Not Just Hitchhikers

Oct 28, 03:43 AM

Current Headlines: By Milius, Susan

Human pathogens make homes on plants Jeri Barak's tomato plants have a weird disease breaking out on them. Not the biggest surprise, perhaps, since she's a bona fide U.S. Department of Agriculture plant pathologist. But what's afflicting Barak's tomatoes isn't some everyday farm ailment-their leaves are colonized with Salmonella enterica, more famous as an animal pathogen. This bacterium leads to about 600 deaths in people each year, along with 40,000 reported cases of illness.

It's the species that everyone's supposed to guard against when handling raw meat, eating undercooked eggs, or petting baby turties. Barak, however, has started studying this animal pathogen's role on plants. And she's not talking about bacteria just passively smearing plants like streaks of dirt.

Evidence has been growing that high-profile human pathogens, such as salmonella strains and the deadly Escherichia coli O157:H7, actively colonize plants. In doing so, the pathogens follow variations on their attack tactics for animals. They grow structures to glue themselves in place. They build defensive shields and fortresses. They set up housekeeping. Barak doesn't go so far as to say that salmonella and E. coli make plants sick, but these human pathogens are definitely up to something on plants.

Understanding what human-disease organisms do when they go plantside could suggest new ways to combat foodborne illnesses, says Barak. Produce is increasingly often the culprit behind disease outbreaks, and cases such as the 3 people killed and more than 200 sickened in 2006 from eating tainted fresh spinach have dramatized that food safety begins with the food, not its handlers.

The people-plant-pathogen interplay inspired a special symposium at last July's annual meeting of the American Phytopathological Society in San Diego. Some pathogen species literally do cause diseases in both plants and people (see sidebar). Other microbes typically don't hurt plants as much as they hurt people. Still, plant pathologists say that discovering how these microbes set up housekeeping on plants could lead to new ways to stop them. Barak, of the USDA in Albany, Calif., told her colleagues at the meeting that although plant pathologists had for decades deferred to human- disease specialists in the study of these pathogens, "now we need to take back salmonella."

PLANT LIFE Just what salmonella and E. coli are up to on plants didn't get much attention until recently. "It wasn't until people started getting sick from fresh produce that [we] started looking at how pathogens do this," says Barak.

According to statistics from the Centers for Disease Control and Prevention (CDC) in Atlanta, only 0.6 percent of disease outbreaks from food in the 1970s could be traced to fresh produce. In the 1990s, however, produce accounted for 12 percent of outbreaks, and since a 1998 revision of surveillance criteria, the percentage has edged up to 14.

Many factors have contributed to the rise in killer fruits and vegetables. People today eat almost a third more fresh produce than they did in the 1970s. And todays produce comes from an international and industrialized system in which it often travels farther than the people who eat it. Outbreak-tracing techniques have improved too.

The increase in outbreaks inspired new research on produce safety and led to disturbing findings. Even a few years ago, Barak says, microbiologists had assumed that human pathogens were merely passengers on plants, and so could be dealt with by soap and water. But in test after test early in this decade, none of the available washes and sanitizers could clean produce completely. The persistence of bacteria prompted researchers to wonder whether pathogens were making more than merely casual contacts with plants.

Tests showed that pathogens, either passively or actively, could infiltrate tissues far beyond the reach of surface washes. For example, Red Delicious apples dunked in water containing E. coli O157:H7 in a lab test ended up with the bacteria inside the core even though the fruit hadn't been cut. The bacteria in this experiment carried a gene that made them fluoresce green, and the glow showed the bacteria near the seeds, Larry Beuchat of the University of Georgia in Griffin and his colleagues reported in 2000.

Other experiments have shown E. coli seeping into uncut, unpeeled oranges through the little break in the skin where the fruit parts from its stem. Mangos and tomatoes likewise are vulnerable to salmonella through their stem scars.

Even pathogens that didn't lurk deep in tissues proved virtually indelible. By 2002, at least four studies had confirmed that neither chlorine treatment nor a good, brisk scrubbing dislodged E. coli from lettuce. Current research is addressing this challenge (SN: 12/ 16/06, p. 394), but the failure of most washing systems has clued researchers in to a secret of E. coli contamination: The bacterium gets a grip on plants and hangs on.

Studies of other pathogens similarly came to the conclusion that pathogens actively colonize plants. Maria Brandl, also of USDA's Albany lab, followed up on a 1999 salmonellosis outbreak in California by testing a bacterial strain of S. enterica called serovar Thompson, which had been isolated from a patient. After inoculating chopped cilantro leaves and even some salsa with the strain, Brandl reported that the bacterium grew quickly to high concentrations in both foods.

Brandl and her USDA colleague Robert Mandrell then took their test to live plants. They inoculated growing cilantro leaves with a pathogenic salmonella strain as well as with two bacterial species common on plants. All three microbes grew and colonized the leaves. The salmonella strain didn't flourish as abundantly as the specialized plant bacteria, but it did establish a presence on the leaf.

When the researchers stressed the bacteria by keeping the leaves dry, the salmonella population shrank but rebounded when Brandl rehumidified the plants. The salmonella strain responsible for the 1999 poisonings was a perfectly plausible leaf colonizer, Brandl and Mandrell reported in 2002.

Ordinary diseases of the plants, which are otherwise harmless to people, may help the human pathogens make themselves at home. Growing in a test tube, E. coli O157:H7 doesn't reliably make a protective biofilm. But when researchers added the E. coli to test tubes along with a regular plant bacterial pest, Erwinia chrysanthemi, the human pathogens readily joined the biofilm of the plant pathogen.

Barak is now working on a study with whole plants. She and her colleagues are finding that salmonella grows more abundantly if a plant is already infected by Xanthomonas campestris pathovar vesicatoria, which causes bacterial spot disease on peppers and tomatoes.

SURVIVOR To tease out the genetic mechanisms behind salmonella's ability to make itself at home on a plant, Barak and her colleagues worked their way through a set of experimentally created mutants of a strain called serovar Newport. The researchers found mutants that couldn't attach themselves to alfalfa sprouts, a common source of disease outbreaks. Analyzing the genetic defects in these bacteria gave Barak a clue as to what genes the bacteria use when confronting a leaf. "It was ironic," says Barak, that 13 out of 20 of these mutants had disruptions in genes that had never been characterized in years of previous work on how salmonella infects animals.

That discovery implies that the bacteria cope with plants by using some of the same genes that power pathogenic attacks on animals-for example, a gene for strands of protein nicknamed Tafi, which attach the bacterial cell to a surface. Tafi have attracted attention from Alzheimer's researchers trying to understand how protein deposits build up outside cells in failing brain tissue.

But as Barak's work shows, salmonella cells apparently also have devices specifically for making solid attachments to plant surfaces. The latest paper from this project, in the September Molecular Plant- Microbe Interactions, details two such fasteners. The molecular gadgets help the microbes hold on to each other as well as to plant tissue. The interconnected bacterial cells form a solid biofilm, which offers protection to its members from external hazards.

Such capacity for living on a plant doesn't surprise Barak. "I think colonizing plants may be vital for salmonella to live out their life cycle," she says. Any such animal pathogen has to survive occasional periods when it's outside the warm, plush world of an animal host. By comparison, a plant leaf is harsh, with no temperature regulation or protection from drought, deluge, or ultraviolet blast. Landing on a plant "is like Salmonella going to the moon," Barak says. So it makes sense that pathogens maintain equipment for emergency landings on plants.

The hardships of life in a crop field have other effects important to people, according to Karyn Meltz Steinberg of Emory University in Atlanta. People who get sick may just be collateral damage in a war between bacteria and their protozoan predators. She and her Emory colleague Brace Levin have been musing about what benefit E. coli, a microbe with hoofed mammals as its natural host, gets from killing people.

The pathogen's toxin might defend the bacteria against grazing protozoa typically encountered outside one of those hoofed hosts, such as in soil. Meltz Steinberg and Levin tested the idea by tracking E. coli's survival with and without the presence of the rapacious, bacteria-hunting species Tetrahymena pyriformis. An E. coli strain carrying a toxin-making factor outperformed a harmless strain only when stalked by the protozoans, the researchers reported in the Aug. 22 Proceedings of the Royal Society B. Even though life on plants may be a stretch for human pathogens, they can be very hard to kill there. A better strategy, Barak says, is to improve farming practices so as to limit the spread of bacteria to crops. To keep food safe, "we've got to give farmers the tools", she says.

The Albany research program includes efforts to find possible routes of agricultural contamination. Barak's been studying the life of salmonella in soil, where other research has suggested that the bacteria can survive at least for a while. In the lab, she mimicked a farmer discovering a contaminated crop of tomato plants, plowing them into the soil, waiting a week, and then planting new seeds. Even 6 weeks after reseeding, she found salmonella on the second crop.

The way in which water is used on farms could also be a problem, according to several labs. For example, the USDA lab in California has recovered E. coli O157:H7 from lettuce seedlings days after experimentally irrigating them with tainted water. And when the Beuchat group painted a salmonella solution onto tomato blooms, 2 of the 8 fruits that formed from those flowers carried the pathogen. Farmers don't go around dabbing their crops with dirty paintbrushes, but they have felt free to use untreated water from the farm for spraying pesticide mixes. Barak speculates that someday farmers might have to make sure they use clean drinking water in their sprayers. These and other routes of contamination need investigating, she says.

In the meantime, she offers comfort-with some cautions-to fans of fresh fruits and vegetables. "Flare-ups of food pathogens are rare events," she says. To minimize her own chance of encountering worrisome bacteria, she avoids damaged fruits and vegetables. They leak moist innards of the plant and offer fertile ground for pathogen picnics. And she minimizes store-to-fridge time as much as she can. "I tell people: "Treat your produce like ice cream.'"

Even with her research on the dark side of salad, she hasn't given up. "Oh, I eat this stuff," she says. "I'm a vegetarian."

BUT WE WASHED - Alfalfa sprouts (ghostly blue ribbons) rinsed with sterile water still show the green spots indicating the presence of E. coli 0157:H7 that has been engineered to fluoresce under laser illumination.

BAD TIP - Ends of alfalfa sprouts, washed with sterile water, still reveal spots of salmonella dyed to appear yellow.

Copyright Science Service, Incorporated Oct 20, 2007

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