Brief Summary of Minutes of Annual Meeting

Minutes of November 12-13, 2009 meeting attached

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Objective 2: Develop and validate mathematical modeling to gain understanding of pathogen behavior in macro and micro-environments. 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. 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. Objective 3: Investigate factors leading to the emergence, persistence and elimination of antimicrobial resistance in food processing and animal production environments 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.

The attached publication list includes funded grants and projects