Research Projects 2008-09
• Unversity of Arkansas
• Iowa State University
• Kansas State University
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| Steven C. Ricke |
During the year of 2008 continuation of the Food Safety Consortium at the federal congressional level was supported at a reduced budgetary level. Prospects for 2009-10 congressional funding appear to be much more promising to sustain the research activities of the Food Safety Consortium in the near future. Because of limited funds in this carryover year, no formal FSC research meeting was held for the fall of 2009. However, the FSC was able to hold a partial meeting of FSC directors and UA researchers in conjunction with the newly formed Arkansas Association for Food Protection – a branch society of the International Association for Food Protection. This meeting brought together speakers from the food industry, commodity groups, universities, and regulatory agencies in a forum titled “Retail Food Safety – A Catalyst for Change.” This meeting was held at Tyson Foods in Springdale, Ark. In the evening a tribute dinner was held in honor of our recently-retired esteemed colleague, Dr. Mike Johnson, the former technical director of the UA Food Safety Consortium. A newly-created food safety graduate scholarship in his name was announced.
The book chapters generated from the papers presented at the October 2006 meeting that were previously written, peer reviewed and accepted for publication in a book are now in the proof stage. Galleys of these manuscripts are being reviewed by the editors prior to final processing and publication by the University of Arkansas Press. It is anticipated that this book of comprehensive reviews will be published as both a hardbound and softbound book in early 2010.
Summarized in the following sections are some of the brief updates on research projects directed toward enhancing and improving the safety of poultry products from “farm to table.” These are excerpts from investigators’ reports based on their pre- and post-harvest research projects funded by multi-year grants, as reported for the fiscal year 2008-09 funding cycle.
Pre-Harvest Projects
Campylobacter: Poultry-derived Campylobacter continues to be a leading cause of human foodborne illness. Young Min Kwon and co-workers studied the genetic diversity of Campylobacter populations within the ceca of individual chickens to better understand the nature of intestinal colonization and diversification of colonizing Campylobacter species. Genotyping was conducted based on the DNA sequence of Short Variable Regions (SVR) in the flaA gene. Cecal samples were collected from 10 market-age broilers and used for isolation of Campylobacter genomic DNA. Sequencing results of SVR fragments obtained from 86 clones (approximately10 clones/bird) showed that on an average 21.9 % of clones had mutational variations within individuals. The mutations revealed consistent patterns, suggesting that the mutations were fairly random. When translated SVR sequences were analyzed, there was on an average 18.5 % of strains carrying altered amino acid sequences in SVR within individuals, and four translated sequences had nonsense mutations to produce truncated proteins. These results suggest that multiple genotypes can colonize in a chicken cecum, suggesting possible adaptive or random genetic diversification during colonization or co-colonization of multiple genotypes.
Kwon and his group also conducted molecular survey of protozoan species biodiversity within the same cecal samples by PCR-amplification and DNA sequencing of 18S ribosomal RNA gene. The majority of the protozoa in the chicken cecal samples were Cryptosporidium species. In the second year of this project, they plan to expand the scope of the research to investigate the diversity of Campylobacter genotypes and protozoan populations in poultry environments using a novel molecular typing method they are developing currently and high-throughput 454 sequencing method, respectively.
Salmonella: Over 50% of foodborne salmonellosis cases are associated with poultry and poultry products. Clearly more intervention steps are needed to limit Salmonella colonization at the pre-harvest stage of production, and vaccines represent an economical approach because they can provide immunization protection in the young chick’s life that will potentially carry over during the entire production cycle. Steven Ricke and co-workers began construction of a Salmonella vaccine that aimed at deleting two genes: the lysA gene and the hilA gene. The purpose of this research was to develop a double deletion mutant Salmonella Typhimurium that is deficient in its ability to synthesize lysine and is defective in its virulence properties.
We completed the transformation of the Salmonella LT2 strain and construction of the cassettes with homology arms in order to create this live attenuated vaccine. The deletion of the lysA gene is expected to turn Salmonella Typhimurium into a lysine auxotroph unable to grow in media lacking exogenous lysine. The hilA gene is a transcriptional activator and regulator of expression multiple genes in response to environmental conditions including pH, osmolarity, oxygen tension and low nutrient concentrations. To delete the genes of interest, homology arms specific to 50bp upstream and 50bp downstream of the target gene were added to an insertion cassette coding for tetracycline resistance. Using electroporation, they inserted a plasmid into Salmonella coding for the enzyme recombinase which will allow the replacement of the hilA or lysA gene with the cassette that was constructed (i.e. homolgous recombination). They have successfully completed the construction of the two cassettes (one specific to delete each gene of interest). In addition, the transformation of Salmonella Typhimurium LT2 with the recombinase encoding plasmid was successfully accomplished. The final step in the construction of the vaccine will be to perform the homologous recombination.
The primary protection against pathogens that the relatively immunocompetent newly hatched chick has is through transmission of antibodies from the mother via the egg. The yolk of immunized chickens is a rich and inexpensive source of polyclonal antibodies and when administered orally can provide passive immunization against infectious diarrheal diseases in a variety of animals. Ricke and co-workers were interested in whether the administration of polyclonal egg yolk antibodies specific for Salmonella spp. may be one possible strategy for preventing the colonization of the bacteria in the gastrointestinal tract. Polyclonal antibodies derived from egg yolks may be particularly useful in clinical applications if the EYA derived from one serovar of Salmonella could cross-react with other serovars. The most frequently reported serotypes in layer flocks in 2002 were S. Enteritidis (57.7%) and S. Typhimurium (9.6%). Ricke and co-workers investigated the cross-reactivity of egg yolk antibodies of S. Enteritidis ATCC 13076 and S. Typhimurium ATCC 13311 to other serovars/strains of these bacteria. EYA that had been produced against purified antigens of SE strain ATCC 13076 and ST strain ATCC 13311 were obtained from Canadian collaborators. The antigens were fimbrae (FIM), flagella (FLA) and lipopolysaccharide (LPS) for S. Enteritidis and FLA, LPS, and outer membrane proteins (OMP) for S. Typhimurium. S. Enteritidis and S. Typhimurium cross-reacted with other serovars of Salmonella and to some extent with other gram-negative bacteria in vitro. The titer of the EYA preparation may be dependent on the strain of bacteria used for immunization.
Likewise, the specificity of the resulting antibodies was dependent on the antigens used in immunization. With judicious choice of targeted antigens, these differences in serological activity could be harnessed for applications in diagnostics and therapeutics. In diagnostic tools (immunological assays, biosensors, etc.) for specific pathogens, high specificity is needed. In this case, anti-fimbrial antibody’s specificity for the homologous serovar could prove useful. In contrast, antibodies that exhibit wide spectrum of activity against multiple pathogenic enterobacteria would be welcome in therapeutic applications. In this study, the anti-OMP and anti-LPS EYA exhibited strong cross-reactivity to 12 different Salmonella serovars/strains and E. coli K-12. Both OMP and LPS are determinants of virulence, and the two antigens are often found as co-contaminants in purified forms. Furthermore, the two antigens synergize the activity of the other in activating macrophages and the production of inflammatory cytokines. A cocktail of OMP and LPS antigens could be used to inoculate laying hens to produce egg yolk antibodies that could protect against pathogenic strains of enterobacteria.
Previous FSC-supported research conducted in Billy Hargis’s laboratory elucidated an effective in vitro screening technique for identification of candidate probiotic organisms and development of a defined Lactobacillus (LAB) culture. Despite their success with the development of the LAB probiotic for poultry commercial use, there is increased need to have commercial probiotics that are shelf-stable, cost-effective and feed-stable (tolerance to heat pelletization process) to increase compliance and widespread utilization. Hargis and co-workers are currently focused on identifying Bacillus probiotic candidates. Bacillus bacterial species are spore formers and as spores can be stored indefinitely on the shelf and be used under extreme heat conditions. Currently, they are collecting environmental samples that are subsequently pasteurized, plated and evaluated for anti-microbial activity using soft agar overlays containing target bacteria. Colonies which produce anti-Salmonella activity are selected for isolation and then evaluated for in vitro anti-clostridial and anti-Campylobacter activity using similar soft agar overlays under appropriate atmospheres. Polyvalent isolates are speciated, and GRAS or non-pathogenic species are further evaluated for resistance to high temperatures (survival of spores in boiling water for 10 minutes with only minimal loss of viability) and for ability to grow to high numbers with high sporulation efficiency (1011 spores per gram or greater).
The results thus far have demonstrated that one Bacillus subtilis spores (BS) isolate was as effective as their previously developed FM-B11TM on S. Enteritidis horizontal transmission in turkey poults. Importantly, since improved efficiency of amplification and sporulation is absolutely essential to gain widespread industry acceptance of a feed-based probiotic for antemortem foodborne pathogen intervention, the ability to grow Bacillus to huge numbers of stable and cost effective spores could have a tremendous impact on broiler health and productivity. Their preliminary studies indicate that screening methods utilized by their laboratory provided effective potential DFM candidates. In particular, isolate NP122 is a candidate DFM for commercial turkeys and may improve growth and reduce Salmonella infection.
Post-Harvest Projects
Campylobacter: Campylobacter is a leading cause of bacterial diarrhea illness in industrialized nations around the world. Most cases of campylobacteriosis can be linked to careless handling and/or consuming raw or undercooked poultry and poultry products. Increasing prevalence of antibiotic resistant bacteria is a growing predicament. Effective Sept. 12, 2005, the FDA withdrew approval for the use of fluoroquinolones in poultry production partly due to multiple studies finding links between antibiotic resistance of Campylobacter and fluoroquinolone use in live poultry. In recent years, there has been a dramatic increase in the popularity of organic poultry partly due to the fact that consumers are attracted to poultry raised without antibiotics. However, little published data is available on the microbiological quality of these organic poultry products.
In addition, even less data is available characterizing the antibiotic resistance in pathogens isolated from these organic poultry products. Michael Johnson, Ricke and co-workers collected samples from two farms (N=178; feed, water, drag swabs and insect traps), retail carcasses (N=48) and one processing plant (N=16). A total of 105 Campylobacter isolates were obtained from 53 (30%), 36 (75%) and 16 (100%) of the samples from the farms, retail carcasses and processing plant, respectively. Of the 106 isolates collected, 65 were C. jejuni, 31 were C. coli and 9 were Campylobacter spp. The isolates were assessed for antibiotic resistance to five antibiotics (ciprofloxacin, erythromycin, nalidixic acid, tetracycline and clindamycin) using a disc diffusion assay. In addition, they used flaA SVR typing to determine the genetic diversity of the isolates. None of the isolates were resistant to ciprofloxacin, erythromycin, tetracycline or clindamycin. Only one isolate that was obtained from a processing plant had resistance to nalidixic acid. The results of this survey indicate that the prevalence of Campylobacter on organic retail carcasses is similar to data reporting Campylobacter on conventional retail carcasses. However, there was a large difference in antibiotic resistance when comparing isolates obtained from organic poultry with isolates obtained from conventional poultry.
One of the highest priority research needs for Campylobacter is to develop practical low cost laboratory methods for quantifying total C. jejuni loads persisting on raw poultry products in order to develop meaningful baseline data for this pathogen to aid in risk assessment and to evaluate the efficacy of intervention and control strategies. Conventional methods for Campylobacter detection in poultry or other foods involve lengthy selective cultural enrichment. Another complication is that traditional confirmative tests based on hippurate hydrolysis test have been recently brought into question with the isolation of C. jejuni strains incapable of hydrolyzing hippurate. Also, the need for user-friendly methods for generating quantitative data – not just presence/absence tests – from raw poultry for HACCP and USDA/FSIS regulatory purposes is another serious concern, pointing out the desirability to develop enumeration methods. New immunological and molecular probes are needed to determine the mechanisms of colonization, pathogenesis and importance of strain variation of C. jejuni. Hence, there is an urgent need for the development of highly specific (immunological or molecular) probes capable of distinguishing/quantifying the C. jejuni load from that of other Campylobacter that may be present.
Johnson’s group is focused on developing efficient MAb probes for C .jejuni cells in various physiological states for devising quantitative monitoring technologies for C. jejuni in poultry products. Once developed, these monoclonal antibodies will be useful in improved detection and quantitative methods for C. jejuni and other Campylobacter on raw poultry and other foods. They have developed several C. jejuni monoclonal antibodies producing cells known as Campy-hybridoma cells. The monoclonal antibodies produced by these Campy-hybridoma cells will exhibit different reactivity patterns to Campylobacter and C. jejuni antigens. These MAbs are classified into two distinct groups based on current evaluation: (1) MAbs specific for C. jejuni without reacting with C. coli or other Campylobacter antigens; (2) MAbs reacting with all C. jejuni and other Campylobacter species. In this step, they have grown the Campy-hybridoma cells and evaluated the ability to produce MAbs by ELISA. They have also characterized complete specificity and sensitivity of these new MAbs against a wide range of C. jejuni and other Campylobacter species and against other gram-negative and gram+positive bacteria commonly encountered in chicken carcass rinses. Once developed, these monoclonal antibodies will be useful in improved detection and quantitative methods for C. jejuni and other Campylobacter on raw poultry and other foods.
Listeria: Studying L. monocytogenes gene regulation under conditions of energy/nutrient availability can reveal insights into how L. monocytogenes is able to persist through the food chain as well as survive passage through the intestinal tract and cause disease. Johnson’s group has continued to focus on mechanisms of energy metabolism of L. monocytogenes using genetic methodologies. Their experimental approach for this phase of the project was designed to reveal how exposure to specific metabolic inhibitors effects L. monocytogenes gene regulation. Competitive hybridizations were performed by comparing RNA from L. monocytogenes cells exposed to a given metabolic inhibitor with RNA from unexposed L. monocytogenes cells. The two metabolic inhibitors used were 2,4-dinitrophenol (DNP) a proton motive force (PMF) dissipater and sodium arsenite (SAs), an inhibitor of pyruvate dehydrogenase. The potential impacts of DNP’s disruption of the PMF include reduced: ATP synthesis, flagellar rotation and ion transport. Impacts of pyruvate dehydrogenase inhibition by SAs include reduced: energy production (ATP/NADH synthesis), fatty acid biosynthesis, and production of adenine, valine and leucine. In addition, the contribution of the alternative sigma factor associated with stress response, sB, to gene regulation in exposed cells was inferred (sB-dependent gene transcription was defined as a fold change of zero in the absence of sB).
New insights from their continued data analysis reveal that while the same level of growth inhibition was observed following exposure to DNP and SAs, fewer genes were influenced by DNP (555 vs. 148). A comparison of the effects of the two metabolic inhibitors showed somewhat surprisingly that most of the major categories affected in both are the same (hypothetical, energy metabolism, protein synthesis and unclassified). However, most of the genes in common between the two treatments are not regulated similarly (i.e. gene X would be upregulated in response to SAs but downregulated in response to DNP). No pattern was identified among the sB-dependent genes regulated in common.
Remaining questions to be addressed include: what does it suggest about the genes that are sB-dependent in both treatments but are regulated differentially; why is sB-dependent transcription much greater following exposure to SAs than to DNP (87% vs 41%); what does this imply about the role of sB in L. monocytogenes; how can it be explained that the SAs treatment affects more genes than DNP given that DNP probably effects energy acquisition while SAs effects nutrient metabolism; and lastly, what do these results say about L. monocytogenes energy acquisition and nutrient metabolism?
Multiple hurdle approaches such as combining different antimicrobials to limit Listeria and other pathogens during processing have become popular as better strategies to reduce overall levels and limit survivors. Navam Hettiarachchy and co-workers examined low fat chicken (2.56% fat) hot dogs formulated with and without potassium lactate (4.8%) and sodium diacetate (0.25%) alone and in combination to determine their antimicrobial properties against Listeria monocytogenes (V7 serotype ½ a) when inoculated at 104 cfu/g level. The combination of lactate and diacetate at their maximum permissible levels demonstrated higher antimicrobial effects than alone. Potassium lactate and sodium diacetate at their maximum permissible levels demonstrated antimicrobial activity by 0.3-0.5 log cfu/g reduction within the first four weeks of storage. The combined effect of both antimicrobials had maximum inhibitory effect on the growth of Listeria monocytogenes at 60 days of storage at 4 C by 0.7 log cfu/g reduction. The control sample was completely spoiled with odor and loss of tissue integrity by 45 days of inoculation. The findings of their investigation indicated that lactates and diacetates when added in a formulation have higher combined effects than alone. Furthermore, lactates and diacetates demonstrated maximum antimicrobial effects on the 60th day of storage (0.7 log cfu/g reduction).
Salmonella: Hetteriarachchy and her group examined 9 different phenolics (epicatechin, catechin, caffeic, protocatechuic, gentisic, benzoic, vanillic, syringic and gallic acids) that are naturally present in plant foods for their effects on L. monocytogenes, S. Typhimurium and E. coli O157:H7 model systems. Minimum inhibitory concentrations of individual phenolics were determined using a 96-well plate technique. The wells were inoculated with 100 μl of S. Typhimurium/ E. coli O157:H7/L. monocytogenes (5.0 log CFU/ml) and incubated at 37°C. Gentistic, benzoic and vanillic acids were inhibitory to S. Typhimurium, E.coli O157:H7 and L. monocytogenes at 5000 μg/ml (reduction of 2.7 log CFU/ml of S. Typhimurium and E. coli O157:H7 each and 3 log CFU/ml of L. monocytogenes). These phenolic compounds have potential to act as potential antimicrobials for reduction of Salmonella, E.coli O157:H7 and L. monocytogenes in foods. Their future studies will be aimed at improving the delivery of these phenolics to enhance their antimicrobial activities.
Conclusions
The food safety research conducted by FSC researchers at the University of Arkansas reflect a combination of mechanistic understanding followed by developing and applying technologies that will help limit initial pathogen establishment in live poultry and inhibit their proliferation in post-harvest settings. As the mechanisms that these pathogens use to overcome the adversity of food production and processing environments are better understood more effective strategies to limit foodborne pathogen contamination in food production can become a reality.
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| James S. Dickson |
The Iowa State University component of the Food Safety Consortium funded four research projects and two diversity graduate assistantships in 2008-2009. Research scientists representing several academic departments at the university, as well as USDA-ARS and HHS-FDA, were involved. The primary basis for research under this research program has been the enhancement of the safety of pork and pork products. The research projects encompassed many aspects of food safety as it is currently viewed, from the farm to the consumer. Since the Food Safety Consortium was not funded by special grants during this time period, the funding to continue the program was made available from Hatch funds and other administrative funds within Iowa State University.
Diversity Graduate Assistantships: Recognizing the need to enhance diversity in the field of food safety, the Iowa State University portion of the Food Safety Consortium funded two diversity assistantships in 2008-2009. This support was half of the typical assistantship, with the major professor supplying the matching funds and tuition funding. The two students receiving support were Ph.D. students, one working in the area of risk assessment and the other in the microbiological safety of processed pork products.
Research Grants
Preharvest Interventions
The Iowa State University component of the Food Safety Consortium funded one research project relating to Antimicrobial Resistance of Campylobacter in Swine.
Dr.’s Zhang and Wesley (USDA-ARS, NADC) evaluated the antimicrobial resistance of Campylobacter found in live pigs. In the second year of a previously funded project, they determined the contribution of various efflux pumps to antibiotic resistance in Campylobacter jejuni and Campylobacter coli. By constructing gene-specific mutants, they found that the multidrug efflux pump CmeABC plays an important role in antibiotic resistance in C. coli. Finally they determined the dynamic changes in the expression of the efflux genes under stepwise selection with macrolide antibiotics. All three efflux genes including CmeABC, Cj1375, and Cj1687 were upregulated in the mutants selected with erythromycin or tylosin. The upregulation was especially obvious in the intermediate mutants. Together, these results demonstrate that efflux transporters are important for antibiotic resistance and overexpression of these transporters is involved in the development of Campylobacter mutants that are highly resistant to macrolides.
Post-Harvest Interventions
The Food Safety Consortium funded two post-harvest research projects, one on decontamination and the other on the safety of naturally cured pork products. Ready-to-eat (RTE) cooked meat products such as hams, sausages and rolls have been the subject of large product recalls and multistate outbreaks linked to Listeria monocytogenes in recent years. Several post-cooking treatments, other than irradiation, have been tested to control L. monocytogenes in RTE meat products, but only with limited successes. Irradiation has shown to be very effective in eliminating L. monocytogenes in RTE meats. However, irradiation causes quality changes in raw and cooked meat products. The major quality changes in irradiated meat include off-odor production, color changes, flavor and taste changes, which can influence consumer acceptance significantly. Among the quality parameters, aroma and color are important that determine consumer acceptance of raw meat, while taste and flavor determine the acceptance of cooked meat. Studies to characterize off-flavor/taste compounds, to elucidate the mechanisms that produce such changes, and to develop methods to prevent quality changes in irradiated RTE cooked meat products is necessary because consumers would not buy irradiated products again when they found strange taste or flavor in their products no matter how safe the product is. The objectives of this research were to characterize taste/flavor changes in cooked ham by irradiation, and to identify and quantify off-flavor/taste compounds in irradiated cooked ham.
The rapid growth in consumer demand for natural and organic foods has resulted in a proliferation of processed pork products that are marketed as natural and
organic. However, in the case of cured pork products like hams, bacon and frankfurters, the traditional curing agents, nitrite and nitrate, are not permitted in natural and organic products because the regulations for natural and organic products do not permit the use of “preservatives.” Because these products cannot be produced with typical cured meat properties without nitrite or nitrate, the industry has developed a new approach that utilizes vegetable juice powder as a natural source of nitrate which is then used to provide the typical cure.
However, this approach results in significantly less nitrite in the products. It has
been suggested that these products represent a greater risk of bacterial pathogens and will require additional antimicrobial measures to provide consumers with the degree of safety that is expected of these products. Therefore this project was proposed to determine the relative risk of bacterial pathogens in natural and organic “cured” pork products and to suggest means of assuring safety of these products. This study first involved collection of commercially available natural and organic hams, bacons and frankfurters to compare the chemical properties of these products to conventionally cured commercial products of the same type. Traditionally cured commercial products were included as controls for the comparison. All of the products were first analyzed for nitrite, cured pigment content, salt concentration and available water. After chemical analyses were complete, another set of samples of each of the commercial products were collected and each was separately inoculated with bacterial pathogens (Listeria monocytogenes and Clostridium perfringens)to assess the potential risk of pathogen growth. Following the inoculations, additional antimicrobial treatments that might be used to provide for improved safety were investigated and evaluated for effectiveness. The results showed that the most significant chemical difference between the natural and organic products and the controls was for residual nitrite with the controls containing a higher concentration in most cases, as expected. The inoculation challenge resulted in faster growth by both pathogens on almost all of the natural and organic products, confirming our hypothesis that these products represent a greater safety risk. A screening of a variety of natural antimicrobial ingredients suggested two commercially available products with significant potential for safety improvement. Therefore, use of natural blends of acetic acid (vinegar) and lactate or acetic acid (vinegar) and citric acid (lemon powder) offer the meat industry a means by which the safety of natural and organic processed pork products can be improved to a level that appears to be equivalent to that of conventionally cured products. Subsequent experiments will assess effectiveness of additional natural antimicrobial ingredients to offer the meat industry more options for meeting the safety needs of these products.
Consumer Information
It is imperative that the information developed in the research laboratory be transferred to the general public, in a format which is accessible to them. The Consortium continues to help fund the food safety Web page designed and maintained under the direction of Dr. Catherine Strohbehn. The Food Safety Consortium consumer Web site project continues to receive a significant number of visits and be recognized for its work. The Web site developed as part of the Food Safety Project at Iowa State University (http://www.extension.iastate.edu/foodsafety and http://www.iowafoodsafety.org) is one of the leading food safety web sites in the world with about two-thirds of its visitors from the United States. More than 3 million page views were recorded this past year from close to 9 million hits (average hits per day of 23,461). Frequently visited pages included Food Safety News, SafeFood© Lessons, and the Spanish version of Guide to Food Safety for Retail Operations.
The Food Safety Consortium and USDA have enabled the Food Safety Project team to reach millions of consumers with up-to-date food safety information. Providing consumers, foodservice workers, educators and other groups with access to reliable, timely, unbiased food safety information via the World Wide Web is one part of the process to increase knowledge with the goal of improving safe food handling behaviors.
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| Curtis Kastner |
Kansas State University continues to focus on hazard detection and hazard control strategies as mandated by Congress. However, other efforts relating to policy, trade, and education are also summarized. The Consortium continues to leverage USDA funding 2 to 1 with non-federal support. That leveraging significantly enhances research outputs relative to the following projects.
Hazard Detection
Research to speed the Fung Double Tube (FDT) system from overnight (10 hours) incubation of sample tubes to four hours after sampling to obtain real time viable colony counts of Clostridium perfringens in meat and other foods was conducted. After only four hours small black fuzzy growth can be seen and in five hours distinct colonies can be counted making the FDT the fastest possible viable cell count method to date.
There are many different environmental sampling methods that are currently used in the industry. They include swab, sponge, flocked swab, direct agar contact, and M-Vac. Several studies have been conducted to determine the benefits and drawbacks of each method. Viable cell count results show that the sponge sampling method, in general, recovered the highest number of microorganisms. The swab was normally shown to recover the least number of microorganisms. This information can be used to insure appropriate sampling strategies that will maximize the ability to detect pathogens.
Proper microbiological examination of foods involves proper sample preparation in terms of mixing the solid or liquid food with a suitable sterile diluent (usually a 1:10 dilution) in a sterile bag and homogenizing them manually or by means of an instrument. The effectiveness of Stomacher®, Pulsifier®, Bagmixer®, and Smasher® instruments in terms of: 1) number of viable cell counts/g of ten food types, 2) noise level of the four instruments ascertained by a) human and b) decibel meter at five feet (1.52 meters) from each instrument, 3) ease in cleaning the instruments after used, and 4) ergonomics was evaluated. All four instruments had similar performance with regards to viable cell counts. However, in regards to noise level, the Smasher® and the Bagmixer® were quietest compared to the Stomacher® and then the Pulsifier®. The Smasher® was also the instrument with the highest ranking in ease of cleaning and ergonomics. This research provides scientists with information to make equipment selection decisions to maximize the efforts of hazard detection.
ECA Check® Easygel® Plus (ECA) is a pectin-base gelling system that reacts with calcium ions bound to a pre-treated Petri dish, eliminating autoclaving prior to use. It can chromogenically and/or fluorogenically distinguish three organisms: Escherichia coli, Salmonella spp., and coliforms. This study compared the recovery of these organisms to conventional media using stock culture, inoculated, and non-inoculated ground beef and ground turkey. ECA Check® Easygel® was efficient, less labor-intensive, comparable to, and, in some instances, better than conventional media at recovering target organisms. Again, this information can be used to insure appropriate sampling and culturing strategies.
The Food and Drug Administration’s Bacteriological Analytical Manual recommends two methods of enumeration for Bacillus spp.: 1) standard plating method using mannitol egg-yolk polymyxin (MYP) agar and 2) most probable number (MPN) method with tryptic soy broth supplemented with 0.1% polymyxin sulfate. The research compared the MYP and MPN method for detection and enumeration of B. cereus in raw milk, high-temperature-short-time pasteurized skim, 2%, and whole milk stored at 4 degrees C for 94 hours. Even though the MPN method gave more B cereus than the MYP method, the MYP method should be used by industry for enumeration of B. cereus due to its ease of use and rapid turnover time (two days compared to five days with MPN). However, the MPN method should be used for valuation research due to its greater population recovered.
Hazard Control
Floor drains in processing environments harbor Listeria spp. due to the continuous presence of humidity and organic substrates. The cleaning and washing activities undertaken in the processing facilities may translocate the bacterial cells from the drain into the surrounding environment thus contaminating food products being produced. The potential for translocation of Listeria monocytogenes from drains to food contact surfaces in the surrounding environment using Listeria innocua as a surrogate was evaluated. Results show that L. monocytogenes may translocate from drains to food contact surfaces via aerosols generated due to cleaning and washing, thus contaminating food products. This research provides information that can be used to reduce contamination during processing and ultimately the incidence of foodborne disease.
Heterocyclic amines (HCAs) are mutagenic and suspected carcinogenic compounds that are produced in protein-rich muscle foods that have been barbecued, grilled, broiled or fried. Epidemiologic studies suggest that dietary intake of HCAs through meat consumption increases the risk factor for colo-rectal, stomach, lung, pancreas, mammary, and prostate cancers in humans. Although total avoidance of exposure to HCAs is difficult, reducing the exposure levels is possible. Adding antioxidants from natural sources such as rosemary have been very effective in inhibiting HCA formation.
Our research has shown that some commercial rosemary extracts can inhibit the formation of HCAs in cooked beef patties in the range of 61-79%. Further research using antioxidant containing spices is important in order to elucidate additional and potentially even more effective strategies for HCA inhibition. One such study was to investigate the influence of spices on the inhibition of HCA formation in cooked beef patties. The spices, namely rosemary, turmeric, and fingerroot contain high phenolic contents; thus are strong free radical scavengers. The addition of these spices in ground beef showed significantly reduced HCA levels. Thus spices containing natural antioxidants could be easily used to provide safer meat products.
The metabolism of 2-dodecylcyclobutanone (2-DCB, a compound formed during irradiation) by rat liver S9 mitochondrial enzymes and to identify possible end metabolites was evaluated. The results showed that 2-DCB was metabolized by rat liver microsomes and that the metabolism was energy and protein dependent. The results also indicated that 2-DCB was reduced by NADP (H) dependent reductase to an alcohol, which is not considered a toxic product. These results further support the position of FDA and WHO regarding the safety of irradiated foods.
Consumption of frozen, pre-browned (but raw), single-serving stuffed chicken products prepared in the home has recently led to several confirmed Salmonella cases. Products appear fully cooked and package labels do not always effectively inform consumers of proper preparation procedures. The USDA-FSIS has advised processors to validate cooking instructions and modify package labels to state that a minimum 165 degrees F internal temperature must be achieved. In an affiliated study, consumers were observed preparing representative products according to label instructions, with frequent variations in microwaving procedures being witnessed.
Microwave cooking of frozen, raw breaded poultry products is unpredictable in achieving uniform target end-point temperatures; however, ovens with wattages greater than or equal to 1,000 provide substantially reduced pathogen survival risks if validated cooking instructions are followed.
High Hydrostatic Pressure (HPP) processing has a tremendous potential for controlling and eliminating pathogenic microorganisms in ground beef and beef products. The use of high pressure processing (HPP) at 600 MPa for 3 min to destroy E. coli O157:H7 and Salmonella spp. in coarse ground beef was evaluated. Greater than a 5 log reduction of natural and antibiotic resistant E. coli and Salmonella spp. by a high pressure treatment of 600 MPa for 3 min was observed. High pressure treatments may be an applicable alternative to ensuring the safety of ground beef.
To validate how packaging and storage reduces Listeria monocytogenes (Lm) on whole muscle beef jerky, shelf stable meat snacks, and smoked sausage sticks, four packaging systems, including heat sealed (HS), heat sealed with oxygen scavenger (HSOS), nitrogen flushed with oxygen scavenger (NFOS), and vacuum (VAC), and four ambient temperature storage times were evaluated. Processors could use packaging in HSOS or VAC in conjunction with a 24-48 hours holding time as an antimicrobial process to ensure a greater than 1 log CFU/cm2 Lm reduction. Since growth of L.m. was inhibited as a result of packaging and storage, processors may be able to use selected packaging and storage combinations as an antimicrobial process.
The antimicrobial effectiveness of persimmon puree and phenolic compounds commonly found in the persimmon and plum was evaluated. The objectives were to evaluate the antimicrobial effects of persimmon puree on Bioball™ Listeria monocytogenes and Escherichia coli 0157. Persimmon puree appears to be an effective antimicrobial agent against Gram-positive bacteria in a liquid medium. However, incorporation of persimmon puree into ground beef did not yield an antimicrobial effect.
Policy and Trade
An outbreak of H5N1, or merely its detection within the country, would have major implications for the U.S poultry industry. The economic impact would have three major components – loss of export markets, costs associated with flock destruction and carcass disposal, and reduction in domestic demand. The cost associated with loss of export markets would depend on the length of time markets remained closed. That time frame is hard to predict but could range from a few months up to several years as has been the case with beef exports following the discovery of mad cow disease in 2003.
The border is important as a physical space, but the best way to secure the border is to focus on countries’ regulatory systems and harmonize them. Research has found that targeting specific geographical regions within nations is one way of streamlining trade between nations where food safety issues might otherwise cause problems.
Education
Credit courses in food science and food safety have been developed and continue to be updated for distance education. These courses integrate interactive components to create an experimental learning environment similar to on-campus courses. Many of these courses have been captured with an enhanced media system while faculty are teaching students on campus. Student evaluations of the courses have been positive and supportive for the future creation of additional interactive components and courses as part of a food safety and security curriculum. The courses integrate results from the Consortium along with research from other sources. This helps meet the information and technology transfer mandate for the Consortium.