July 24, 2008

Outmaneuvering Foodborne Pathogens

By Wood, Marcia

If pathogens like E. coli O157:H7 or Salmonella had a motto for survival, it might be: "Find! Bind! Multiply!" That pretty much sums up what these food-poisoning bacteria do in nature, moving through our environment to find a host they can bind to and use as a staging area for multiplying and spreading.

But ARS food-safety scientists in California are determined to find out how to stop these and other foodborne pathogenic bacteria in their tracks, before the microbes can make their way to leafy greens and other favorite salad ingredients like tomatoes and sprouts.

The research is needed to help prevent the pathogens from turning up in fresh produce that we typically eat uncooked. That's according to Robert E. Mandrell, who leads the ARS Produce Safety and Microbiology Research Unit. His team is based at the agency's Western Regional Research Center in Albany, California.

The team is pulling apart the lives of these microbes to uncover the secrets of their success. It's a complex challenge, in part because the microbes seem to effortlessly switch from one persona to the next. They are perhaps best known as residents of the intestines of warmblooded animals, including humans. For another role, the pathogens have somehow learned to find, bind, and multiply in the world of green plants.

Sometimes the pathogenic microbes need the help of other microbial species to make the jump from animal inhabitant to plant resident. Surprisingly little is known about these powerful partnerships, Mandrell says. That's why such alliances among microbes are one of several specific aspects of the pathogens' lifestyles that the Albany scientists are investigating. In all, knowledge gleaned from these and other laboratory, greenhouse, and outdoor studies should lead to new, effective, environmentally friendly ways to thwart the pathogens before they have a chance to make us ill.

A Pathogen Targets Youngest Leaves

Knowing pathogens' preferences is essential to any well-planned counterattack. So microbiologist Maria T. Brandl is scrutinizing the little-understood ability of E. coli O157:H7 and Salmonella enterica to contaminate the elongated, slightly sweet leaves of romaine lettuce. With a University of California-Berkeley colleague, Brandl has shown that, if given a choice, E. coli has a strong preference for the young, inner leaves. The researchers exposed romaine lettuce leaves to E. coli and found that the microbe multiplied about 10 times more on the young leaves than on the older, middle ones. One explanation: The young leaves are a better nutrition "buy" for E. coli. "These leaves exude about three times more nitrogen and about one-and-one-half times more carbon than do the middle leaves," says Brandl.

Scientists have known for decades that plants exude compounds from their leaves and roots that bacteria and fungi can use as food. But the romaine lettuce study, published earlier this year in Applied and Environmental Microbiology, is the first to document the different exudate levels among leaves of the two age classes. It's also the first to show that E. coli can do more than just bind to lettuce leaves: It can multiply and spread on them.

Adding nitrogen to the middle leaves boosted E. coli growth, Brandl found. "In view of the key role of nitrogen in helping E. coli multiply on young leaves," she says, "a strategy that minimizes use of nitrogen fertilizer in romaine lettuce fields may be worth investigating."

In other studies using romaine lettuce and the popular herb cilantro as models, Brandl documented the extent to which E. coli and Salmonella are aided by Erwinia chrysanthemi, an organism that causes fresh produce to rot.

"When compared to plant pathogens, E. coli and Salmonella are not as 'fit' on plants," Brandl says. But the presence of the rot- producing microbe helped E. coli and Salmonella grow on lettuce and cilantro leaves.

"Soft rot promoted formation of large aggregates, called 'biofilms,' of E. coli and Salmonella and increased their numbers by up to 100-fold," she notes.

The study uncovered new details about genes that the food- poisoning pathogens kick into action when teamed up with plant pathogens such as soft rot microbes.

Brandl, in collaboration with Albany microbiologist Craig Parker, used a technique known as "microarray analysis" to spy on the genes. "The assays showed that Salmonella cells- living in soft rot lesions on lettuce and cilantro-had turned on some of the exact same genes that Salmonella uses when it infects humans or colonizes the intestines of animals," she says. Some of these activated genes were ones that Salmonella uses to get energy from several natural compounds common to both green plants and to the animal intestines that Salmonella calls home.

A One-Two Punch to Tomatoes

Salmonella also benefits from the presence of another plant pathogen, specifically, Xanthomonas campestris, the culprit in a disease known as "bacterial leaf spot of tomato." But the relationship between Salmonella and X. campestris may be different than the relation of Salmonella to the soft rot pathogen. Notably, Salmonella benefits even if the bacterial spot pathogen is at very low levels-so low that the plant doesn't have the disease or any visible symptoms of it.

That's among the first-of-a-kind findings that microbiologist Jeri D. Barak found in her tests with tomato seeds exposed to the bacterial spot microbe and then planted in soil that had been irrigated with water contaminated with S. enterica.

In a recent article in PLoS ONE, Barak reported that S. enterica populations were significantly higher in tomato plants that had also been colonized by X. campestris. In some cases, Salmonella couldn't bind to and grow on-or in-tomato plants without the presence of X. campestris, she found.

"We think that X. campestris may disable the plant immune response-a feat that allows both it and Salmonella to multiply," she says.

The study was the first to report that even as long as 6 weeks after soil was flooded with Salmonella-contaminated water, the microbe was capable of binding to tomato seeds planted in the tainted soil and, later, of spreading to the plant.

"These results suggest that any contamination that introduces Salmonella from any source into the environment- whether that source is irrigation water, improperly composted manure, or even insects- could lead to subsequent crop contamination," Barak says. "That's true even if substantial time has passed since the soil was first contaminated."

Crop debris can also serve as a reservoir of viable Salmonella for at least a week, Barak's study showed. For her investigation, the debris was composed of mulched, Salmonellacontaminated tomato plants mixed with uncontaminated soil.

"Replanting fields shortly after harvesting the previous crop is a common practice in farming of lettuce and tomatoes," she says. The schedule allows only a very short time for crop debris to decompose. "Our results suggest that fields known to have been contaminated with S. enterica could benefit from an extended fallow period, perhaps of at least a few weeks."

Ordinary Microbe Foils E. coli

While the bacterial spot and soft rot microbes make life easier for certain foodborne pathogens, other microbes may make the pathogens' existence more difficult. Geneticist Michael B. Cooley and microbiologist William G. Miller at Albany have shown the remarkable effects of one such microbe, Enterobacter asburiae. This common, farm-and-garden-friendly microorganism lives peaceably on beans, cotton, and cucumbers.

In one experiment, E. asburiae significantly reduced levels of E. coli and Salmonella when all three species of microbes were inoculated on seeds of thale cress, a small plant often chosen for laboratory tests.

The study, published in Applied and Environmental Microbiology in 2003, led to followup experiments with green leaf lettuce. In that battle of the microbes, another rather ordinary bacterium, Wausteria paucula, turned out to be E. coli's new best friend, enhancing the pathogen's survival sixfold on lettuce leaves.

"It was the first clear example of a microbe's supporting a human pathogen on a plant," notes Cooley, who documented the findings in the Journal of Food Protection in 2006.

But E. asburiae more than evened the score, decreasing E. coli survival 20- to 30-fold on lettuce leaves exposed to those two species of microbes.

The mechanisms underlying the competition between E. asburiae and E. coli are still a mystery, says Cooley, "especially the competition that takes place on leaves or other plant surfaces."

Nevertheless, E. asburiae shows initial promise of becoming a notable biological control agent to protect fresh salad greens or other crops from pathogen invaders. With further work, the approach could become one of several science-based solutions that will help keep our salads safe.-By Marcia Wood, ARS.

In a greenhouse, microbiologist Maria Brandl examines cilantro that she uses as a model plant to investigate the behavior of foodborne pathogens on leaf surfaces.

Research assistant Danielle Goudeau inoculates a lettuce leaf with E. coli O157:H7 in a biological safety cabinet to study the biology of the human pathogen on leafy greens.

Using a confocal laser scanning microscope, microbiologist Maria Brandl examines a mixed biofilm of Salmonella enterica (pink) and Erwinia chrysanthemi (green) in soft rot lesions on cilantro leaves (blue). -By Marcia Wood, ARS.

This research is part of Food Safety, an ARS national program (#108) described on the World Wide Web at www.nps.ars. usda.gov.

To reach scientists mentioned in this article, contact Marcia Wood, USDA-ARS Information Staff, 5601 Sunnyside Ave., Beltsville, MD 20705-5129; phone (301) 504-1662, fax (301) 504-1486, e-mail [email protected]

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