Progress 10/01/98 to 09/30/03
Outputs
1. Colitag is a simple, rapid, and sensitive way to detect and enumerate the fecal indicator bacterium Escherichia coli in raw sprouts. This result is important because raw sprouts are an important source of fecal pathogens, and a major cause of foodborne outbreaks. Colitag can be used to monitor the microbiological safety of sprouts. 2. Aeromonas can interfere with coliform tests for fecal contamination, but this interference can be eliminated in two ways: A) Use of an elevated incubation temperature such as 44.5 degrees C. This is the preferred way to use Colitag medium. B) If one is forced by regulations or tradition to use lower temperatures in the 35-38 degree C range, one may use chemical inhibitors. Two inhibitors are especially promising: the antibiotic cefsulodin and the herbicide 2,4-D. Cefusulodin does not interfere with Colitag's ability to resuscitate chlorine-injured E.coli, but the antibiotic is unstable in solution. The herbicide 2,4-D is stable, but it
interferes with Colitag's ability to resuscitate chlorine-injured bacteria. These results are important because lactose-fermenting aeromonads are ubiquitous and quite capable of giving false positive tests for fecal contamination, which may result in needlessly discarding food or holding food for retesting, which is costly. 3. In laboratory experiments with both artificial seawater and authentic seawater from Bodega Bay, we found that the inclusion of sodium pyruvate results in improved resuscitation of seawater-injured E.coli. However we were not able to test sodium pyruvate on naturally-contaminated seawater because we could not locate sufficiently contaminated material. We have just located a source of contaminated seawater, and in our next AES project we plan to study this material. These studies are important because about 40% of America's wastewater is discharged into saltwater. Thus seawater must be tested for fecal contamination. Unfortunately conventional bacteriological
tests for fecal contamination appear to have serious false-negative problems in salt water, thus giving a false sense of security. We hope to reduce the false-negative problem by improving the resuscitation of seawater-injured E.coli. 4. The ability of acidic Colitag 3 to resuscitate indicator bacteria that have suffered other forms of injury. We know that acidic Colitag 3 efficiently resuscitates chlorine-injured E.coli, but we do not know if it will aid the resuscitation of bacteria that have suffered other types of injury. So far we have found that Colitag does NOT improve resuscitation of E.coli that have been injured by heat or freezing and thawing. These results are important because chlorination is a major method for disinfecting water and food handling surfaces. Heating and freezing are major methods of food preservation. If bacteria are not killed, but merely injured by these processes, they may live to infect humans and cause disease. These injured bacteria are not
detectable by conventional bacteriological methods. However Colitag 3 will detect survivors of chlorine treatment. Thus it can be used to improve monitoring of chlorine-treated waters and surfaces.
Impacts
All of our research is focused on improvement of methodology for detection of fecal contamination in food and water. Improved methodology can be used to reduce the incidence of gastrointestinal infection form these sources.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Detection of the fecal indicator Escherichia coli in alfalfa sprouts: We have started to explore the limits of sensitivity of the Colitag method for detecting E. coli in alfalfa sprouts. In our latest experiments, we are inoculating five (5) grams of sprouts in 45 ml of Colitag with as little as one E. coli cell, incubating at 44.5 deg C, and observing indole production. Positive indole tests indicate the presence of E.coli. (The more conventional ONPG test for fecal coliforms is not useful here because sprouts have a large natural coliform population. Nor can the popular MUG for E. coli test be used, because sprouts often contain fluorescent substances that give false positive MUG tests.) Because our high-sensitivity Colitag tests can be conducted in disposable bags, we have nicknamed the test "Tag in a Bag." False Positive coliform tests due to non-target Aeromonas: As mentioned in previous reports, the ubiquitous aeromonads can generate false positive total
coliform tests at 35 degrees C. Last year we speculated that this was why the Standard Methods includes a tedious two-medium procedure for presumptive and confirmed coliforms. A search of the older literature and a few experiments indicate that this is indeed the case. We conclude that IF scientists of the day knew that the interference was from a single genus of bacteria, and IF they knew (as we found a decade ago) that 2,4-dinitrophenol is a selective inhibitor of aeromonads, they could have formulated an effective one-tube coliform medium in the 1930s. Such a medium would have saved half the labor and testing time of every coliform test done for nearly 70 years! Detection of E. coli O157:H7 with Colitag: Pathogenic E. coli O157:H7 cannot be detected by the popular MUG tests because the bacterium lacks a MUG degrading enzyme. In principle, O157:H7 could be detectable by the indole test of Colitag. However this cannot be done at the fecal coliform incubation temperature (44.5 deg C)
used with Colitag. We are beginning to explore the use of lower incubation temperatures. We are trying to find a temperature low enough to permit single cells of O157:H7 to grow, but high enough to eliminate false positive interference from indole positive bacteria such as Klebsiella oxytoca and Aeromonas. In preliminary experiments, temperatures in the 42-43 deg C range seem to be suitable.
Impacts
Waterborne and foodborne illnesses are major causes of morbidity and mortality. Our experiments with Colitag will help to protect the public from these diseases.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Defense of the Colitag patent. Much of our effort in 2001 was expended in the defense of the Colitag patent of the Regents of the University of California. In a long series of experiments and written materials, we demonstrated that, contrary to the charges of the plaintiff, Colitag is a rich, nutritive medium that supports the luxuriant, non-specific growth of a great variety of coliform and non-coliform bacteria. After considering the data, the US Superior Court of Connecticut issued a summary judgement dismissing the plaintiffs' suite. In addition, we pushed Colitag to its limits for detecting E.coli in decomposing alfalfa sprouts. These sprouts have massive populations of coliform bacteria that can swamp out a few cells of the target E.coli. We found that when the coliforms outnumber the E.coli by roughly six logs, the target E.coli are no longer detectable unless the medium is subcultured. This is the same detection limit obtained with the more tedious Standard
Methods LTB+EC/MUG medium. Curiously, we could not reproduce this swamping-out effect in model experiments with pure cultures of Klebsiella pneumoniae isolated from the sprouts. Decreasing interference in Colitag from non-target Aeromonas and other oxidase-positive bacteria. As mentioned in previous reports, we are interested in preventing the ubiquitous aeromonads from generating false positive coliform tests at 35 degrees C. (This interference was probably the major reason that the Standard Methods includes a tedious two-media procedure for presumptive and confirmed coliforms.) As we extended our studies to foods of plant origin, we find that Pseudomonas and other oxidase-positive bacteria were also sources of interference. In our ongoing search for suitable inhibitors of all these interfering bacteria, we found that in addition to dinitrophenol, the herbicide 2.4-D is a promising compound. Service activities. Over the years we have become a resource for those who have questions
about water testing. In 2001 we helped the university Department of Environment and Health Services conduct sanitary monitoring of Strawberry Creek which runs through the campus. As a result of our collaboration, they decided to use Colitag as a convenient in-house monitoring tool, rather than sending their samples out for coliform analyses.
Impacts
Waterborne and foodborne illnesses are major causes of morbidity and mortality. By defending the Colitag patent, we helped assure that it will be available to protect the public from these diseases
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
There is excellent agreement between our Colitag INDOLE TEST and more tedious classical methods. Plant materials are full of fluorescent chemicals and bacteria that interfere with the popular MUG tests for E.coli. The Colitag INDOLE test is free from such interference. The popular Petrifilm filter test for E.coli is easily swamped and inactivated by the huge coliform populations. The Colitag INDOLE test is free from such problems. We can detect as few as one E.coli per gram of sprouts (we chose sprouts because they tend to have very high bacterial populations, and to pose an inordinate risk of foodborne illness). Even though we can detect E.coli easily by the multiple fermentation tube method, using Colitag medium, it is not so simple to isolate viable cultures of the bacterium. However we have found that by promptly subculturing our positive tubes under selective conditions, we can obtain pure cultures of our one target bacterium. This problem is much more severe in
plant materials than it is in other foods. Colitag test works very well with shellfish and other seafood. Plant materials present formidable problems, and we are starting with alfalfa sprouts. G W Chang and R L Tung. Simultaneous Enumeration of E.coli and Total Coliforms. US Patent #6,146,840. November 14, 2000.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
In the the last year we found: 1) The Colitag indole test for E.coli is very effective with alfalfa sprouts if it is incubated immediately at 44.5 degrees C. This eliminates interference from the massive natural coliform microflora of the vegetable. 2) Alfalfa sprouts have an intrinsic fluorescence, even when incubated at 44.5 deg. This makes the popular MUG test unreliable in the most concentrated tubes. 3) When compared to the 3M Petrifilm, Colitag is thousands of times more sensitive to E.coli in the presence of natural coliforms.
Impacts
(N/A)
Publications
- No publications reported this period
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