Bacteria_Foodborne.jpgThe Science Picture Company 2011

Fever, muscle ache, and diarrhea—perhaps even confusion, loss of balance, and convulsions. These were some of the symptoms suffered by the 33 Americans who died of listeriosis in 2011 as a result of eating cantaloupes tainted with the bacteria Listeria monocytogenes.

The outbreak — the deadliest since 1985, affecting more than 100 people in 26 states — eventually was traced to a farm in Littleton, Colorado.

Officials said that the likely cause of contamination was improperly cleaned and sanitized washing equipment, and that the bacteria might have spread throughout processing and storage areas by workers walking through puddles of Listeria-contaminated water.

Soon after the outbreak ended, farmers Eric and Ryan Jensen pleaded guilty to six counts of introducing adulterated food into interstate commerce.

According to the Centers for Disease Control and Prevention (CDC), listeriosis primarily affects older adults, pregnant women, newborn babies, and adults with weakened immune systems. In the United States, around 1,600 people contract the disease each year and approximately 260 die from it.

Through research and education, food safety scientists in the College of Agricultural Sciences are working to prevent illness and death from foodborne pathogens such as Listeria among consumers. They also are working to prevent financial ruin and legal consequences among farmers and food processors.

The Biology of Bacteria

In September 2011, officials spent about 10 days attempting to track the origin of the Listeria outbreak before they were able to identify its source. In the meantime, the number of people who fell ill increased by the day.

According to Edward Dudley, associate professor of food science, part of the reason that tracking food-related disease outbreaks takes so long is that traditional methods relying on first isolating the organism from human and food samples, followed by DNA analysis using a method called pulsed field gel electrophoresis, can take several days to complete.

To help speed things up, Dudley is investigating methods based on DNA sequencing, which capitalizes on the fact that different strains of the same bacterial species have small differences in their DNA.

"This technique can give DNA sequence results within 24 hours and allow you to conclusively say that the organism that was found in a farm environment is the same exact one that ended up in the food and in a patient," said Dudley, who conducts most of his molecular subtyping research on the bacterium Salmonella.
In addition to distinguishing specific strains of bacteria, molecular subtyping also may be useful in predicting the antibiotics to which certain strains are resistant.

"This is incredibly useful information for the physician who is trying to treat a bacterial disease," said Dudley.

Dudley explained that he is able to distinguish different strains of bacteria by examining the prokaryotes’ CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) regions. These regions, he said, protect bacteria from viruses that attack them.

"Bacterial viruses — called bacteriophages — attach to bacteria, inject their DNA, and turn bacteria into machines that manufacture more viruses," said Dudley. “A bacterium counters this attack by recognizing that a virus has injected its DNA and digesting the DNA. In the process, the bacterium clips out a small portion of the virus’s genome and it incorporates it into its own genome. This new ‘DNA marker’ is a reminder to the bacterium that if it ever encounters the DNA sequence again, it should know that it belongs to a virus and it should trigger a response to inactivate it immediately. The process is basically an immune response.”

Dudley noted that this immune response takes place within the CRISPR region of bacterial DNA, which is why the region changes at a rapid pace.

“When groups of bacteria become separated—in different processing plants, for example—the CRISPR regions of these new strains of bacteria become unique and trackable,” he said. “We have shown that we can use these regions to quickly distinguish among strains of bacteria.”

Persistent Prokaryotes

Professor of Food Science Stephen Knabel also conducts research on the molecular epidemiology of foodborne pathogens. In a 2011 study conducted with Sara Lomonaco, adjunct professor in the Department of Food Science, and his colleagues at the Centers for Disease Control and Prevention, the Food and Drug Administration, and the U.S. Department of Agriculture, Knabel described the use of a novel molecular subtyping method developed in his laboratory, called Multi-Virulence-Locus Sequence Typing, to identify one novel outbreak strain and two novel epidemic clones associated with the cantaloupe outbreak.

In addition to investigating ways to track foodborne pathogen outbreaks, Knabel also works to understand why bacteria are so pervasive and difficult to eliminate in the first place.

In particular, he is examining why some strains of bacteria persist in processing plants despite meticulous cleaning and sanitation. The reason, it turns out, may be the same reason that the organisms can be so tough to eliminate from human bodies.

"In the 1940s, when scientists first began to use penicillin to treat Staphylococcus aureus, they noticed that there was a small fraction of the Staph population that was impervious to penicillin," said Knabel. "First they thought maybe these were antibiotic-resistant Staph mutants, but it turned out they were just organisms in the population that had gone dormant. Because of their dormancy, they had become temporarily tolerant of the antibiotics."

According to Knabel, these dormant cells—called persister cells—also may be responsible for the persistence of bacteria in food processing plants.

"If you go into a food processing plant, and years later you go back, you are likely to find the same exact same strain persisting," he said. "They are almost impossible to get rid of. We believe that they are forming persister cells in the plants and, therefore, are able to survive some of the methods people use to kill bacteria."

Knabel explained that sometime during the course of their 3.5 million years of evolution, bacteria have figured out how to survive long term by shutting down and becoming dormant when the environment becomes unfavorable.

"In our laboratory, we have seen bacteria remain dormant for up to a year,” he said. “During this time, the bacteria are harder to kill. They not only become more tolerant to antibiotics, but they also become more resistant to sanitizers, dry conditions, starvation, and other stressors. We think this may be what causes persistence within food processing plants."

Knabel and his colleagues have conducted numerous experiments to determine whether various eradication treatments—such as pasteurization, UV light, and high pressure—will kill persister cells. The researchers have focused much of their effort on Listeria, but they are beginning to investigate this same concept in pathogenic Escherichia coli, Staphylococcus aureus, and other organisms that are important human pathogens.

Knabel said that certain bacterial strains may also be persistent in food processing plants because they have adapted through natural selection to the plants. "Each processing plant seems to have its own unique strain," he said. "We have found that plants that make the exact kind of food have the exact kind of strain. Ones that have slightly different foods or different types of foods have different strains."

Most recently, Knabel—along with colleague Luke LaBorde, associate professor of food science, and Ph.D. student Latha Murgesan—have discovered a particular persistent strain of Listeria in a specific mushroom processing plant. They currently are investigating whether that strain has adapted to the unique conditions in the facility with the goal of making recommendations for improved cleaning and sanitizing procedures.

"Our research is helping us to better understand how pathogens like Listeria persist in food processing plants so we can then prevent problems from occurring," he said.

Counteracting Contamination

Knabel and LaBorde’s research may one day lead to foods leaving processing plants with significantly less bacterial contamination. In the meantime, however, the foods that enter the consumer realm inevitably contain some bacteria. For example, some 30 percent of the chicken in grocery stores contains Salmonella and Campylobacter, according to Catherine Cutter, professor of food science.

Cutter spends much of her time investigating techniques to reduce bacteria on food products just before they go to consumers. In one of her latest projects, she is examining the use of edible films—like the ones LISTERINE® uses in its breath strips—to kill bacteria on meat, poultry, and seafood.

"We are incorporating antimicrobial agents into edible pullulan films and applying them to whole cuts of meat, like steaks, slices of deli meat, and shrimp, and we are finding that they are successful at killing bacteria," she said. "A visiting scholar from Egypt incorporated harmless nanoparticles, like silver and zinc oxide, as well as essential oils from rosemary and oregano, into the films, and they worked well at inhibiting E. coli O157:H7 on beef and Listeria monocytogenes on deli meats."

Another of Cutter’s visiting scholars, this one from Thailand, found success incorporating an antimicrobial peptide, isolated from a lactic-acid bacterium found in catfish, into the pullulan films. When the edible films were combined with lauric arginate (another naturally occurring antimicrobial compound), the method inhibited several foodborne pathogens on meat, poultry, and seafood surfaces.

Cutter said that right now the cost of treating meats with these types of films may be too high to do commercially, but she is continuing her research in this area and looking for ways to make the technique more cost effective.

At the same time, she is also conducting a project to help farmers who sell their meats at farmers’ markets ensure that their products are safe for consumers. One of Cutter’s graduate students purchased 100 chickens from farmers’ markets across Pennsylvania and found that they often contained higher amounts of Campylobacter and Salmonella than grocery store samples.

The researchers then conducted a needs assessment of the farmers’ market vendors and found that there were gaps in their knowledge about food safety regulations. For example, some of the farmers’ market vendors did not know if their processors use an antimicrobial, such as lactic acid or peroxyacetic acid, during processing to control pathogens.

"Many of these vendors may have been producing unsafe food without realizing it,” said Cutter. "We currently are developing food safety educational materials to help these vendors navigate the maze of complex and increasing regulations so they can develop products that are safe for the consuming public. This approach will enable them to keep their livelihoods by preventing recalls or outbreaks that can shut them down and put them out of business."

In another area of research, Cutter and colleagues have investigated ways to kill E. coli in fresh ground beef while maintaining the eating quality of the meat. By using high-pressure processing (HPP), the researchers significantly reduced pathogenic E. coli in experimentally inoculated, fresh ground beef patties.

"We were excited because it worked so well to control the pathogens," she said. "We then evaluated uninoculated ground beef products that were subjected to HPP and cooked thoroughly in a series of consumer taste panels. We found that the panelists could detect slight differences in juiciness and texture of the cooked HPP-treated patties. So, while the process worked great for killing the pathogens, the quality aspects mean we have some work to do. We would like to marry food safety with quality aspects. Otherwise, processors won’t be able to sell their products."

Helping Producers and Consumers

Helping farmers and owners of packaging plants in Pennsylvania stay in business is part of what motivates Luke LaBorde as well. LaBorde, an associate professor of food science, pursues this goal by offering education about and assistance in meeting the latest food safety regulations. Lately, this means gearing up to help translate the requirements of the Food and Drug Administration’s (FDA’s) Food Safety Modernization Act (FSMA), which was signed into law by President Obama in 2011 but has not yet been finalized. The law aims to ensure the U.S. food supply is safe by shifting the focus from responding to contamination to preventing it.

"Congress and the FDA have been pretty late in getting to this," said LaBorde. "Already in place were commercial mandates. Food stores and distributors have already had stringent food safety requirements in place and have already required audits."

According to LaBorde, these organizations, in cooperation with the Pennsylvania Department of Agriculture, send inspectors to farms and packaging plants. If the buyers decide that the conditions within the farm or plant meet their safety criteria, they will continue to do business with the farm or plant. In addition to passing an inspection, these buyers often require farms and plants to keep food safety plans.

LaBorde helps the farmers and packaging plant owners by teaching them what they need to do to meet the safety requirements imposed by buyers. "For example, we can help them determine how far away their beef cattle area should be from their lettuce fields," he said. "There’s no real right answer to this question, but farmers should be aware that this is a potential hazard. We try to help them lower their risks and meet the requirements of food buyers, so they can stay in business." LaBorde also helps farmers and packaging plant owners write food safety plans.

Martin Bucknavage, senior food safety extension associate, also keeps his finger on the latest commercial mandates and the upcoming FSMA regulations. Like LaBorde, he serves as a point of contact for farmers, packaging plant owners, and members of the public who have questions about regulations and food safety, more generally.

"As the FSMA regulations are being written, they will certainly change the way food is produced and processed, so it is important for us to be able to communicate those changes to companies in Pennsylvania and beyond," said Bucknavage.

According to him, the information available on the Internet is not always accurate. "Our information is science based, and people know that they will get the best answer available when they call us," he said.

For consumers, Bucknavage and his colleagues recently implemented a process for the county extension offices to respond to questions posed by constituents in their respective counties. Questions are considered by the food safety team, and Bucknavage calls the consumers back with answers.

Bucknavage also is involved with teaching a number of classes each year that focus on helping a variety of groups be more savvy regarding food safety. Classes include a food microbiology course for food processors, a "ServSafe" course for restaurant and retail food managers, and a volunteer food safety training program for nonprofit organizations, such as fire halls, churches, and food banks.

In addition, Bucknavage works with hunters on game meat safety; with home processors on canning, drying, and cooking; and with people in crisis because their electricity has been out, among other groups.

Whether by educating or conducting research, faculty and staff members in the college aim to protect Pennsylvania’s businesses and consumers from the effects of outbreaks like the one in 2011 that killed 33 people and sickened more than 100.

"Despite numerous outbreaks in this country, none so far have been traced to Pennsylvania’s farms,” said LaBorde. "We want to keep it that way."