Brief Summary of Minutes of Annual Meeting

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

The attached publication list includes funded grants and projects