Microbial tools for tracking the source of E. coli contamination
By Nancy Hall, University Hygenic Lab

Escherichia coli is found in both human and animal intestines.  It is the predominant fecal coliform bacterium, and an indicator of fecal pollution.  Its presence indicates that disease producing organisms may be present, but does not determine whether the source is animal or human.

Watershed management can reduce fecal loading.  This is a prerequisite for remediation plan formulations. In the past there have been beach closures in 5 of 35 Iowa state parks.

She described the methods that have been used for detection, and the early attempts to differentiate the source. One was a fecal coliform to fecal streptococci ratio. This is inaccurate because one of the organisms persists longer in the environment.  There is a need for source (animal species) tracking methods.

She then talked about the methods of source tracking.

Bactgeriophage typing a virus that infects bacteria.  This typing scheme has four groups: serotypes 1 and 4 indicate animal feces, serotypes 2 & 3, human and pig feces. 

Genotyping is the nucleic acid probe technique.  Group 1 includes both human and animal, Group 2 humans in North America, Group 3 includes humans in other parts of the world and Group 4 is primarily animal.  The University of Massachusetts has received a grant to use this method to test waterways in Massachusetts and it will be interesting to see the results.

The Toxin Gene Biomarker was developed by Betty Olson of the University of California in Irvin. It detects the occurrence of toxin genes in E coli using PCR technologies.

There are two kinds of E. coli that cause disease, others do not cause disease and it is important to distinguish between them.  There are three biomarkers for toxin genes in E. coli, for human, pig and cattle.  A biomarker for bird feces is pending. 

Ribotyping of E. coli automatically generates genetic fingerprints.  It is a useful tool for tracking outbreaks and for identifying human and non human pollution. E. coli cells are lysed and release their DNA which is cut into fragments using special restriction enzymes.  The fragments are separated by size through electrophoresis and run through a gel.  Because these fragments have a charge they will travel at different speeds and intensity through the gel.  The pattern of the fragments in the gel is transferred from the gel membrane, mixed with a DNA probe and chemi-luminescent chemicals to produce a visible band pattern from which a DNA analysis can be made. The computer software can do isolates in 8 hours.

Eight  human pathogens have been collected from all over the state.  Some are the same but the animal isolates are all distinct so that they can tell which animal they came from.

In order to do source identification they need to have a  database of known patterns they can work from to compare with the sample they are testing.  A small sample size is not as accurate as a larger sample because it is prone to bias. The process is expensive. The cost is a minimum of $100 per isolate and some want to screen 500 isolates.

Recent published Ribotyping studies indicate that correct classification of samples is in the 90% range, but when all sources are grouped the percent came down to 74%.

In a park service study half of the E. coli were not identified, however they were able to identify seven waste sources.

In summary she said that all source tracking methods have strengths and limitations, we need better criteria and the methods continue to evolve.

A tool box approach to the problem is advocated, using watershed evaluation, key monitoring parameters, strategic monitoring sites and microbial and chemical tracking tools.

Cephalosporin resistant E. coli 
Patricia Winokur, Dept. of Internal Medicine, Iowa City

A project studying Cephalosporin Resistant E coli in Iowa started in 1999 and has continued for three years.

84.2% of antibiotic use is for livestock and only 12.8% for human use.  In livestock, therapeutic uses account for  24.5  million, therapeutic uses, 2 million.

Data around the world suggests that antibiotic resistant organisms are being transferred from animals to humans.  In Europe Vancomycin resistant Enterococci were found in animals.  Then humans were found to be carrying the same antibiotic resistance.

A story in the New England Journal of Medicine told of a Quinolone resistant Campylobacter in Missouri from poultry products and humans with identical molecular typing.  There are absolutely identical products in poultry products, so it has been shown that these bacteria can transfer back to meat products.

There are now Quinolone resistant salmonella .

These bacteria can be transferred by direct contact between animals and man.  There is also food-borne transmission of resistant bacteria, and environmental contamination.

How the environment may play a role is unknown Data is being acquired to trace this transfer from the farm to the human.
 
Tetracycline resistance has been found in swine production facilities.  The resistant genes have been identified in lagoons and traced to ground water up to 250 miles downstream from the lagoons.  The resistance has also been found in soil bacteria and groundwater is a potential source of transmission.

In 1999 Cephalosporin resistant E. coli and salmonella were found in hogs, cattle and humans.  There are also multiple additional antibiotic resistances. 

A map of E. coli isolates shows that they are found all over Iowa.  There is a clustering of human isolates in  North East Iowa and some in or near the Quad Cities.

All of these isolates had the CMY2 resistant gene.  It is present in plasmids which are easily transferred from organism to organism because the plasmid doesn’t have a species boundary.

This same human Celphalosporin resistant isolate has been identified in 14 different states.  Retail ground meat products with Cepahlosporin resistance have been found in Washington, D.C. stores

In addressing the question of whether Iowa waterways are  a source for transferring resistance from farm to farm, she collaborated with Nancy Hall and the Iowa DNR.  They sampled 62 sites, concentrating on eastern Iowa.  They found that between 2 and 7.1% of the E. coli  were resistant.  The prevalence of resistant bacteria in sick animals is 15.6%.

Over 70% of the resistant bacteria encode the CMY2 gene and determined that it is being spread through waterways.

These bacteria demonstrate co-resistance.  In addition to being resistant to Cephalosporin, they are 100% resistant to Chloraphenicol, Sulfamethoxazole and Streptomycin.  They were resistant to other antibiotics at a lower percentage.

Then they began the molecular typing of CMY2 plasmids.

They want to know how persistent the contamination is.  They plan to target sites with multiple episodes and go back and sample on a daily basis.  They would like to find particular facilities and look at farm soil samples, and learn what the climatic events were.

Conclusion: Cephalosporin resistant E. coli is present in Iowa’s surface waterways.  The expanded spectrum Cephalosporins are the drugs of choice for empiric therapy of gram negative infections
Some CMY 2 organisms are now resistant to most classes of antimicrobial agents

Food-borne transfer of ant microbial resistant organisms continues to be a concern. Water contamination may be an important mechanism from spreading this resistance from site to site.

A question was asked about fish. She said she does not know about fish but would expect it to be in the gut. The reason it is in ground meat is because of fecal contamination.  With non-ground meat if you sear it on the grill that will kill it.

Fresh manure will transfer this to the water system.  If it dries out it will die.  Getting rid of it will be difficult.  In the absence of antibiotics they will persist for a year.