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Researchers Unlock One E. coli Mystery

By following a “guiding light,”  USDA researchers were able to unlock one piece of the baffling puzzle about how the potentially fatal E. coli O157:H7 can contaminate spinach and other salad greens.

The good news coming from the research is that while spinach roots can take in E. coli from contaminated soil or irrigation water, the pathogen likely doesn’t go any farther than that. In other words, in all likelihood, the pathogen doesn’t travel up from the roots into the plants’ leaves.

The research, funded by California-based Fresh Express, the leading supplier of fresh produce in the United States, was triggered by concerns that the roots of spinach and other leafy greens could transport the pathogen into the plant’s internal vascular system and thereby increase the incidence of foodborne illness.

The goal of the research was to track the pathogen’s journey in spinach from field to harvest.


That’s where the “light” comes in. By modifying several strains of E. coli — some of them highly pathogenic and others non-pathogenic — to contain a gene for fluorescence,  the researchers were able to use a specialized microscope to see where the pathogen was actually going — in this case, from the soil into the roots.

They achieved this feat by placing the fluorescence gene at a specific location within the chromosome structure of the bacterium where it wouldn’t interfere with any essential metabolic functions or stress responses of the cells. That made the cells more likely to survive and fluoresce under stressful conditions within the plant. This, in turn, gave the researchers a higher level of confidence about their observations.

The sleuths in the USDA research team were microbiologist Manan Sharma; post doctoral research associate David Ingram; food technologist Jitu Patel; and microbiologist Patricial Millner, all who work at USDA’s Environmental Microbial and Food Safety Laboratory in Beltsville, MD.  In developing the  strains containing the gene for fluorescence, they worked in collaboration with researchers at the University of Maryland School of Medicine.

Putting A Concern About Greens to Rest

They began by confirming that the pathogenic E. coli could survive in the soil for up to 28 days, although at different levels.  In doing that, they also observed that the fluorescent E. coli cells had been able to migrate from the soil into the roots of the spinach plants.

The research team also examined baby spinach plants over the course of 28 days after they had germinated to see whether any of the E. coli strains traveled past the roots and up in the plants’ interior structures. To do that, they grew baby spinach in pasteurized soil and hydroponic media.

At day 28, the researchers could find no evidence that E. coli had gone into the leaves or the shoots of the baby spinach plants grown in pasteurized soil.

They did, however, detect the pathogen in hydroponically grown spinach samples that were analyzed 14 and 21 days after the plants had germinated. However, only sporadic, very low levels of the pathogen’s survival were observed in shoot tissue after 28 days.

USDA researcher Sharma told Food Safety News that the research eliminates one potential route of contamination in leafy greens.

“The uptake and internalization of E. coli through the roots seems an unlikely way for the greens to get people sick,” he said. “We wanted to investigate this, because it was one of the questions out there: “Is this a likely route of E. coli in foodborne illnesses?”

The conclusion: “We’ve taken something that has been of concern for 8 or 9 years and put it to rest,” Sharma said.

Although the research focused on spinach, Sharma said he thinks it’s relevant to other salad greens as well.


By eliminating one route of contamination in spinach, the results of the research are important, said Sharma, because they “will  allow us to focus on more likely routes of contamination.”

He also pointed out that if the plants’ defenses didn’t have a way to keep the pathogen from traveling up in the plants’ leaves, “we’d probably see a lot more contamination and a lot more sick people.”


Results from this research will be published in the Journal of Food Protection.

When asked about two research papers — both of them also published in the Journal of Food Protection, one in 2002 and the other in 2003 — showing that E. coli O157:H7 can travel into leafy greens either from their roots or infected seeds, Sharma pointed to an important difference in that research and his.

“In most cases, those two papers report studies that were conducted with very high numbers of pathogenic bacteria,” he said, pointing out that those high numbers are unlikely to be encountered in a true field setting where leafy greens are being grown. 

Even so, he said the findings of those research papers are important because they show the susceptibility of seeds and seedlings to pathogen contamination at very early stages and the need to have procedures in place to prevent seeds from being contaminated. 

He also pointed out that his recent research used both high numbers of contamination, which would be unlikely in a field setting, and low numbers, which would be more likely in a field setting.

“We feel that our results are comprehensive and reveal that the risk of uptake of E. coli O157:H7 cells into spinach plants grown in soil is unlikely,” he said.

Like Greens, Like All Vegetables?

When asked what his research means for root crops such as carrots, parsnips and beets, and whether this means those crops can contain E. coli internally,  Sharma said he doesn’t think that the team’s results can be used to answer that question.

“I think different crops will have different root systems and microflora, transport systems, etc.,” he said. “I think applying results done with spinach plants to root crops like carrots and such may not translate. Each crop system should receive its own evaluation of the risk of the uptake of foodborne pathogens through root systems.”

Sharma was quick to say that the research results don’t mean that growers shouldn’t take precautions when using raw manure, or improperly composted manure, in their fields. That’s because the leaves can become contaminated by coming into contact with the manure.

Fresh manure may contain Salmonella, Listeria and E. coli bacteria.

Under USDA’s organic standards, the time between applying raw, uncomposted manure and harvesting a crop whose edible portion has come into contact with the soil is 120 days — 90 days before harvest for crops that don’t come into direct contact with the soil.


Washington State University microbiologist and consumer food-safety specialist Karen Killinger told Food Safety News that pathogens are naturally present at low levels in the environment, including soil and irrigation water. In addition, many livestock, wild and domestic animals and birds are natural hosts for pathogens, so raw manur
e can be a source of pathogens. Other potential sources of pathogens can include sick farm workers and unsanitary equipment.

“Programs, like Good Agricultural Practices (GAPs), can assist farmers in managing on-farm pathogen risk and improve food safety,” she said, pointing out that good agricultural practices include irrigation water-quality assessment, documentation of manure-management practices, worker health and hygiene programs, and sanitation programs.


Ed Loyd, spokesman for Fresh Express, a subsidiary of Chiquita Brands, told Food Safety News that USDA’s research findings about E. coli and leafy greens is very important to the company.

“We need to understand more about pathogens and how they affect fresh produce,” he said. “We need to learn as much as we can about these organisms so we can enhance food-safety practices. Food safety should never be a static program.”

Referring to the solid growth of sales of fresh spinach and other salad greens, Fresh Express assistant spokesman Andrew Ciafardino said that it’s obvious that people are seeking healthier and fresher options.

“As a company promoting fresh fruits and vegetables, we want to  help improve world nutrition  in a safe and effective way,” he said.


With the invention of the “Keep Crisp” bag in the early 1980s, Fresh Express pioneered the now multi-billion-dollar retail packaged-salad category and was the first company to make the bagged salads available nationwide.

In 2007, shortly after a major E. coli outbreak was linked to bagged fresh spinach — but not to any Fresh Express products —  the company awarded grants of $250,000 each to nine research teams to study the E. coli O157:H7 pathogen. The goal was to advance science-based practices to prevent its occurrence in fresh produce.

Sharma’s research, which explored whether E. coli could travel up the roots and into the leaves of spinach, was one of the projects funded under that round of grants.

The need for continuing research on preventing E. coli contamination in fresh leafy greens is readily apparent. By the time the 2006 E. coli bagged spinach outbreak was over, for example,  204 people had become ill across 26 states and Canada, 104 had been hospitalized, 31 had developed the serious complication of hemolytic-uremic syndrome (HUS), and 3 had died, according to information from the USDA.


In information that was updated in 2009, USDA said the contamination was traced to a load of spinach from a 2.8-acre field that was packed at one processing facility on August 15. Potential environmental risk factors at or near the field included the presence of wild pigs and irrigation wells near surface waterways exposed to feces from cattle and wildlife.

The outbreak strain of E. coli O157:H7 was identified in samples of river water, cattle feces, and wild pig feces on the ranch. The closest contaminated sample was just under 1 mile from the spinach field. But the precise means by which the bacteria spread to the spinach remains unknown.


Photos courtesy of the USDA’s Agriculture Research Service.  Top: Microbiologist Manan Sharma (center) and student Sean Ferguson (left) observe whether fluorescent E. coli cells are internalized into roots of baby spinach plants.  Lower:  E. coli, magnified about 7,000 times.   


© Food Safety News
  • R. R. Ihrig

    Intuitively, it appears that pathogens or any microscopic organism could travel throughout the circulatory system and any part of any vegetable or fruit tree–any kind of plant, for that matter. If not, what prevents it? The fluorescent gene approach seems an efficient method to learn the answer. I would like to see studies funded by a disinterested entity, even though the USDA is, or should be, above reproach,
    Also, how would one sterilize the inside of an apple that contains a pathogen, after sterilizing its outer surface?