The Food Research Institute (FRI) hosted its annual meeting on May 16–17, 2001 in Madison, WI with over 120 scientists from university, industry, and government in attendance. Updates on various food safety issues were presented. Special sessions addressed: (a) Lessons learned from dealing with high-profile issues, (b) Mycotoxins, and (c) Antibiotic resistance. A poster session highlighted results of recent research projects. A brief summary of the meeting is presented here. Further information can be obtained from our web site or by contacting FRI.

Risk Perception and Risk Assessment — Lessons Learned
Novel and potentially controversial research and technological developments must be conducted with an eye on ultimate consumers, global markets and politics, as well as with close attention to the scientific evaluation of the product's performance and safety. A foundation of impeccable science is a necessary basis for any innovations. But this is not sufficient to ensure acceptance of new products or processes.

A farm demonstration project involving growth and sale of genetically modified (GM) and conventional corn and potatoes, undertaken with intensive local publicity, demonstrated that consumers were glad to have information available. Many were willing to try the GM vegetables, and the GM corn outsold conventional corn by 3 to 2. Providing such information prevented development of a risk communication vacuum and promoted better understanding of the issues by consumers.

BSE (bovine spongiform encephalopathy) in the UK was originally a livestock problem but became a food safety issue as more than 100 human cases of vCJD (variant Creutzfeldt Jakob disease), a related disease, were diagnosed. The prions responsible for this type of disease are usually species specific. But this is not true for BSE. Boiling and autoclaving only partially destroy infectivity of prions so the safety of bovine tissues is in question.

Presence of Listeria monocytogenes in ready-to-eat foods has prompted the FDA/FSIS to develop a risk assessment model. Data on amounts of foods consumed, history of different foods as vehicles for listeriosis, frequency of contamination of foods, numbers of bacteria in the food, and estimates of microbial growth during refrigerated storage were put together to generate predicted relative risks for contracting listeriosis from consuming different foods.

Antimicrobial Resistance
Antibiotic resistance genes in bacteria may be spontaneous mutations or may be acquired from other bacteria. The complete genomes of E. coli K-12 (a harmless laboratory strain) and of the infamous strain E. coli O157:H7 have recently been sequenced. A comparison of the two sequences revealed nearly 25% of the O157:H7 genome was not present in K-12. Many of the novel genetic elements in strain O157:H7 appear to have been acquired from bacterial viruses, and others were apparently transferred from other gram negative enteric bacteria and/or from unrelated gram positive bacteria. These data demonstrate the capacity of bacteria to acquire new virulence or antibiotic resistance genes.

High levels of antibiotic use in human medicine are believed to have caused the development of antibiotic resistant bacteria. The widespread incorporation of subtherapeutic antimicrobial agents in animal feed to improve feed efficiency and stimulate growth has also increased antibiotics levels in the environment. Antibiotic-resistant foodborne pathogens have been isolated from farm animals and meat, and there are reports that these bacteria have caused human illness. Cross resistance can occur with structurally similar antibiotics, such as synercid and virginiamycin. Up to 60% of Enterococcusfaecium isolated from chickens were synercid-resistant because of the use of virginiamycin in chicken husbandry. These resistance genes may be passed to Salmonella or Campylobacter, and thereby compromise treatment of serious human infections.

Surveys of chicken carcasses have shown that as many as 80% may be contaminated with Campylobacter. Quinoline-resistant human isolates of C. jejuni in Minnesota increased from 1.3% in 1992 to 17.3% in 1999. Many cases were acquired abroad — particularly in Mexico. Domestically acquired quinoline-resistant C. jejuni infections also increased, and in 1999–2000, 14% of C. jejuni isolated from chickens were quinoline-resistant. (Fluoroquinoline use in poultry began in 1995.) Molecular subtyping indicated that some quinoline-resistant C. jejuni from chickens were identical to some isolates from human infections.

For many Europeans the issue of antibiotics in feeds is linked not only to bacterial resistance but also to food safety and to the industrialization of food animal production. In 1986, Sweden banned the practice of adding subtherapeutic levels of antibiotics to animal feed as growth promoters, and other European countries have prohibited the use of many antimicrobial feed additives. A holistic approach to food animal production affects European politics and trade proposals to the World Trade Organization. It is likely that these developments will impact the use of such feed additives in the USA.

Mycotoxins and Food Safety
Fumonisins are very toxic to some animals and have been classified as a class 2b carcinogen for humans (associated with liver and esophageal cancer). Based on reported toxic effects, regulatory guidelines for fumonisins in feeds were developed with the recommended maximum level of 1 ppm in feed for the most sensitive animals, and 30–50 ppm for less sensitive animals

Recent research on biosynthesis of fumonisins and trichothecene mycotoxins indicates that both are produced by clusters of 10 or more genes. Fumonisin synthesis is not well understood. Trichothecene synthesis is required for disease in wheat, and three mutant genes which confer trichothecene resistance have been identified. Experiments are under way to transfer these genes into tobacco plants to determine whether this could be an effective way of producing trichothecene-resistant crops.

Inhibition of mycotoxin synthesis is the goal of research on proteins involved in the relay of environmental signals through the cytoplasm to the nucleus where toxin-producing genes are turned on. Some proteins inhibit sporulation and production of aflatoxins, but may increase synthesis of other mycotoxins. Other proteins are required for aflatoxin production. Some polyunsaturated fatty acids in seeds stimulate and others inhibit asexual spore production and mycotoxin synthesis.

Recent Research on Foodborne Pathogens
Viruses caused nearly 20% of outbreaks of foodborne illness with known etiology in Wisconsin during the past 10 years. Improved virus identification methods and epidemiological studies demonstrated that viral foodborne illness can often be traced to an infected food handler. This was the case with two separate outbreaks of Norwalk virus in 2000: in both, the most probable vehicle was a pasta salad and ill food workers prepared the salads.

Using tools of chemical reaction engineering and computer modeling, the interplay of many molecular processes coded by the genome of a simple virus has been simulated. Based on rates of enzymatic reactions occurring during viral replication, one can simulate growth of a virus under different conditions and test the effects of different antiviral strategies. This research on viruses in silico (on computers) is expected to improve understanding of how to design new drugs to inhibit them.

Several global regulators affect expression of the stress tolerance genes that code for proteins enabling survival at high temperatures, low pH, and other limiting conditions. Two such regulators in E. coli O157:H7 are the leucine responsive regulatory protein, which binds to DNA in the presence of leucine, and the histone-like nucleoid-structuring protein, which increases acid resistance in log phase. By understanding the regulation of stress-tolerance genes in E. coli O157:H7 it may be possible to devise methods of controlling this bacterium in cattle, or to enhance the effectiveness of decontamination processes during slaughter and processing.

Bacillus cereus causes both diarrhetic and emetic types of foodborne illness and also a very destructive eye infection (endophthalmitis). Recent research has identified some toxins involved in these illnesses. Hemolysin BL, a pore-forming diarrheal toxin, is highly toxic to the retina and is likely to be one virulence factor in endophthalmitis. Data also implicate a collagenase enzyme and phosphatidylcholine phospholipase C as other virulence factors. Effective treatments may require direct neutralization of the toxins.

Causes of autism are unknown but some parents report that children developed symptoms after receiving antibiotic therapy accompanied by diarrhea. This suggests the possibility of an intestinal clostridial infection with neurotoxin production (analogous to development of infant botulism). Treatment of 10 autistic children for 8 weeks with vancomycin (effective against clostridia) caused significant improvements in 8 of them. Fecal cultures from affected children indicate that the species most likely to be involved is C. perfringens. Further studies utilizing the "Sugiyama" mouse model are underway to determine effects in laboratory animals.

Biofilms formed by pathogenic and spoilage bacteria are a common problem in food-processing environments. Cold plasmas, such as glow discharges, contain highly reactive species which can be deposited on stainless steel surfaces to form an antifouling layer. Efficient decontamination and disinfection of water was also carried out using dense medium plasma (DMP) conditions.

Outreach and Education
Education is an important function of FRI. Collaboration of FRI with the Environmental Toxicology Center (ETC) has facilitated exchange of scientific information and training of the next generation of specialists. Other departments on campus also participate in ETC courses and research on health related toxicology and ecotoxicology (which includes study of safety of genetically modified organisms).

Outreach to government, industry and the public is another essential component of the FRI mission. This takes many forms including direct interactions, meetings, short courses and our web site with links to current food safety research. Scientific literature reviews such as one evaluating the safety of microbial enzymes used in food processing are also published.

• Molecular Genetic Regulation of Mycotoxin Biosynthesis
• Seed Lipids Affect Aspergillus Growth and Mycotoxin Biosynthesis
• A Mechanical Antibacterial Approach
• Combined Fluorescence In Situ Hybridization and Tetrazolium-based Viability Estimation of Salmonella
• Plasma and Biofilms

  ---

We are using the genetically accessible ST producing fungus A. nidulans as an experimental model to clarify the molecular genetics of AF production in A. flavus and A. parasiticus. Our current focus is in understanding how AF and ST production is correlated with asexual sporulation in these Aspergillus spp. For example, we have described the role of a G  protein-dependent signaling pathway in simultaneously inactivating sporulation and mycotoxin production in A. nidulans, A. flavus and A. parasiticus. We have recently found that this signal is partially transmitted through protein kinase A and that this transduction pathway is conserved in other fungi including the trichothecene-producing Fusarium spp. Interestingly, the trichothecene mycotoxins are up regulated by the same G  protein that down regulates AF and ST production. This clearly demonstrates that advances made in understanding AF biosynthesis in A. nidulans are applicable not only to the agronomically important aflatoxin-producing fungi, A. flavus and A. parasiticus, but to other mycotoxic genera.

We have determined that 13S hydroperoxylinoleic acid (13S-HPODE), the product of soybean seed lipoxygenase 1, suppresses AF/ST production and AF/ST mRNA expression. Conversely, 9S hydroperoxylinoleic acid (9S-HPODE), the product of a corn embryo lipoxygenase, extended the time AF/ST mRNA was expressed. These findings are significant since linoleic acid is a major component of the seed attacked by Aspergillus spp. and lipoxygenase enzymes are natural constituents of seeds which we have shown to be activated by Aspergillus infections. This suggests that these enzymes could play an important role affecting Aspergillus colonization of seed. Current studies include characterizing Aspergillus genes required for psi formation and to determine how the fatty acids signal sporulation and AF/ST production in Aspergillus. We also are characterizing the seed lipoxygenase genes involved in the seed/Aspergillus interaction.

The researchers hypothesized that the mechanism by which the coating killed bacteria was by a direct disruption of the cell membranes and cell wall, which in turn resulted in leakage or autolysis. If the mechanism is correct, one could speculate that eventual bacterial resistance to the coating may be lost. The potential use of the coating on a wide variety of surfaces, including those in contact with food, could be compelling. Additional work on the ability of the coatings to prevent biofilm formation could add another useful product in reducing foodborne pathogens.

We have successfully combined our rapid FISH assay with use of the respiratory substrate 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a red fluorescent viability indicator. This has enabled the simultaneous and quantitative detection and viability assessment of Salmonella in mixed populations by flow cytometry. Ultimately, we wish to examine the application of this combined protocol in foods. The ability to correlate target organisms at the single-cell level with their viability status would provide greater information on which to base important food safety decisions.

Cold-plasma-aided manufacturing has found many applications, including surface modification of materials to produce desired properties. Surface modification by plasma can be achieved in many ways, including etching, introduction of functional groups, and deposition of thin-layer macromolecular layers. Numerous polymers, such as those used in food packaging, have been treated with plasma for various purposes, including enhancement of wettability, adhesion, printability, improvement of barrier properties and increase in mechanical resistance and shear strength.

One of the research areas we have focused on includes developing anti-bacterial layers on surfaces encountered in food processing. We have employed cold plasma to deposit coatings containing polyethylene glycol (PEG)-like structures onto stainless steel, which resulted in inhibition of attachment and biofilm formation by foodborne pathogens such as Listeria monocytogenes. This can be extended to other types of materials such as rubber, polyester, and polyurethane that are used in gaskets, conveyors, etc. Another potential application is the use of plasma to incorporate colloidal silver into surfaces to inhibit bacterial deposition. In addition, we are developing plasma-aided technologies to sterilize food contact surfaces and to disinfect water.

Nancy P. Keller, Ph.D.,  Associate Professor	The long-term goal of my research program is to reduce mycotoxin contamination of food and feed crops. My program focuses on identifying the molecular genetic processes governing mycotoxin gene expression in Aspergillus and Fusarium spp., and host metabolites which effect fungal growth and mycotoxin biosynthesis. These areas of research are directed toward designing rational mycotoxin control strategies and should contribute to control of other fungal pathogens. I received my graduate training at the Plant Pathology Department at Cornell University. For both my Masters and Ph.D. work I focused on fungal pathogens of corn. My interest in plant pathology first stemmed from my Peace Corps Volunteer work in Lesotho, Africa, where the issue of quality food looms large every year. After graduating from Cornell University in 1990, I did my postdoctoral research at the USDA-ARS laboratory in New Orleans, LA. This was my introduction to mycotoxigenic fungi. There I purified an Aspergillus enzyme required for aflatoxin production; this project was in collaboration with Dr. Fun Sun Chu working at FRI. I enjoyed this work so much that I actively sought a position where I could start my own mycotoxin program. In November 1991 I accepted a position as Assistant Professor in the Department of Microbiology and Plant Pathology at Texas A&M University where I focused on the molecular mechanisms governing genetic control of aflatoxin and sterigmatocystin biosynthesis in Aspergillus spp. The year 2000 brought many changes to my life including my acceptance of a joint position as associate professor in FRI and the Department of Plant Pathology at the University of Madison, WI. I moved to Madison in 2001 with my daughter Nina (2 in November) and we are having fun exploring the city and surrounding area.
.

In March of this year I joined Dr. Johnson's lab as an Associate Research Specialist. I earned my B.S. in Bacteriology at UW–Madison in 1999. Following graduation, I worked for Genetic Visions as a Research Specialist, performing genetic testing on cattle. I now perform food challenge studies on a wide variety of foods. We challenge with many different pathogens, including Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum. I have already worked on several studies and I enjoy the variety. I have been married to my husband Ryan, a fellow Bacteriologist, for a year now. We are currently getting ready to move into our first home and we are very excited. We are both big sports fans and enjoy following the Packers, Badgers, Brewers and Bucks. We also try to be athletic by playing softball in a summer league.	Lindsey McDonnell, B.S.,  Associate Research Specialist