegetables and nutmeats. <br /> <br /> Researchers at Iowa State University are investigating thermal tolerance of acid adapted Escherichia coli O157:H7 and Salmonella in ground beef during refrigerated (4°C) and frozen storage (-20°C). Both the pathogens were adapted to acidic conditions by growing in Tryptic Soy Broth with 1 % glucose (TSB+ 1%G). Five-strain cocktail of both bacteria were grown separately in TSB and TSB+1%G for 24h at 37°C to provide cells with or without acid adaptation. Irradiated ground beef was inoculated with a five strain cocktail of either acid adapted or non-adapted E. coli O157:H7 and Salmonella. Inoculated ground beef was then subjected to heat treatment at 62 and 65°C on day 1, 7, 14, 21, and 28 (4°C) and on day 1, 30, 60, 90, and 120 (-20°C). Decimal reduction time (D-values) of the pathogens was determined as an indicator of thermal tolerance. Significant differences (P<0.05) were observed in the D62-values of acid adapted and non-adapted E. coli O157:H7 on day 21 and 28 at 4 °C and on day 120 at -20°C. For Salmonella significant difference (P<0.05) in the D62-values was observed on day 21 and 28 at 4°C and on day 30, 60, and 90 at -20°C. No statistical differences (P>0.05) were observed in the D65-values of the acid adapted and non-adapted strains on E. coli O157:H7 and Salmonella throughout the storage period, irrespective of the storage temperature. <br /> <br /> In 2001, Salmonella Enteritidis Phage Type (PT) 30 was isolated from drag swabs of 17 61-hectare almond orchards on three farms linked to an outbreak of salmonellosis associated with consumption of raw almonds. At the University of California researchers evaluated the long-term persistence of Salmonella Enteritidis PT 30 in one of the almond orchards associated with the outbreak. All 53 Salmonella isolates over 5 years were identified as Salmonella Enteritidis PT 30. These data demonstrate the potential for long-term environmental persistence of Salmonella in almond orchards. Data on prevalence and populations of pathogens in individual foods are critical to the development of product-specific quantitative microbial risk assessments. Raw almonds arriving at almond processors were sampled over 5 years to determine the overall prevalence and levels of Salmonella in raw almonds and to characterize the Salmonella isolates obtained. An isolation frequency for Salmonella of 0.87% ± 0.2 % (81 of 9,274 100-g samples tested) was determined for raw almonds sampled from throughout California over the 5-year period. Salmonella was not isolated upon retesting in 59 of 65 positive samples. When detected, levels were very low: 1.2 to 2.9 MPN/100 g. Of 81 total isolates, 35 different serotypes of Salmonella were represented. <br /> <br /> During the last 10 months, researchers at University of Delaware have studied the physiology and genetics of S. e. Kentucky, and determined that, with respect to most characteristics, this serovar is indistinguishable from other serovars isolated from poultry sources; however, one physiological difference between most of the S. e. Kentucky serovars and the other serovars studied was found. Salmonella enterica Kentucky is more sensitive to low pH, and it does not adapt well to low pH following pre-exposure to mild acid. PCR-based profiling of 29 virulence genes indicated that the S. e. Kentucky isolates possessed similar profiles as S. e. Enteritidis and S. e. Typhimurium, but genes usually associated with a virulence plasmid such as spvB, spvC, and pefA (Nolan et al., 1995; Swamy et al., 1996; Bakshi et al., 2003) were absent. This observation together with the low acid resistance of most S. e. Kentucky isolates could explain the virtual absence of these isolates from human sources. The response of the serovar Kentucky isolates to heat (survival at 55oC), cold (growth at 4oC), desiccation and storage at 37oC, hydrogen peroxide, organic acids, ammonium, quarternary amines, sodium hypochlorite, egg white (24 h survival), and high salt was also explored, but no differences between serovar Kentucky isolates and other poultry isolates were found. <br /> <br /> Researchers at Alabama investigated if Salmonella-targeted bacteriophage treatment can be integrated with traditional pathogen reduction strategies to further reduce colonization of chickens. Five Salmonella-targeted bacteriophages were chosen for use in chickens from a phage library of 36. One of these bacteriophages showed exceptional in vitro killing; each of the 5 phages showed unique plaque-forming abilities on 7 Salmonella serovars. In vitro bacteriophage: Salmonella multiplicity-of-infections (MOI) of 1000:1 showed more rapid killing than an MOI of 10:1. Bacteriophage survived in deionized water and in tap water supplemented with skim milk (1:400), as well as in the feces of phage-treated chickens. Chickens orally challenged with Salmonella and orally treated with either bacteriophage alone or in combination with a commercial probiotic showed significantly fewer Salmonella in their ceca compared to challenged, untreated chickens. Comparison of cecal Salmonella numbers by standard culture versus quantitative PCR showed good correlation.<br /> <br /> Efforts to minimize foodborne illnesses through proper refrigeration of post harvest oysters may be compromised if Vibrio strains exhibit significant differences in survival rates at refrigeration temperatures. There is limited research currently available on whether there are growth and survival differences between various strains of V. vulnificus and V. parahaemolyticus. Researchers at Louisiana State University Ag Center determined if strain-to-strain differences exist in the growth and survival of V. vulnificus and V. parahaemolyticus at low temperatures. Ninety-nine ml of TSBN2 was added to specimen cups which were then prechilled to 5oC. The cups were inoculated with one ml of washed overnight cultures of the eight strains of V. vulnificus and eight strains of V. parahaemolyticus that had been grown at 37oC in TSBN2. This gave an initial count of about 106 log CFU/ml. The cups were then stored at 5, 8 or 10oC for 10 days. Bacterial counts were determined every other day by plating on TSAN2. At 5°C by day 10, V. vulificus strain 515-4C2 had the highest counts, with 1.97 log CFU/ml; strain 541(O) 49C had counts of 0.65 log CFU/ml while strain 33815 reached non-detectable levels at day 8. At 8°C by day 10 V. vulnificus Strain 515-4C2 had the highest counts, with 2.23 log CFU/ml; strain 29306 had counts of 1.15 log CFU/ml while strain 33815 reached non-detectable levels. At 10°C by day 10 V. vulnificus Strain 541(O) 49C had the highest counts, with 8.02 log CFU/ml; strain 0106-14 had counts of 7.00 log CFU/ml while strain 33815 reached non-detectable levels. V. vulnificus strains cultured at 37°C and then held at 15°C (cold temperature adaptation for four hours, before storage at 5 or 8°C for 9 days, exhibited better survival rates (but no growth) than cultures transferred directly from 37°C to 5 or 8°C. Cultures similarly held at 15°C before storage at 10°C resulted in better growth rates than cultures transferred directly to 10°C. The duration of either the growth or survival responses differed for various V. vulnificus strains. Some strains showed increased survival rates throughout the experimental storage times and temperatures investigated, while others exhibited significant increases in growth or survival for shorter time periods. <br /> <br /> Researchers at University of Delaware are looking at high pressure processing to control V. vulnificus in shucked oysters. Nine strains of V. vulnificus were tested for their sensitivities to high pressure. The most pressure-resistant strain of V. vulnificus, MLT 403, was selected and used in the subsequent experiments to represent a worst case scenario for evaluation of the processing parameters for inactivation of V. vulnificus in oysters. To evaluate the effect of temperature on pressure inactivation of V. vulnificus, oyster meats were inoculated with V. vulnificus MLT 403 and incubated at room temperature for 24 h. Oyster meats were then blended and treated at 150 MPa for 4 min, and 200 MPa for 1 min. Pressure treatments were carried out at -2, 1, 5, 10, 20, 30, 40, and 45°C. Cold temperatures (< 20°C) and slightly elevated temperatures (> 30°C) substantially increased pressure inactivation of V. vulnificus. For example, a 4-min treatment of 150 MPa at -2 and 40°C reduced the counts of V. vulnificus by 4.7 and 2.8 log, respectively, while at 20°C the same treatment only reduced counts by 0.5 log. Temperatures of -2 and 1°C were used to determine the effect of pressure level, temperature, and treatment time on the inactivation of V. vulnificus infected to live oysters through feeding. <br /> <br /> Listeria monocytogenes and Salmonella spp. are two of the most prevalent microbial pathogens associated with recalls of meats, including pork products. Because traditional cultural methods for detection of these pathogens in foods may take as long as one week, these methods represent a critical bottleneck in todays otherwise rapid and highly efficient food production and distribution networks. Fluorescence in situ hybridization (FISH) is a rapid molecular method for the detection of specific pathogens. In the FISH technique, fluorescently labeled oligomer probes are reacted with ribosomal RNA present in whole microbial cells, leading to the selective phylogenetic staining of target pathogens. As a whole cell fluorescent method, FISH can be combined with flow cytometry to enable the rapid detection and enumeration of specific cells in complex samples. Researchers at Iowa State University are evaluating the utility of combined FISH and flow cytometry for the rapid detection of Salmonella and Listeria monocytogenes in contaminated pork products, including raw, cubed pork and pork franks. Both pathogens were directly detectable at high levels of contamination (106 CFU/g) in cubed pork (Salmonella) and pork franks (Listeria). Unambiguous detection of both pathogens at low levels (102 CFU/g) was also possible after only 8 hours of non-selective pre-enrichment in either buffered peptone water or universal pre-enrichment broth. <br /> <br /> Researchers at the University of Arkansas study 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 were measured. Survival of both 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 4oC: slightly reduced (10-15% reduction) and significantly reduced (20-30% reduction). When examined for survivability at -20oC and -80oC, 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. <br /> <br /> Ultraviolet light (UV) may be an effective tool for control of Listeria monocytogenes in brines used to cool Ready-to-Eat (RTE) meat products. At Virginia Tech researchers are investigating the minimum dose of UV (peak: 253.7nm) required to inactivate L. monocytogenes in water and a 9% NaCl solution using uridine as a chemical actinometer. L. monocytogenes strains N1-227 (hot dog batter), N3-031 (turkey franks), and R2-499 (RTE meat) were mixed in equal proportions and suspended in water and 9% NaCl solution, each containing 10-4 M uridine. Fourteen ml of suspension was placed into a sterile quartz cell, and irradiated for 1, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, or 120 min using an Oriel photoreactor (model 66901) fitted with a filter to allow only UV light in the 253.7 +/- 10 nm range to pass to the sample. The sample was held at 8°C (+/-2°C) and continuously stirred during UV exposure. Inactivation was evaluated by serially diluting samples in 0.1% peptone, surface plating onto Modified Oxford Agar (MOX) and Trypticase Soy Agar with Yeast Extract (TSAYE), and by enrichment in Brain Heart Infusion Broth (BHI), followed by incubation at 35°C for 24 h. The absorbance of each sample was measured before and after irradiation, using a Shimadzu spectrophotometer (model UV-2101PC). L. monocytogenes irradiated in water decreased to below the detection limit (1 log CFU/ml) at UV doses greater than 23 mJ/cm2, but was detected via enrichment after exposure of up to 95 mJ/cm2. L. monocytogenes irradiated in 9% salt decreased to below the detection limit after exposure to UV in the 28 and 54 mJ/cm2 range, but was detected via enrichment after exposure to UV at doses greater than 54 mJ/cm2. <br /> <br /> Researchers at Michigan State University studied the prevalence of Listeria spp. in a commercial deli establishment. A total of 790 environmental samples including five each from five slicers (3 Hobart and 2 Bizerba) were collected at deli/restaurant during 14 monthly visits. One hundred-seventy samples (21.5%) tested positive for Listeria with 90 (11.4%), 50 (6.3%), and 30 (3.8%) yielding L. seeligeri, L. innocua and L. monocytogenes, respectively. Listeria spp. were most frequently recovered from the basement (floors, drains, walk-in coolers) (29%) followed by the kitchen (floors, drains, walk-in cooler) (21%) and sandwich line (12%). The contamination routes will be determined following PFGE analysis. Deli slicers used for slicing meats were more frequently contaminated with Listeria spp. than those used for slicing cheeses and produce. These results also indicate that significantly greater Listeria contamination occurred by mid-afternoon compared to shortly after start-up in the morning, confirming that sanitation every 4 hours is necessary for deli establishments. Upper management attended a training program for preventing L. monocytogenes in deli operations that was administered by us midway through the 14-month survey. Following this training program, a 40% decrease in the incidence of Listeria spp. was observed, however, the frequency of L. monocytogenes isolation increased five times. The reason for this change is unknown at this time. <br /> <br /> Objective 2: Develop and validate mathematical modeling to gain understanding of pathogen behavior in macro and micro-environments.<br /> <br /> Researchers at the University at Minnesota have been working on modeling the growth of Listeria monocytogenes in growth media containing deli meats ingredients to eventually provide the basis for a safety-based shelf life model. The specific aim of their research is to identify the best fitting primary growth model using the F-test and then to select the fastest growing strain(s) of L. monocytogenes based on growth kinetic parameters. They compared the performance of four primary mathematical models to study the growth kinetics of Listeria monocytogenes ribotypes grown at low temperature so as to identify the best predictive model. The parameters of the best fitting model were used to select the fastest growing strains with the shortest lag time and largest growth rate. Nineteen food and animal L. monocytogenes isolates with distinct ribotype were grown at 4, 8, and 12 °C in tryptic soy broth and slurries prepared from cooked uncured sliced turkey breasts (with or without potassium lactate and sodium diacetate, PL/SD) and cooked cured frankfurters (with or without PL/SD). Separate regressions were performed on semi-logarithm growth curves to fit log-linear (Monod) and non-linear (Gompertz, Baranyi-Roberts, and Logistic) equations and performance of each model was evaluated using an F-test. No significant differences were found in the performance of linear and non-linear models, but the Baranyi model had the best fit for most growth curves. The maximum growth rate (MGR) of Listeria strains increased with increase in temperature. Similarly MGR was found significantly greater when no antimicrobials were present in the initial formulation of turkey or frankfurter products. The variability in lag times and maximum growth rates in all media as determined by the Baranyi model was not consistent among strains. No single strain consistently had the fastest growth in all media, but strains DUP-1044A, DUP-1030A and DUP-1042B grew faster under most growth conditions than the rest of the ribotypes. The lack of association between serotype and fastest strain was also observed in the slurry media study. <br /> <br /> It is common practice in microbiology to use a spectrophotometer to detect and assess microbial growth in liquid cultures. The phenomenon involved is actually light scattering and a photometer senses this as a decrease in transmitted light. For low growth conditions this measurement is the difference between a large amount of light (no absorbance or scattering) and a slightly smaller amount of light (slight growth). Results obtained by subtracting one large number from another generally provide poor metrology because they have a relatively large variation compared to the result. Measuring actual scattered light with a turbidimeter, on the other hand, is more akin to seeing a flashlight beam on a dark night (a small signal against a low background). In principle light scattering should be more sensitive than photometry and therefore preferable for detecting low level growth. Researchers at Cornell University conducted experiments to test this hypothesis. Turbidimetry was more sensitive than photometry but this did not appear to be greater than one order of magnitude; turbidimetry also appeared to be more precise. <br /> <br /> Objective 3: Investigate factors leading to the emergence, persistence and elimination of antimicrobial resistance in food processing and animal production environments<br /> <br /> Identification of the major routes in antibiotic resistance gene dissemination is critical for establishing effective counter strategies to combat this problem. Researchers at The Ohio State University have revealed for the first time the prevalence of antibiotic resistant commensal bacteria in many ready-to-eat retail food products. Various antibiotic-encoding genes were detected by PCR, the size of the representative tetracycline resistant gene pool in cheese was quantified by real-time Taqman PCR, and resistance-encoding genes from food isolates were transmitted to human pathogens via natural gene transmission mechanisms led to increased resistance in these organisms. The finding of high magnitude of resistance bacteria in many popular food items strongly supports the notion that food chain might have played an important role in directly disseminating antibiotic resistance genes to the general public, independent from clinical settings.<br />

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Impact Statements

1. Objective 1: Our Research group is developing ways to control foodborne pathogens in animals and the food supply. Researchers at Alabama found that most of the Salmonella-targeted bacteriophages can kill multiple serotypes, indicating that phage cocktails used to reduce Salmonella in chicken flocks would remain active even as serovars entering the flock changed. Large-scale delivery of bacteriophages via drinking water is feasible. Combining traditional intervention strategies such as probiotic treatment with phage treatment results in reduced pathogen load compared to probiotic treatment alone. Quantitative PCR may be an alternative method to standard culture techniques, reducing the labor required to enumerate Salmonella in chicken ceca.
2. Objective 1: In September 2007 pasteurization of almonds (to reduce Salmonella by 10,000-fold (4-log)) sold in North America became mandatory. Research conducted at the University of California validated that oil roasting, blanching and propylene oxide treatments could be used by the almond industry to comply with the regulation. Where data were not available, methods developed by there laboratory for inoculating almonds and recovering Salmonella after treatment were used by industry to validate specific pieces of equipment. Data on the survival of Salmonella in almond orchards has been used to further refine almond-specific good agricultural practices.
3. Objective 1: Researchers at the University of Delaware achieve a > 5-log reduction in the counts of V. vulnificus in oysters for a relatively short treatment time (£ 4 min) and that pressure treatment needs to be conducted at pressure levels of e 250 MPa at -2 or 1°C. These results point to the potential of using temperatures above or below room temperature (20 - 30°C) to lower the pressure needed to cause the desired level of V. vulnificus inactivation in oysters.
4. Objective 1: Research conducted at Virginia Tech showed the importance of the UV dose required to control L. monocytogenes in brines used during RTE meat processing. This knowledge will aid manufacturers in establishing appropriate food safety interventions for these products.
5. Objective 1: The ability of foodborne bacteria to survive under adverse conditions and what implications this can have in food products was investigated. Acid adaptation of foodborne pathogens provides a certain level of protection against heat treatment at lower cooking temperatures. 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. V. vulnificus and V. parahaemolyticus strains vary significantly in their ability to survive and grow at refrigeration temperatures. This may be useful in updating risk assessment models for these pathogens.
6. Objective 1: Methods for rapid detection of foodborne pathogens in food products are being developed by researchers in our multi-state project. Researchers at Iowa State University have clearly demonstrate the promise of combined FISH and flow cytometry for the rapid molecular detection of Salmonella and Listeria monocytogenes in pork-based meats and show that detection of even low levels of these pathogens is possible within a days work using this approach.
7. Objective 2: The fastest growing strains resulting from a study conducted by Michigan State University can be recommended for future use in L. monocytogenes challenge studies in delicatessen meat and poultry food matrices, so as to develop conservative pathogen growth predictions. Researchers at Cornell University applied the principles of signal detection theory to measurement bacterial growth assessment using light scattering. This presents firm statistical ground for distinguishing between a baseline signal and one differing significantly from the baseline level.
8. Objective 3: Researchers at The Ohio State University have determined that antibiotic resistance genes may be transferred by bacteriophage among strains of Salmonella, that not Escherichia coli O157 are of equal pathogenic potential, and that management factors (antibiotic use, stocking density) on farms may impact the prevalence of Salmonella in pigs. In addition the distribution of pathogenic organisms in wildlife was reported. Understanding the major routes leading to antibiotic resistance in humans and the role of commensal bacteria in the spread of these resistance genes will most likely will lead to changes in medical, agriculture and food industry practices. The social and financial impacts are tremendous.

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Objective 1: Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments including meat, poultry, seafood, fruits and vegetables and nutmeats. <br /> <br /> At Clemson University in South Carolina, several projects which focus on Helicobacter pylori Listeria monocytogenes, and Salmonella spp. are underway. The effects of muscadine grape skin extract on the in vitro inhibition, AGS cell proliferation, and in vivo antimicrobial activity against H. pylori attachment to mice stomach are being investigated. In addition, biological strategies for controlling Salmonella contamination in rendering plants are being tested. Novel techniques (nanoparticle-based immunomagnetic separation (IMS) with real-time PCR) for a rapid and quantitative detection of Listeria monocytogenes is being developed. Our results demonstrated that both the use of nanoparticles and the choice of anti-L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples. <br /> <br /> Alternatives to antimicrobials used in food processing are being explored at the University of Delaware. Using Alamar Blue assay, minimum inhibitory concentrations (MICs) were determined for thymol, carvacrol, cinnamaldehyde, cinnamon bark oil, and thyme oil, respectively, against Salmonella enterica Typhimurium. However, the addition of thymol at its MIC to poultry feed showed no inhibition of Salmonella as compared to the control feed in a chicken feeding trial. In contrast, the addition of thymol at its MIC in washing water for tomatoes inhibited the contamination of the tested Salmonella.<br /> <br /> Researchers at the University of Georgia have investigated the efficacy of electrolyzed (EO) water and chlorinated water treatments at various temperatures and for various lengths of time and in conjunction with ultrasonication to inactivate E. coli O157:H7 on strawberries and broccoli. Dipping strawberries and broccoli into EO water or chlorinated water significantly reduced the E. coli O157:H7 counts. Dipping inoculated strawberries with chlorinated water or EO water with ultrasonication for 1 or 5 min reduced E. coli O157:H7 cells by 0.7 to 1.9 log CFU/g. Dipping inoculated broccoli into chlorinated water or EO water with ultrasonication for 1 or 5 min reduced the bacterial population by 1.2 to 2.2 log CFU/g. Significant reductions in populations of the pathogen were observed when produce was treated with EO water in conjunction with ultrasonication. <br /> <br /> At Michigan State University, low-energy X-ray irradiation was assessed as a means of eliminating E. coli O157:H7 on dip- inoculated lettuce. When individual leaves were irradiated, a D10-value of 0.040 kGy was obtained, which is 3.4 times lower than the previously reported value using gamma irradiation. When ten stacked leaves were irradiated from both sides, a dose of 0.2 kGy was achieved at the center of the stack, corresponding to a ~5 log reduction of E. coli O157:H7. <br /> <br /> Escherichia coli O157:H7 continues to cause outbreaks of produce-associated foodborne illness. Contaminated water may facilitate transfer of E. coli O157:H7 to leafy greens during irrigation. Rapid assays should be developed to detect bacterial pathogens in irrigation water, and should be able to detect the viability of the pathogens. Light scattering spectroscopy (LSS) is a powerful technique that has been applied to qualitatively and quantitatively distinguish internal structural changes in cells upon perturbation by chemical/biological agents. When combined with bacteriophage infection of a target bacterial cell, the method can distinguish between viable and non-viable bacterial cells. Appropriate E. coli O157:H7 and Salmonella strains were seeded into individual water samples at several concentrations. 1 ml aliquots were withdrawn and subjected to immunomagnetic separation (IMS) using E. coli O157-specific IMS beads. Following IMS and wash steps, the beads (with any bacteria attached) were resuspended in 1 ml of lambda diluent, and one half (500 µl) of each sample was added to 10 ml of Tryptic Soy Broth (TSB) that contained 1 ml of bacteriophage AR1 (1010 PFU/ml). The other half of the samples was added to TSB that did not contain phage AR1, and these samples served as controls. The samples were incubated for up to 8 h. Following incubation, 100 µl aliquots were removed from each sample, and separately assayed using a light scattering spectrometer. E. coli O157:H7 was detected within 6 h in all samples that contained this pathogen. An algorithm was developed to evaluate the area under the curve of each spectra. Researchers at Colorado State University found when compared to the light scattering spectra of the non-phage treated controls, the spectra of phage infected E. coli O157:H7 cells differed markedly. In contrast, the spectra of samples that contained Salmonella were identical, due to the fact that phage AR1 does not infect Salmonella spp. The detection limit after 6 h of incubation was an initial concentration of 100 CFU/ml.<br /> <br /> Work related to microbial contamination of nuts and fresh fruits and vegetables is also being conducted at University of California, Davis. The survival of E. coli O157:H7 in growing lettuce plants was assessed. Our objective was to evaluate the impact of irrigation method on the survival of E. coli O157:H7 in field inoculated-lettuce. A split plot design with three replicates was used to evaluate two main treatments: drip and overhead irrigation in three separate field trials. Rifampicin-resistant attenuated (non-pathogenic) E. coli O157:H7 (ATCC 700728), was inoculated by spraying onto 4-week old lettuce plants at target inoculum concentrations of 7 log CFU/plant. E. coli O157:H7 was recovered by stomaching, standard enumeration and filtration on TSA with 50 mg/ml of rifampicin. When counts were below the limit of detection, prevalence was determined by enrichment of entire plants. Recovered levels of E. coli O157:H7 were 5 log CFU/plant immediately after inoculation and 3 log CFU/plant 2 h after inoculation. After 7 days E. coli O157:H7 was recovered on some plants by plating and on most plants by enrichment. By day 28 7% or more plants remained positive (120 to 150 plants tested per time). Differences were not detected between irrigation methods. A better understanding of factors that influence E. coli O157:H7 survival in lettuce fields will help design strategies to reduce pre-harvest contamination.<br /> <br /> In other studies, the thermal resistance of Salmonella on almonds heated in oil was determined. Whole almonds were inoculated with Salmonella Enteritidis PT 30 or Salmonella Senftenberg 775W and heated by immersion in hot oil. After heating, almonds were drained, transferred to tryptic soy broth, and stomached prior to plating onto tryptic soy and bismuth sulfite agars. Survivor inactivation curves were upwardly concave with rapid reductions of 2.9, 3.0, or 3.6 log CFU/g of Salmonella Enteritidis observed in 30 s of exposure to oil at 116, 121, or 127 degrees C, respectively. Thereafter, the reduction was at a much slower rate of decline. Similar reductions were observed at 127 degrees C for Salmonella Senftenberg. The Weibull model was used to predict 4- and 5-log reductions of Salmonella of 2.4 and 1.3 min at 127 degrees C, respectively. Neither Salmonella serovar could be recovered by enrichment of 1-g samples after almonds inoculated at 5 log CFU/g were exposed to oil at 127 degrees C for 1.5 min. Standard almond oil roasting times and temperatures that achieve acceptable kernel color and texture should result in much greater than 5-log reductions of Salmonella in almonds. <br /> <br /> Accomplishments from the University of Delaware towards the fulfillment of this aim include the following projects related to the biology of noroviruses, Salmonella and the response of foodborne pathogens, notably Bacillus cereus and Listeria monocytogenes, to high hydrostatic pressure and refrigerated storage. Transmission and genetic stability of norovirus and other enteric viruses has been studied in association with the use of manure and biosolids on vegetable crops. Virus attachment to leafy green vegetables increases when they are bound to colloids in biosolids or contaminated water, but viruses can be killed by subsequent use of high pressure or UV light. <br /> <br /> Microarray analysis of a collection of Salmonella enterica Kentucky isolates obtained from poultry sources has been initiated to compare gene expression profiles of a S. enterica Kentucky and Enteritidis isolate in response to acid exposure. Current data indicate that, although most acid-responsive genes are expressed similarly in both isolates in response to pH change, some differences were observed. Experiments to verify these differences by quantitative PCR are being initiated. <br /> <br /> The application of high hydrostatic pressure technology as a seed decontamination technology was evaluated for alfalfa seeds. Soaking seeds prior to pressure treatment was found to play a critical role on enhancing the pressure inactivation of E. coli O157:H7; seeds soaked in water for 60 min followed by treatment at 600 MPa for 2 min at 20 degrees C were decontaminated and had a germination rate of 91% which was 4% lower than that of the untreated seeds. In terms of their impact on the seed viability, the process of 550 MPa for 2 min at 40 degrees C was the most desirable achieving final germination percentages and sprout sizes statistically similar to control untreated seeds (P > 0.05). When applied to Sensitivity 30 strains of L. monocytogenes, no correlation between pressure tolerance and heat, acid or nisin resistances was found. Nucleotide sequence analysis of the ctsR region in these 6 strains demonstrated that this gene codes for a CtsR protein with identical predicted amino acid sequences. The sequences of the 200-bp region located immediately upstream of the ctsR start codon of the different strains were virtually identical, and it is therefore likely that differences in pressure tolerance are based on factors other than the stress gene regulator CtsR.<br /> <br /> Virginia Tech scientists are also actively involved in working on this aim. Specifically, the retention of pathogenic bacteria, including Salmonella spp., on food contact surfaces increases the risk of transmission to food products. We compared the recoveries of an inoculation of Salmonella enterica serotype Typhimurium from stainless steel, using three recovery methods including a standard rinse, a one-ply composite tissue (Kimwipe®), or a sonicating toothbrush. Findings were used to design an additional project to compare recovery method efficacy for a similar inoculated study with fluorescent microspheres. Marked difference in recovery were obtained based on the sampling method used. A sonicating brush method was the most effective method for recovery of Salmonella Typhimurium from stainless steel coupons. The mean percent recovery of S. Typhimurium was only 1.2% for this method, and only 0.8% for a KimWipe® tissue method. A standard rinse method resulted in recovery that was significantly lower and yielded half the number of cells as the sonicating brush method. <br /> <br /> Shrimp is among the most common seafood and a favorite among consumers. Like any other food there are safety concerns about shrimp. Louisiana State University Ag Center has taken leadership in improving the safety of this nutritious food. Listeria spp., Salmonella spp., Clostridium spp. and Vibrio spp. are among the pathogens of prime importance. Most of these pathogens can be eliminated by cooking. However, the extent of cooking and temperatures can greatly influence the safety of seafood. The current study is focused on the determination of minimum cooking temperatures for reducing Listeria spp., Salmonella spp. and Vibrio spp to non-detectable levels on the surface of shrimp. Shrimp were surface inoculated with the three different species mentioned above to about 5 Log CFU/g of shrimp and then incubated for two days. Shrimp samples were treated at five different temperatures on 0, 1 and 2 day by boiling in a water bath. The effects of temperature on bacterial counts were determined by plating and calculating the log CFU/g reduction for each temperature. The experiment was repeated with different temperatures for each bacterium until the bacterial load in the shrimp was at non-detectable levels. The internal temperature of 85 degrees C was the least minimum temperature that was needed to kill all the bacteria tested. Vibrio spp. was less resistant to heat with bacterial counts reaching non-detectable levels at 55 degrees C. For Salmonella spp. the minimum temperature required to reduce bacterial counts to non-detectable levels was 75 degrees C, while Listeria spp. showed highest resistance up to 85 degrees C. <br /> <br /> Iowa State University (ISU) researchers recently developed an approach combining fluorescence in situ hybridization (FISH) and flow cytometry for detecting low levels of Salmonella spp. (~103 cells/ml sprout wash) against high levels of naturally occurring sprout flora (~107  108 CFU/g sprouts). Although this FISH and flow approach provided rapid presence/absence testing for Salmonella in this complex food system, it was not capable of more nuanced tasks, such as probing the phenotypic complexity of the microbes present in sprouts or determining the physical interactions of Salmonella with these microbes, or with sprout debris. In the present study, we have combined rapid FISH-based labeling of Salmonella spp. in sprout washes with flow-through imaging cytometry (FT-IC), using the ImageStream® 100, a commercial FT-IC instrument. This approach enables image-based characterization of various subpopulations of interest occurring within these samples. Here, we demonstrate the ability of FT-IC to unambiguously identify cells, cell aggregates and other events within these subpopulations based on both cell morphology and hybridization status after reaction with a Salmonella-targeted probe cocktail. Our ability to directly explore the nature of these events expands the layers of information possible from cytometric analyses of these complex samples and clearly demonstrates that a picture is worth a thousand dots.<br /> <br /> Additional experiments at ISU quantitatively and qualitatively analyzed the impact of of airborne contamination of Listeria monocytogenes on ready-to-eat meats. Three strains of L. monocytogenes were attached to sterile sand and dusted into vessel containing bologna slices and hot dogs. Three quantities of sand were used (1.0, 5.0 and 10.0 g). Half the samples were evaluated at day zero and the other 28 days later. RTE meat product were evaluated by spiral plating on chromogenic L. monocytogenes agar to determine colony forming units (CFU) per sample and then enriched in Modified University of Vermont broth (UVM) and to 4-Morpholinepropanesulfonic acid buffered Listeria enrichment broth (MOPS-BLEB). The MOPS-BLEB enrichments were plated on Modified Oxford agar (MOX) and also analyzed using a commercially available PCR system. There was a significant difference in the potential contamination between the two product types using both qualitative methods. The quantitative data from the bologna showed no significant differences between the day 0 and 28 and the 5.0g and 10.0g samples. The qualitative analysis found significant differences between the inoculum quantities and percent of positive samples after cold storage. This study illustrates the potential of airborne contamination of RTE meats. Airborne L. monocytogenes is a problem in RTE food production, and more research is needed to fully comprehend the issue, and how it can be prevented or controlled. <br /> <br /> The Ohio State University has focused on pre-harvest prevention of foodborne diseases. Investigations and outreach activities have focuses on E. coli O157, Listeria monocytogenes, and antibiotic resistance. We documented opinions of experts on microbial contamination of fruits and vegetables, as well as beliefs and practices of physicians, veterinarians, and pet owners regarding the control of foodborne and antibiotic resistant pathogens. Original research has explored the household environment as a source of food contamination and acquisition of foodborne pathogens in rural residents. On-farm studies focused the transmission of E. coli O157 among dairy farms and on vegetable farms are continuing and are providing additional information about the ecology of this important pathogen.<br /> <br /> A study conducted by Colorado State University evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (46 degrees C) stresses, sequentially or simultaneously within 1.5 h, in tryptic soy broth with 0.6% yeast extract. While no cross-protection was observed at 52 and 63 degrees C, all treatments induced adaptive responses on L. monocytogenes at 57 degrees C. As determined by a Weibull model, survivor curves at 57 degrees C appeared convex with profound shoulders. Regarding acid tolerance, prior exposure to low pH or a combination of NaCl, pH and heat resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log10 CFU ml-1) than the control cultures. The highest thermotolerance was observed after combined exposure to acid and heat shock, followed by exposure to osmotic shock, and by the combination of osmotic with heat shock. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses resulted in more survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. Further research should assess the effect of sequential or simultaneous application of stresses on the cross-tolerance against a variety of food processing-related stresses in vitro and in situ, and the influence of the physiological stage of cells on their sensitivity to sublethal stresses. <br /> <br /> Objective 2: Develop and validate mathematical modeling to gain understanding of pathogen behavior in macro and micro-environments.<br /> <br /> Modeling of pathogen fate in foods and the environment is currently being conducted at Clemson University and Michigan State University. At MSU, a systems approach is being used to identify and minimize the Escherichia coli O157:H7 hazards associated with leafy greens. During processing of E. coli O157:H7-inoculated heads of iceberg and Romaine lettuce, 90% of the inoculum transferred to the wash water. After processing, E. coli O157:H7 populations were highest on the shredder and conveyor followed by the flume tank and shaker table with 30% of the remaining inoculum lost during centrifugal drying. E. coli O157:H7 was transferred from the contaminated equipment surfaces to the entire next batch of processed product. Based on these findings, a mathematical model is currently being developed to predict the rate of E. coli O157:H7 transfer between product, water, and equipment. <br /> <br /> Clemson University researchers have investigated the fate of pathogens in both pre- and post harvest environments. In the pre-harvest environment, proper composting methods (including field conditions) and pathogen inactivation and control of re-growth is under investigation. In our trials, an indicator microorganism, E. coli, was inactivated at a rate similar to that of E. coli O157:H7. Results indicate that composting, with periodic heap turning, can be a practical approach to inactivating E. coli O157:H7 in cattle wastes on the farm. <br /> <br /> At the post-harvest stages of food production these researchers are developing heat-inactivation models of pathogen destruction in food and quantifying the synergism between pasteurization and antimicrobial additives in food. The combination of heat with nisin + lysozyme or nisin alone treatments significantly reduced the time required to reduce L. monocytogenes populations compared to heat combined with no antimicrobial or lysozyme. The rate of log reduction at each of the three temperatures did not fit a linear model; thus a Weibull model was used to fit data at 65 and 62.5 degrees C while a log-logistic model was used to best describe the log reduction vs. time relationship.<br /> <br /> Objective 3: Investigate factors leading to the emergence, persistence and elimination of antimicrobial resistance in food processing and animal production environments<br /> <br /> Researchers at Auburn University are continuing efforts to incorporate bacteriophage treatment of chickens with traditional intervention methods to augment Salmonella reduction. Daily treatment of S. Typhimurium- infected chicks with a cocktail of four lytic bacteriophages resulted in a decrease in cecal and cloacal colonization of Salmonella during the first 5 days post-challenge compared to untreated chicks. However, after 5 days, numbers of Salmonella in the ceca and cloaca of the treated and control groups were not different. This finding indicates that the Salmonella multiplying in the chicks may be developing resistance to the bacteriophages. Interestingly, the number of B cells in the cecal tonsils of treated chicks was significantly higher (P = 0.0001) than the number of B cells in cecal tonsils of untreated chicks. This finding reflects the delayed increase in Salmonella numbers in treated chicks, resulting in delay in the homing of B cells out of the cecal tonsils to the lamina propria that occurs earlier in untreated chicks. This finding also shows that bacteriophage treatment does have an effect on the inflammatory response to Salmonella infection. Taken together, these findings indicate that bacteriophage treatment may be most effective in reducing Salmonella numbers when administered the last few days before slaughter, resulting in less carcass contamination. <br /> <br /> Iowa State University investigators also documented the effects of grape seed extract (GSE) - a product Generally Recognized as Safe (GRAS), and reported to have wide-ranging bioactive, anti-inflammatory, anti-carcinogenic and antimicrobial properties- against Listeria monocytogenes. They characterized the antilisterial effects of a commercial GSE preparation (Gravinol®-S) alone at low levels (0.00015% - 0.125%) in aqueous solution and tested its possible use as an antimicrobial wash for fresh produce surfaces. Based on broth microdilution tests, the minimum inhibitory concentrations of GSE against L. monocytogenes Scott A and L. innocua ATCC 33090 were as low as 50 and 78µg ml-1, respectively. GSE was evaluated in 0.85% saline against live cells of L. innocua via flow cytometry, using propidium iodide as a probe for membrane integrity. At sub-MIC levels and after only 2 min exposure, treatment with GSE caused rapid permeabilization and clumping of L. innocua, results that we confirmed for L. monocytogenes using fluorescence microscopy and Live/Dead staining. At higher levels (0.125%), GSE reduced viable cell counts for L. monocytogenes by approximately 2 logs within 2 min on tomato surfaces. These results suggest the potential for GSE as a natural means for control of Listeria spp. on low-complexity foods such as tomatoes.<br /> <br /> To further explore alternatives to conventional antimicrobials in food processing and production ISU scientists surveyed a panel of thirteen metal nanoparticle (NP) catalysts for their antifungal activities against Candida albicans ATCC 90028. Initial characterization using SEM suggested that our ability to detect NP binding to Candida surfaces with this method was impeded by preparation artifacts. As an alternative method for visualizing NP binding, we used an enhanced dark field illumination system (CytoViva®) attached to a standard light microscope. When viewed using this system, all NPs produced intense optical signals due to resonant light scattering. To assay binding, NPs were allowed to interact with C. albicans hyphae and cells in spent RPMI broth for 15 min with gentle inversion, followed by viewing with the CytoViva® system. The antifungal efficacy of NP preparations was determined separately using a 24 h broth microdilution test. For single-metal NPs, observations of binding at 15 min made via CytoViva® corresponded to antifungal efficacy at 24 h, with the most antifungal NPs yielding complete coverage of hyphal surfaces. For alloy NPs, the relationship between rapid binding and antifungal activity was not as clear. Our work suggests the utility of visual screening using the CytoViva® system for rapid, simple and artifact-free viewing of NP-cell interactions in support of antimicrobial screening efforts.<br />

Publications

The attached publication list includes funded grants and projects

Impact Statements

1. Muscadine grape may be used as a dietary approach for controlling H. pylori, thereby improving the health of American people in the near future. Adoption of electrolyzed water, addition of thymol to wash water or low-energy X-ray irradiation for the produce industry may decrease the microbial contamination of fresh and fresh-cut produce and thereby increase shelf-life and enhance food safety.
2. Understanding the patterns of how S. enterica serovar Kentucky are destroyed by heat will result in better treatments to ensure consumer safety. The combination and the sequence of application of different stresses have a major influence on the extent of acid cross-protection of L. monocytogenes cells, while its heat tolerance is mainly affected by the combination of pre-heating stresses rather than by the sequence of their application. Moreover, the combination of low pH and insufficient thermal treatment are more likely than other potential stresses to compromise food safety by developing stress hardened L. monocytogenes cells.
3. Improved sampling and testing protocols for Salmonella will provide greater assurance that the food processors sampling plans can detect the presence or enumerate of this pathogenic microorganism. In addition, the use of devices similar to a sonicating toothbrush may lead to a faster and more accurate method of enumerating surface contaminants while enhancing recovery rates. Light Scattering Spectroscopy has the capability to detect viable bacterial cells, following phage infection. When coupled with IMS, this method may be applied to the rapid and sensitive detection of viable E. coli O157:H7 in irrigation water. The capacity to detect foodborne pathogens is necessary for targeting efforts to control or reduce pathogens in the food supply.
4. Finding the temperature that can destroy foodborne pathogens in shrimp will allow us to design a simple, easy and unbiased consumer guide for cooking shrimp to enhance the safety while handling and cooking them at home. This can also serve as a guide for manufacturers of ready-to-eat shrimp products while designing and planning the GMPs and HACCP plans during production. Understanding stakeholders understanding and mental models of disease prevention and control is critical for developing the most effective educational programs. Audience specific media can be developed to target specific behaviors in different populations to yield behavior changes and lower incidence of foodborne illnesses. These messages are supported by the biological data concerning pathogen distribution and dissemination discovered in the laboratory and field investigations.
5. This is the first report to quantitatively define both the role and importance of E. coli O157:H7 transfer from product to equipment surfaces during production of fresh-cut leafy greens. The research on composting has generated very practical and useful information, which can be adopted easily by the farmers to produce safe compost. Consequently, the food safety on farm can be improved by reducing the pathogen loads in animal manure, which is considered as the source of fresh produce, water, and environmental contamination.
6. The use of bacteriophage intervention, coupled with the traditional interventions of competitive exclusion and vaccination, should decrease Salmonella colonization of chickens without increasing antibiotic resistance.

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Participants

Attendees of November 12-13, 2009 meeting included in attached minutes of meeting.

Brief Summary of Minutes

Minutes of November 12-13, 2009 meeting attached

Accomplishments

Objective 1: Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments including meat, poultry, seafood, fruits and vegetables and nutmeats. <br /> <br /> At the University of Alabama, research has focused on the development of a bacteriophage treatment to reduce Salmonella enterica serovar Newport infection and shedding in calves. An experimental Salmonella infection model in cattle was developed that involved oral inoculation of 6-8 week old just-weaned calves with 9 log CFUs of S. Newport. In this model, calves developed signs of fever, diarrhea, and dehydration beginning 24 h post-inoculation, and shed quantifiable numbers of S. Newport for > 7 days. Oral treatment with a cocktail of three S. Newport-targeted bacteriophages (10 log PFUs of each phage) 24 and 48 h post-inoculation resulted in significant reduction of shedding (P < 0.05) of S. Newport on days 4-6. Clinical disease was also reduced in the treated calves as indicated by significant reduction (P < 0.05) in rectal temperature on days 4 and 7 post-inoculation and the presence of normal feces throughout the 11-day experimental period.<br /> <br /> Researchers at the University of Arkansas have investigated methods to control Salmonella. For example, recent studies have centered around the use of acidified 55 degrees C solutions of select organic acid salts, and their effect on Salmonella enterica serovar Typhimurium. Previous studies at UA have shown that exposure to acidified sodium lactate (2.5% SL, pH 4) and sodium propionate (2.5% SP, pH 4) solutions at 55oC led to significant log reductions of S. Typhimurium (~1.5 and 3.5 logs, respectively), and it was hypothesized that this log reduction was due in part to effect(s) on the cell membrane. Therefore, the objective of this study was to characterize the mode of action of this thermal acidified organic acid salt treatment. Osmotic response assays designed to measure cell plasmolysis, a measurement of cell wall/membrane damage, were conducted at 55 degrees C. The results demonstrated that exposure to SL and SP, but not sodium acetate (SA), led to a significantly reduced ability of S. Typhimurium to respond to a change in osmotic pressure (5%, 10%, and 17%, respectively) as compared to the control (pH 4 deionized water [26%]). Using transmission electron microscopy, treated and control cells were visualized. Surprisingly, all treatments including those with no effect on viability, such as exposure to pH 4 deionized water or SA, resulted in visible cellular stress. However, cell damage appeared most severe for the pH 4 organic acid salt treatments. Additionally, initial measurements of S. Typhimurium potassium ion leakage revealed that exposure to SP resulted in the greatest and pH 4 deionized water the least leakage (14.6 ppm and 2.8 ppm, respectively). These data support our hypothesis that following treatment with 55 degrees C 2.5% SP at pH 4 the loss of S. Typhimurium viability is at least in part due to membrane damage. <br /> <br /> Research at Colorado State University has centered around the development of rapid diagnostics based on light scattering spectroscopy for detection of Escherichia coli O157:H7. Appropriate E. coli O157:H7 and Salmonella strains were seeded into individual water samples at several concentrations. 1 ml aliquots were withdrawn and subjected to immunomagnetic separation (IMS) using E. coli O157-specific IMS beads. Following IMS and wash steps, the beads (with any bacteria attached) were resuspended in 1 ml of lambda diluent, and one half (500 µl) of each sample was added to 10 ml of Tryptic Soy Broth (TSB) that contained 1 ml of bacteriophage AR1 (10 log PFU/ml). The other half of the samples were added to TSB that did not contain phage AR1, and these samples served as controls. The samples were incubated for up to 8 h. Following incubation, 100 µl aliquots were removed from each sample, and separately assayed using a light scattering spectrometer. E. coli O157:H7 was detected within 6 h in all samples that contained this pathogen. An algorithm was developed to evaluate the area under the curve of each spectra. When compared to the light scattering spectra of the non-phage treated controls, the spectra of phage infected E. coli O157:H7 cells differed markedly. In contrast, the spectra of samples that contained Salmonella were identical, due to the fact that phage AR1 does not infect Salmonella spp. The detection limit after 6 h of incubation was an initial concentration of 2 log CFU/ml. These results demonstrate the ability of LSS to detect viable bacterial cells, following phage infection. When coupled with IMS, this method may be applied to the rapid and sensitive detection of viable E. coli O157:H7 in irrigation water.<br /> <br /> At Cornell University, the use of UV light (254 nm), acidified sodium hypochlorite (pH 6), and mild heat, was investigated to determine the effectiveness of the various treatments and combinations to inactivate E. coli O157:H7 in green onions and spinach. Green onions and spinach were inoculated by spot or dip-inoculation, in order to compare the decontamination efficacy for both surface and infiltrated E. coli O157:H7 contamination, respectively. UV light (500 ± 20 mJ/sq cm) was shown to reduce E. coli O157:H7 populations by 1 ± 0.2 log CFU/g with dip-inoculated samples, the population was reduced by 1.7 ± 0.2 log at 90 ± 7 mJ/sq cm and 2.7 ± .2 log at 1000 ± 40 mJ/sq cm level for spot-inoculated produce. Chlorine treatments alone were capable of reducing E. coli O157:H7 populations by 0.6 ± 0.3 log CFU/g for dip inoculated samples, whereas spot-inoculated samples treated with chlorine resulted in a 3.5 ± 0.3 log CFU/g log reduction. Even though mild heat treatments alone showed no significant differences, a further 0.5 log reduction was observed when used in combination with chlorine (200 ppm at 50°C). The combination of selected UV exposure (90 ± 7 mJ/sq cm) and chlorine (200 ppm at 50°C) treatments showed a total of 4.7 ± 0.3 log reduction with a five-strain cocktail of E. coli O157:H7 spot-inoculated produce. Additional UV exposure (500 mJ/sq cm), yielded a 2.1 ± 0.3 log CFU/g for dip-inoculated samples. These results indicate that dip inoculated produce required significantly higher levels of UV exposure to achieve the same reduction as for spot inoculated samples. Combination treatments on produce showed additional inactivation compared to the cumulative reductions for individual treatments.<br /> <br /> The University of Delaware has developed procedures using high hydrostatic pressure to decontaminate green onions against E. coli O157:H7 and Salmonella, since green onions have been described as one of five commodity groups that together, make up over 75% of produce-related food-borne-illnesses. Green onions inoculated with a cocktail of nalidixic-acid and streptomycin resistant double mutant strains of E. coli O157:H7 and Salmonella (ca. 5 log CFU/g) were subjected to pressures ranging from 250-450 MPa for 2 min at 20°C in a dry, pre-wet or pre-soaked state. In addition, inoculated samples were also treated at 250-550 MPa for 2 min in the pre-soaked and pre-wet states at reduced (4°C) and/or elevated temperatures (30 and 40°C) as appropriate. The decontamination efficacy of HHP for either pathogen, increased in the order of soaked > wet > dry states at all pressure levels. The pressure-sensitivity of the enteric pathogens was also higher at elevated treatment temperatures achieving complete elimination of the pathogens at pressures > 400 MPa and temperatures > 20°C in the pre-wet and pre-soaked states. High pressure processing of green onions contaminated during cultivation also effectively eliminated a ~ 4-5 log CFU/g burden of Salmonella and E. coli O157:H7. In addition, the application of selected HHP treatment conditions reduced the background microbial load of green onions thereby improving its microbiological quality. <br /> <br /> In addition to green onions, tropical fruit represents another type of fresh produce that has been implicated in outbreaks of foodborne disease. For example, several salmonellosis outbreaks have been associated with the consumption of tropical fruits, including mango, papaya and pineapple. <br /> <br /> At the University of Florida, a study was conducted to evaluate the fate of E. coli O157:H7 and Salmonella on fresh (23°C, 12°C, and 4°C) and frozen (-20°C) cut mangoes, papayas and pineapples. E. coli O157:H7 and Salmonella spp. have the potential to grow on temperature abused fresh-cut mangoes and papayas held at 23°C and 12°C, and survive on fresh-cut pineapples. E. coli O157:H7 and Salmonella spp. can survive for extended periods of time on refrigerated (4°C) and frozen (-20°C) cut mangoes, papayas and pineapples. Our work indicates that both fresh and frozen cut mangoes, papayas and pineapples have the potential to be vectors for E. coli O157:H7 and Salmonella spp. transmission.<br /> <br /> Researchers at the University of Georgia have been investigating the efficacy of electrolyzed (EO) water and chlorinated water treatments at various temperatures and for various lengths of time and in conjunction with ultrasonication to inactivate E. coli O157:H7 on strawberries and broccoli. The results of this study indicated that dipping strawberries and broccoli into EO water or chlorinated water significantly reduced the E. coli O157:H7 counts. Dipping inoculated strawberries with chlorinated water or EO water with ultrasonication for 1 or 5 min reduced E. coli O157:H7 cells by 0.7 to 1.9 log CFU/g. Dipping inoculated broccoli into chlorinated water or EO water with ultrasonication for 1 or 5 min reduced the bacterial population by 1.2 to 2.2 log CFU/g. Significant reductions in populations of the pathogen were observed when produce was treated with EO water in conjunction with ultrasonication. <br /> <br /> Alligator meat is mainly consumed in the southern United States and the industry wants to expand their market. To take advantage of this potential for increased market penetration and industry viability, the industry is also aware that the final product quality of alligator meat needs improvement. <br /> <br /> Faculty at Louisiana State University have evaluated the effects of different antimicrobial agents on alligator meat and to identify effective treatments. In this work, four month old alligators were skinned and gutted, and then the carcasses were treated individually with different antimicrobial agents dissolved in an icy water bath for 5 minutes in order to find the most effective treatment. The antimicrobial agents were Lactic Acid (200PPM), Sodium Benzoate (200PPM), Calcium Lactate (200PPM), Chlorinated water (150PPM of Sodium Hypochlorite), and Acidified Sodium Chlorite (ASC) (50PPM). The two most efficient antimicrobial agents were Lactic acid and ASC which were combined with steam (60 seconds at 2 to 3 inches from surface). For the combined treatments, the samples were steamed before soaking in the antimicrobial solutions. The back, tail and ribs of alligator carcasses were swabbed (2 square inches) and then analyzed for total coliforms, total Enterobacteriaceae, Escherichia coli and Salmonella spp. Treatments with Lactic Acid, Calcium Lactate, Chlorinated water and the combinations of steam and Lactic Acid or ASC, significantly reduced total coliforms, total Enterobacteriaceae and Salmonella spp. counts by 1 log from control levels. Sodium Benzoate did not show significant reduction on any of the bacteria analyzed. The combination of steam and ASC was the treatment that proved to be the most efficient in reducing coliforms, Enterobacteriaceae and Salmonella spp. on alligator carcasses. <br /> <br /> At the University of Nebraska, A computation fluid dynamics (CFD) model was developed to determine the temperature distribution within shell eggs during cooling to control the growth of Salmonella enterica serovar Enteritidis (SE). Experimental tests were conducted to determine the center temperature of an egg placed inside the test chamber of a wind tunnel. The simulated and experimental values of egg center temperature were found to be in good agreement, with root mean square error (RMSE) values ranging from 0.2°C to 0.9°C. The CFD model was then expanded to chilling of multiple eggs placed on an egg tray (6 rows x 5 columns) under forced air convection condition. A single row of the tray having 5 eggs was included in the CFD model. The RMSE for predicting egg temperatures by CFD model was within 1°C. The heat transfer model was integrated with a microbial growth model to estimate the risk of SE growth in shell eggs during storage.<br /> <br /> Researchers at The Ohio State University (OSU) are leading several extramurally-funded, multidisciplinary teams to better understand the impact of vegetable production practices on the ecology of E. coli O157 on plants, in water, and in wildlife and in livestock populations. In addition, other OSU researchers have been identifying novel pathogenicity/colonization factors and drug targets in Campylobacter jejuni. These researchers have uncovered novel pathogenicity determinants [Twin Arginine Translocation (TAT) system and Polyphosphate Kinases (PPK1 and PPK2)] critical for C. jejuni survival, adaptation, and persistence both inside and outside the host environments. Both the TAT system and PPKs represent potential targets for anti-C. jejuni therapeutics innovations. Currently, the TAT system is targeted to identify small molecule inhibitors to control C. jejuni. <br /> <br /> The retention of pathogenic bacteria, including Salmonella spp., on food contact surfaces increases the risk of transmission to food products. At Virginia Tech University, the recoveries of an inoculation of S.Typhimurium, fluorescent microspheres (1.0 ¼m diameter, carboxylate-modified, crimson FluoSpheres, Molecular Probes, Eugene, OR), or a combination of both from stainless steel, were compared. Three recovery methods, including a standard rinse, a one-ply composite tissue (Kimwipe), or a sonicating brush were used. Findings were used to assess the effectiveness of fluorescent microspheres as surrogates for S. Typhimurium. For example, for microspheres and Salmonella, recovery by sonicating brush was the most effective, and recovery by the Kimwipe method was least effective. Additionally, the retention of microspheres on the steel ranged from 16 to 25%. Microspheres yielded a significantly higher recovery rate (11 to 60 %) than Salmonella (approximately 1%) for each recovery method. Since the quantitative recovery of microspheres was significantly higher than the recovery of S. Typhimurium, the microspheres used in this study, may not be appropriate surrogates for the bacteria in recovery studies on stainless steel. However, microspheres may still be useful as a surrogate for bacteria in quantitative studies since a relatively high proportion that are removed or retained may be enumerated, with an opportunity for greater precision and accuracy. <br /> <br /> Beef cattle is the reservoir of E. coli O157:H7. Fecal shedding of E. coli O157:H7 causes beef contamination. Therefore, it is essential to reduce E. coli O157:H7 colonization in the gastrointestinal (GI) tract of beef cattle. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger that mediates a myriad of cellular processes. Researchers at the University of Wyoming hypothesized that c-di-GMP affects expression of virulence genes in E. coli O157:H7 and regulates E. coli O157:H7 colonization in the GI of beef cattle. To test this hypothesis, the yhjH gene was deleted, which encodes one of the potent c-di-GMP phosphodiesterases in E. coli. Results indicated that disruption of yhjH decreased motility and increased biofilm formation. qRT-PCR analysis indicated that deletion of yhjH decreased (p<0.05) expression of Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2A and 2B) genes. However, expression of genes involved in protein translocation and host cell adhesion, i.e. intimin (eae), EspA (espA) and EspB (espB), increased (p<0.05). Further, the attachment of E. coli O157:H7 to cultured epithelial cells was altered due to yhjH deletion. In summary, increased levels of c-di-GMP resulted from deletion of a potent c-di-GMP-specific phosphodiesterase differentially affect expression of virulence genes in E. coli O157:H7 and its attachment to gut epithelial cells, showing that c-di-GMP signaling has a role in the regulation of E. coli O157:H7 virulence and colonization in gastrointestinal tract of beef cattle. <br /> <br /> Objective 2: Develop and validate mathematical modeling to gain understanding of pathogen behavior in macro and micro-environments.<br /> <br /> Using inoculated lettuce to quantify E. coli O157:H7 transfer to a pilot-scale processing line for fresh-cut leafy greens, faculty at Michigan State University showed that 83-97% of the E. coli O157:H7 population transferred from the lettuce to the wash water. After processing, populations of a 4-strain avirulent, GFP-labeled, ampicillin-resistant E. coli O157:H7 cocktail were highest on the shredder and conveyor, followed by the flume tank and shaker table, with 30% of the remaining product inoculum lost during centrifugal drying. Similarly, when E. coli O157:H7 transfer from product-inoculated equipment surfaces to uninoculated lettuce was assessed, E. coli O157:H7 was quantifiable in all 90.8 kg batches of previously uninoculated iceberg and romaine lettuce. Based on these findings, a mathematical model is being developed to predict the extent of E. coli O157:H7 when large quantities of product are processed. In related work, a novel low-energy X-ray irradiator has proven capable of decreasing E. coli O157:H7 populations 5 logs in lettuce at a dose of 0.2 kGy without adversely impacting product quality. These findings will ultimately be invaluable in refining current microbial risk assessments being developed for fresh-cut produce.<br /> <br /> At the University of Nebraska, growth data of Salmonella at nine different isothermal conditions: 10, 15, 20, 25, 28, 32, 35, 37, 42, and 45 degrees C, were first fitted into primary models, namely the logistic, modified Gompertz, Baranyi models. The specific growth rates derived from each model was fitted to the Rajkowski equation, relating the specific growth rate to growth temperatures. These models, if validated, can be used to construct dynamic models to predict potential Salmonella growth in raw ground beef. <br /> <br /> Objective 3: Investigate factors leading to the emergence, persistence and elimination of antimicrobial resistance in food processing and animal production environments<br /> <br /> Salmonella spp. are important zoonotic pathogens in humans and animals. A longitudinal study was conducted at Iowa State University to observe changes in Enterobacteriaceae populations (specifically Salmonella) before and after the placement of dairy livestock. To our knowledge, this is the first study that evaluated environmental changes of Gram-negative organisms in a new dairy farm environment. Environmental samples were taken using drag swabs and immediately processed in the laboratory using phenotypic methods. Genotypic methods were also used (the BAX PCR system " and PFGE). Organisms identified as Salmonella were sent to the National Veterinary Services Laboratory (Ames, IA) for confirmatory serotyping. Resistance to antibiotics (ampicillin, nalidixic acid and tetracycline) was determined from replica plating of Enterobacteriaceae and Salmonella isolates using the guidelines of the National Antimicrobial Resistance Monitoring System (NARMS) and Clinical and Laboratory Standards Institute (CLSI). The microbiota of Enterobacteriaceae changed as cattle were introduced and as time progressed. Additionally, multi-drug resistant (MDR) isolates began to appear immediately after cattle were introduced (MDR isolates were rare prior to introduction of livestock). Variables such as temperature and humidity did not affect the proliferation of bacterial organisms; however this study was completed over a 9-month period. Seventeen Salmonella isolates were identified as S. london and three isolates as S. montevideo. Based on PFGE-generated dendograms, it is likely that the 17 S. london isolates are clonal and the 3 S. montevideo isolates are clonal.<br /> <br />

Publications

The attached publication list includes funded grants and projects

Impact Statements

1. Bacteriophage treatment shows promise as an alternate approach to antibiotic treatment for reducing Salmonella contamination of beef and dairy products, and for treating disease.
2. Combined thermal acidified sodium propionate treatment may provide an effective antimicrobial treatment for Salmonella-contaminated poultry, leading to significant reductions in Salmonella-related foodborne illness cases.
3. Bacteriophage-based light scattering spectroscopy is a novel and promising technique for the detection of viable bacterial pathogens in food and water.
4. High hydrostatic pressure (HHP) is an emerging method to minimally process green onions in order to alleviate the risks of E. coli O157:H7 and Salmonella infections associated with the consumption of this commodity.
5. A ground breaking study that evaluated environmental changes of Gram-negative organisms in new dairy farm environments will lead to increased knowledge regarding dissemination of foodborne pathogens in food animal environments.
6. Research on the safety and quality of alligator meat can help the alligator industry to increase their yield and extend the shelf life of their by-products.
7. Leafy green food safety research is expected to have a major impact on the manner in which leafy greens are processed in the United States with enhanced knowledge of bacterial transfer during the processing of leafy greens leading to a number of carefully targeted intervention strategies. In addition, these findings should lead to a reduction in the incidence of E. coli O157:H7 contamination in fresh-cut commercially produced leafy greens with the current x-ray work to providing a cost-effective means to completely eradicate E. coli O157:H7 and other bacterial pathogens in packages of leafy greens.
8. USDA Food Safety Inspection Services (FSIS) developed risk assessment models for S. Enteritidis growth in shell eggs are based on simplistic exponential cooling rate. In their report, FSIS identified a need for development of a heat transfer model to predict the internal temperature of eggs for estimating growth rates of S. Enteritidis at various storage temperatures. In response to their needs, mathematical models were developed to predict the temperature of eggs during chilling. The heat transfer model was integrated with a microbial growth model to estimate the risk of S. Enteritidis growth in shell eggs during storage. Use of predictive models can help in improving microbial risk assessment and developing appropriate risk management strategies. We have uploaded predictive models in the web so that food processors, extension educators, food safety inspectors, and students can use the developed models to improve the safety.
9. Control strategies targeting the TAT system will circumvent: a) problems associated with emergence of resistant bacteria with the use of conventional antibiotics, b) problems associated with the risk of developing polyneuropathies with the use of live attenuated vaccines and c) problems associated with emergence of antibiotic resistant bacteria including FQ resistant bacteria.
10. E. coli O157:H7 is a major foodborne pathogen associated with numerous beef product recalls. In addition, cattle farm runoffs containing E. coli O157:H7 also contaminates vegetables. The gastrointestinal (GI) tract of cattle is the major source of contamination and it is essential to reduce E. coli O157:H7 colonization in the GI tract. Studies to identify mechanisms regulating E. coli O157:H7 colonization in GI tract will provide molecular targets for future efforts to reduce or eliminate E. coli O157:H7 colonization in beef cattle, ensuring the microbiological safety of beef.

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Report Information

Participants

In attendance were the following members:

Elliot Ryser Michigan State University;
Cathy Cutter Penn State University;
Jeyam Subbiah University of Nebraska;
Matt Taylor Texas A&M University;
Steven Ricke University of Arkansas;
Phil Crandall University of Arkansas;
Marlene Janes Louisiana State University;
Jeff LeJeune The Ohio State University;
Lawrence Goodridge Colorado State University;

Micheal Johnson (University of Arkansas, retired) attended on Sept 12th as a visitor/guest

Brief Summary of Minutes

On the afternoon of September 12, project updates were provided to the group by representatives from each university. Summaries of these updates are attached.

Michelle Danyluk, member from University of Florida arrived on the evening of Sept 12th.

Members of the team also met with Jim Dickson, Iowa State University on the evening of Sept 12th.

Sept 13. The entire day was devoted to discussion and planning for the future of the group. A project resubmission proposal must be completed by January 31, 2012. A list of possible additional participants was established and individuals were contacted via e-mail to determine their interest in participating in this new project.

The need to change the meeting time to a date that is typically outside of the academic year was discussed. It was a consensus decision that having the meeting around the winter academic break or the summer might allow greater involvement of participants.

The issue of group diversity was also raised. Increased representation and participation from minority individuals and minority serving institutions is desired. One way to increase awareness and participation among a more diverse audience that was suggested was to occasionally hold the meeting at or near minority serving institutions (HIS, 1890s, tribal colleges). The possibility of holding the next meeting at the University of Puerto Rico was discussed. Pros and cons of such a move were debated, including potential travel costs (might not really be that much more than some remote locations in continental US) vs. expected increase in attendance from existing members and new members alike.

In preparation for the renewal application, a database of recently completed, current, and planned collaboration among participants was developed. A survey of areas of research focus was developed and sent to potential participants to better catalog the areas of interest and expertise among participants. A new, risk analysis framework was used to outline the proposal re-submission with emphasis given to comprehensive, integrated and interdisciplinary systems approaches to risk assessment, risk mitigation and risk communication. Writing teams for each of these sections were determined

Introduction: Goodridge and Ryser

Risk Assessment: Danyluk and Janes

Risk Management: Taylor and Subbiah

Risk Communications: Cutter and LeJeune

Although outlines for each of these sections have been developed, they are not complete. Outlines are posted on the S-1033 BaseCamp website for further completion and editing, a process that will commence again in earnest after the Oct NIFA deadline.

On the afternoon of Sept 13, a phone conference with Dr. Ram Rao, the projects USDA representative, was held. He had provided, in advance, a PowerPoint overview of NIFA programs and prospects for funding. One program that Dr. Rao mentioned that was still accepting applications was the program for conference grants on food safety.

Following Dr. Raos presentation, the group decided, based on the expertise and work currently being performed, that submission of a conference grant on Emerging Pathogens might be a good idea. Talylor and LeJeune agreed to lead this effort with a target inclusion of a symposium on the subject for the 2012 IAFP meeting.

The meeting adjourned at 6:00 pm on Sept 13, 2011. However, several members of the group (Goodridge, LeJeune, Ryser, Taylor and Dickson) provided research presentations at the Arkansas Association for Food Protection on Sept 14.

Summary Reports

Michigan State University, Elliot Ryser

Dr. Rysers research is currently focused on quantifying the transfer of bacterial pathogens including Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes during production of fresh-cut leafy greens, packing of tomatoes and dicing of celery with work on quantifying pathogen transfer during slicing/dicing of fresh fruits and vegetables to begin in early 2012 as part of a newly funded special emphasis NIFA grant that he is leading. Much of this work has been or will be done using a small-scale commercial processing line which includes a shredder, dicer, conveyor, flume tank, shaker table, roller conveyor, brush roller and centrifugal dryer with this processing line easily modified for different products. Additional studies will be designed to mimic small-scale hand dicing/slicing operations. Dr. Rysers other research interests in collaboration with Dr. Bradley Marks include the efficacy of X-ray irradiation to inactivate foodborne pathogens in various products including leafy greens, ground beef and nuts and the thermal inactivation of Salmonella in meat and poultry products as impacted by various processing conditions.

Penn State University, Cathy Cutter

Research in the Food Microbiology Group at Penn State under the research project S-1033 is being conducted by Drs. Catherine Cutter, Stephanie Doores, Luke LaBorde, and Steve Knabel.

Dr. Cutters research involves the control, detection, and monitoring of foodborne pathogens in meat and poultry. Her lab has developed a multiplex PCR assay for the detection of non-O157:H7 Shiga-toxin producing E. coli and is using it to detect the pathogens in very small plant environments and beef products (carcasses, trim, and ground beef). Additionally, Dr. Cutters lab is surveying poultry from farmers markets in Pennsylvania for Campylobacter and Salmonella and comparing the results to those of conventionally processed poultry from grocery stores. The data from the sampling will be incorporated into a needs assessment that will be used to develop food safety training programs for vendors at farmers markets. Recent research has demonstrated the application of an antimicrobial film, made from pullulan and containing Sakacin A or nisin, for controlling Listeria monocytogenes on ready-to-eat meat and poultry products. Research also has demonstrated that non-O157:H7 E. coli in ground beef (80:20 or 90:10 lean:fat) can be controlled by using high pressure processing. And finally, recent research in Dr. Cutters lab has demonstrated that attachment of Listeria monocytogenes and ability to form biofilms on food surfaces is affected by a combination of several factors, including water activity, pH, temperature, nutrient availability, and bacterial load.

Drs. LaBorde and Doores are collecting data that that will contribute to the development of a simple, economical test kit that can be used by produce growers to submit surface water samples for microbial testing. Levels of indicator microorganisms and pathogens in Pennsylvania surface water sites used for produce irrigation have been determined. Data on chemical and physical factors are also being collected in an effort to determine the extent to which these may serve as indicators of the microbial status of the water sources. The goal is to determine optimal holding conditions to minimize microbial changes and design a suitable package that will allow overnight, or longer, shipping of surface water samples to remote testing laboratories. Preliminary results indicate that 67% of the samples we tested would be over the fecal coliform limit of 200 CFU/100 ml established in the Pennsylvania recreational water standards and that 44% would be above the irrigation water limit required for GlobalGap certification. If samples were evaluated against California leafy greens standards for generic E. coli allowed in irrigation water, 57% would be in violation.

Dr. LaBordes laboratory also has conducted random retail product sampling that has revealed that Listeria monocytogenes and Salmonella spp. can be recovered from whole and sliced fresh mushrooms. Current research is focusing on pre-harvest hurdles that might prevent microbial contamination and growth of human pathogens on mushrooms. Specific objectives are to: compare levels of indigenous microflora found in light peat casing soils and dark peat casing soils; determine the fate of human pathogens inoculated into light and dark peat casing soils while held at commercial conditions; determine the fate of human pathogens in a model mushroom growing system. A laboratory scale growing system that modeled commercial growing protocols was developed to follow microbial changes during a complete growing cycle. Casing soils, essentially lime neutralized peat, makes up the thin top layer of the 2-level mushroom growth substrate. Unlike the heat treated manure based compost below, it is not typically pasteurized. Recent trends in the commercial formulation of casing soil is to supplement light peat with dark peat which is known to have different physical and microbial properties. Casing soils, prepared from one light peat and two dark peat types, were inoculated with Agaricus bisporus (common white mushroom) and with Listeria monocytogenes and Salmonella spp. Pathogen levels were monitored over the course of a mushroom growing cycle. As the fruiting bodies emerged, mushroom samples were taken to determine if soil to mushroom pathogen transfer can occur. Confirming earlier studies, light peat had significantly higher levels of total aerobic bacteria, Actinomycetes, and yeasts and molds. Each of the casing soils had a suppressive effect on L. monocytogenes and Salmonella spp. populations. However pathogen reductions were significantly greater using the light peat compared to either dark peat casing soils. This suggests that microbial competition plays a role in the effect of peat type Combinations of light and dark peat that might be used commercially were added to the model mushroom growing system in ratios of 100:0, 80:20, and 60:40 light:dark. Each was inoculated with L. monocytogenes and Salmonella spp. Pathogen levels decreased by at least 3.18 logs for each peat type between inoculation and harvest. However, pathogen populations were lowest in the 100:0 light:dark peat and were highest in the 60:40 light:dark peat. Average frequency of
pathogen transfer from soil to mushroom was between 45 and 66 percent for Salmonella spp. and between 53 and 56 percent for L. monocytogenes and did not differ significantly based on peat type. Mushrooms were spot-inoculated with pathogens and irrigated with water supplemented with chlorine dioxide, sodium hypochlorite, hydrogen peroxide, and peroxyacetic acid. Log10 CFU/g reductions of both pathogens ranged between 2.5 and 3.9 and however there were no significant differences between the control (water) and the sanitizers. Results of this research have demonstrated that peat casing soils provide a natural microbial hurdle that helps to maintain the safety of mushrooms. Furthermore, although microbial reductions in dark peat soils occur significantly more slower than light peat soils, differences among light:dark ratios do not appear to markedly increase food safety hazards.

Dr. Knabels lab is developing novel molecular subtyping methods for tracking Listeria monocytogenes and Salmonella, as well as understanding the genotypic and phenotypic mechanisms underlying the persistence and transmission of Listeria monocytogenes in food processing plants. Some methods employed include Multi-Virulence-Locus Sequence Typing (MVLST), comK prophage and CRISPRs in Listeria monocytogenes and Salmonella, respectively, to accurately detect outbreak clones of these pathogens. Additionally, Dr. Knabels lab has demonstrated that Listeria monocytogenes regulates its cell density as it transitions to the long-term-survival (LTS) phase, where it forms cocci resistant to heat and high pressure.
University of Nebraska , Jeyam Subbiah

At the University of Nebraska-Lincoln, we have a multi-disciplinary approach integrating microbiological and engineering expertise for addressing food safety issues. To address the safety of meat and egg products, we have developed heat transfer models for chilling of shell eggs in pallets and cooked meat products. The developed models have been validated by conducting chilling experiments in wind tunnel. Pathogen growth models were developed to estimate the growth of pathogens in these food products for varying time-temperature profiles (such as chilling) and validated. Heat transfer models and pathogen models were then integrated to estimate the growth of pathogens during a chilling process or a process deviation such as power breakdown. The developed models have been deployed on the web at http://numodels4safety.unl.edu/. The processors can upload a time-temperature data from a data-logger and can estimate the growth of various pathogens in different food systems.

Currently, we have a USDA-NIFSI project on improving the safety of microwaveable food products. A coupled electromagnetic and heat transfer model has been developed to understand the interaction of microwaves with various food components and how they heat during microwave cooking and standing time. The model will then be integrated with the pathogen destruction model to estimate the lethality of pathogens during microwave cooking.

Texas A&M University,Matt Taylor

Research efforts in the Taylor laboratory focus primary on the utilization and functionalization of food antimicrobials via nano-encapsulation. Research efforts are designed to enhance the usefulness of antimicrobials for bacterial pathogen inhibition on food items, particularly fresh and minimally processed produce (e.g., leafy greens, netted melons, smooth). In addition, research into the antimicrobial mechanism(s) of approved and novel food antimcirobials for the inhibition of microbial organisms are also explored and determined. Research recently completed detected the fermentation of organic acid(s) and an unidentified bacteriocin in a commercially available, FSIS-approved Lactic Acid Bacteria-derived food antimicrobial. One research manuscript has been submitted from this research, with two more manuscripts in preparation. Ongoing funded research (USDA-NIFA NIFSI, AFRI) will seek to develop and validate the utility of nano-encapsulation systems for food antimicrobial delivery to fresh produce commodities pre- and post-harvest.

University of Arkansas, Steven Ricke/Phil Crandall

Consumers purchase organic meats for superior taste, better nutritional value, long-term health benefits, enhanced product freshness and curiosity about the differences between organic and non-organic meats. Many consumers also believe organic poultry is safer than conventional. However, reports comparing conventional to organic poultry have demonstrated that organic poultry may have a higher rate of Salmonella contamination. Organic poultry products may have higher contamination rates of Salmonella because the use of antimicrobials is restricted in both live production and at the plant. This is also true for natural poultry production where antibiotics are not used. In addition, organic and all-natural poultry are characterized by production and processing in smaller facilities. Birds are processed in small, independent facilities in states that permit small-scale exemptions to federal inspection. Small production is usually not integrated, providing less opportunity for control of product quality, including food safety, as in large-scale, integrated production. Salmonella levels for small pasture flock facilities are not known. Therefore, it is absolutely essential to further USDAs goals of reducing Salmonella contamination by developing an integrated approach for natural and organic poultry in both the preharvest and postharvest areas, to fill in critical gaps in determining Salmonella contamination and to develop effective measures to minimize it. Key food safety and Salmonella control points in preharvest must be identified and intervention strategies developed. However, almost no University research has focused on small-scale poultry production systems or their food safety issues. We are comparing natural live production and processing systems and conveying these findings in a series of implementation steps by: 1) Monitoring foodborne pathogen appearance during production and processing 2) Characterizing strains and serotypes of foodborne pathogen isolates. We are collecting environmental samples for both cultural and molecular analysis. These results and the corresponding profiles will provide us with a better idea where foodborne pathogens are occurring and what factors contribute to their prevalence

Louisiana State University Agricultural Center, Marlene Janes

Dr. Janes spoke about determining the consumer safe cooking temperature for blue crabs. Results of the heat treatment experiments were: boil four crabs for 10 minutes and cool five additional minutes for an internal temperature of at least 85° C and a total cooking time of 15 minutes; steam four crabs for 15 minutes and cool five additional minutes to reach an internal temperature of at least 85° C with a total cooking time of 20 minutes. These results will be presented to consumers as easy, concise instructions for safe preparation of Louisiana blue crabs.

The Ohio State University, Jeff LeJeune

Dr. LeJeune summarized several ongoing projects in which he is involved at The Ohio State University. These included the following: Assessment of the association between irrigation water quality and microbial contamination of fresh produce with the indicator organisms E. coli and coliforms; determination of the effects of distillers grain feeding on the colonic microbial ecology of feedlot cattle; the characterization of Shiga toxin-producing E.coli recovered from cattle at slaughter; the role of Clostridium difficile as a potential foodborne pathogen and its epidemiology in feedlot cattle; and the measurement of cold chain interruption of foods during the transport from retail to the home environment. Dr. LeJeune indicated that his laboratory had received recommendation for funding from Foreign Agricultural Service to funding to support a 5 person team (Four of which are S-1033 members) to participate in a scientific exchange program with the Peoples Republic of China. In addition, in collaboration with Dr. Larry Goodridge at CSU (another S-1033 collaboration), their team was also recommended for a special emphasis NIFSI award to study the ecology of antimicrobial resistant organisms in the food supply.

Colorado State University, Lawrence Goodridge

Dr. Goodridge spoke of recent work on water quality and diagnositics: Economically and environmentally sustainable wastewater treatment options are important tools in the reuse of greywater for food crop irrigation. Constructed wetlands (CWs) are an effective, low cost system for the remediation of bacterial contamination in greywater. However, current construction methods for CWs can incur large capital costs, prohibiting the implementation of this technology in water stressed communities. Therefore, alternative methods of CW construction should be investigated. The purpose of this study was to develop and evaluate CWs with low surface area requirements, and low capital construction costs, which would achieve biologically acceptable contaminant removal efficiencies. A total of four 1 m2, portable, recycled vertical flow constructed wetlands (RVFCW) were built for this study. Two RVFCWs were built with recycled, polyethylene terephthalate (PET) plastic as the primary wetland bed media, and two more were constructed with traditional volcanic tuff. The wetlands were dosed with 350 l d-1 of greywater six times during a three month period. Water samples were taken at four different locations within the treatment stream, and analyzed for nine parameters including: total plate count (TPC), fecal coliforms (FC), and total organic carbon (TOC). The RVFCWs achieved 2 log reduction for TPC (p< 0.0001), and 3 log reduction for FC (p < 0.0001), while no significant differences were observed between the RVFCWs constructed with recycled PET and volcanic tuff (p>0.05). In addition, the RVFCWs achieved 51.5% removal of TOC (p < 0.0001), with no statistical differences found between RVFCW types (p>0.05). The results of this study indicate that RVFCWs can achieve appreciable removal efficiencies for TPC, FC, and TOC. Therefore, RVFCWs may be a viable, low cost, minimal technology, polishing step for treating greywater to reuse as irrigation water. In addition, RVFCW construction cost can be drastically reduced by utilizing recycled PET plastic as a primary wetland bed media without compromising treatment efficacy.

Accomplishments

Publications

Impact Statements

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