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