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LINCOLN, Neb. -- A new genetic "fingerprinting" method developed by University of Nebraska food scientists is revealing surprising insights about potentially deadly E. coli 0157:H7 bacteria.

The new technique shows that there are two genetically distinct E. coli 0157:H7 populations found in cattle -- one that causes sometimes fatal food poisoning in people and a second that is not commonly isolated from food poisoning cases. Previously, relationships of different E. coli 0157:H7 populations weren't well-understood.

Genetic results suggest that the population most commonly found in cattle either is non-virulent, meaning it is incapable of causing disease, or it is not easily transmitted to people, said Food Microbiologist Andy Benson, whose laboratory developed the technique that led to the findings.

"Our method gives a very high-resolution snapshot of the genome and lets us see things we couldn't see before," Benson said. The technique, called octamer-based genome scanning (OBGS), allows researchers to pinpoint where genetic differences exist on E. coli's DNA and offers a means for rapidly cloning and identifying the genes at those DNA sites.

Because the OBGS technique will be broadly applicable to many organisms, this Institute of Agriculture and Natural Resources research is generating great interest among genetics researchers. A paper detailing the work of Benson and food science department colleagues Jaehyoung Kim and Joseph Nietfeldt was published this month in the Proceedings of the National Academy of Sciences.

Benson was struck by the idea for the genome scanning method while attending a research conference. After spending eight months working out the bugs, Benson tested the technique on E. coli samples, called isolates, drawn from humans and cattle. All the isolates originated in a three-county region of Wisconsin. Researchers expected that the isolates, when illustrated on an evolutionary tree-type structure called a dendrogram, would be dispersed throughout the structure.

"We really weren't prepared for what we saw," Benson said. Most of the human isolates were clustered together in one part of the dendrogram, and most of the cattle isolates were clustered together, indicating that there were significant genetic differences between the two groups.

"That could mean two things," Benson said. Either their data were biased because all were collected from the same region, or -- surprisingly -- the E. coli population infecting cattle in Wisconsin was distinct from the population making people sick.

To test for bias, Benson's team used the method again on a large group of isolates gathered from 16 states. Again, two distinct populations -- human and bovine -- appeared on the dendrograms.

Benson's hypothesis: the cattle population either is very inefficient at causing human illness or it is a weak population that does not survive the necessary hurdles to infect people and cause E. coli related illness.

This could be good news for the public health and for cattle producers, especially following data recently released by U.S. Department of Agriculture researchers at the Meat Animal Research Center (MARC) at Clay Center, Neb., that found E. coli 0157:H7 to be present in more than half of cattle tested.

"This fits well with the MARC data," Benson said. "Only about 20,000 cases of human infection with E. coli 0157:H7 are reported each year. You would expect a much higher number, given the number of cattle infected." In comparison, an estimated 2 million cases of Salmonella poisoning occur annually, many caused by eating contaminated poultry and beef, Benson said.

"The key for us now is to get a good fix on the numbers," Benson said. "Our numbers now suggest that two-thirds of the isolates from cattle are genetically related to the apparently non-virulent population -- the population that doesn't cause illness. But we need to do a much larger sample size to get a better idea of exactly what that number is. We're doing that now."

Benson's team analyzed 78 E. coli 0157:H7 samples to reach the current results. He plans to test hundreds to get his "good fix on the numbers."

As with all significant research, Benson's work has opened many new avenues of study.

"We have our work cut out for us. There are a lot of different directions we can go with this," he said.

The researchers already are fine-tuning the OBGS method, making it faster and easier. Another ongoing project is cloning and sequencing the DNA of a specialized virus, called a bacteriophage, that Benson believes is associated with the genetic divergence between the E. coli populations. Sequencing work on the bacteriophage may provide clues to the mystery of why the prevalent bovine 0157:H7 population isn't commonly found in humans.

Perhaps the most practical application of the OBGS method will be in developing a more sensitive test of E. coli 0157:H7 isolates, Benson said.

"We're using OBGS to pinpoint what the exact genetic differences are between populations so that we can develop a much simpler test that can discriminate the two. This would make it much easier to do extensive testing, such as in feedlot populations," he said.

Benson cautions that researchers must be careful not to over-interpret the data at this point. The newly-discovered population must be tested to determine if it is, in fact, non-virulent. The most powerful experiment is one that can't be done, Benson said.

"The only way to be absolutely sure the non-virulent 0157:H7 population won't cause human illness would be to test it on people. Obviously, this isn't an experiment that we could do or would do, so we have to come at it from other ways, such as using model systems, epidemiology studies and in vitro studies," Benson said.

This IANR Agricultural Research Division research was funded by a USDA National Research Initiative competitive grant and by Nebraska Legislative Bill 1206, which appropriated $250,000 annually for five years for E. coli research.

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11/22/99-MN
Contact: Andrew Benson, Ph.D, assistant professor, NU Department of Food Science and Technology, (402)472-5637
Vicki Miller, science writer, IANR Â鶹´«Ã½ and Publishing, (402)472-3030 [email protected]