An official website of the United States government.
Our overall objective is the development of improved methods for the detection and enumeration of fecal indicators and pathogens in food and water. Our narrower objective is the use of what we call the "Colitag approach" to develop improved tests for bacteria such as Escherichia coli and Salmonella. By "Colitag approach," we mean using microbial physiology and genetics in order to devise faster, simpler, less costly test methodology. Examples of the "Colitag approach" include: <OL> <LI> Use of low carbohydrate, peptone-based media to assure induction of catabolite-repressible metabolic pathways such as those for ONPG and MUG cleavage, and indole production. <LI> Exploitation of our discovery that a slightly acidic (pH 6.2) medium will massively enhance detection of injured but viable bacteria. <LI> Use of substrates such as trimethylamine-N-oxide (TMAO), which occurs naturally in seafood, to enable bacteria to neutralize their own environment and change its pH from 6.2 to near neutrality. <LI> Judicious control of incubation temperatures to eliminate the growth of competing bacteria without inhibiting target pathogens or indicators.
</ol> We propose to address the following goals in this project: <ol> <LI> Modification of "Colitag 3.0" medium for use in a variety of foods. <LI> Use of the "Colitag approach" to improve methods for detecting pathogenic bacteria such as E.coli O157:H7 and Salmonella. <LI> Re-examination of the history of microbiology. Reassessment of classical methods in the light of our more modern understanding of microbial physiology and population ecology. While this will not yield scientific breakthroughs, it will give students a much greater awareness of and insight into applied science.
NON-TECHNICAL SUMMARY: Food and water borne illness is a major cause on morbidity and mortality throughout the world. Much of this illness is transmitted by the fecal-oral route. We are developing ever better methods for detecting fecal contamination and pathogenic bacteria in food and water.
<P>
APPROACH: 1) Modification of Colitag 3.0 for use in a number of foods. We have already found that for shellfish meat, Colitag medium is fastest and most economical when the enzyme substrates ONPG and MUG are omitted. These expensive substrates are useless because of their high false positive rate in shellfish. The indole test of Colitag is quite enough. We have also found that for sprouts of alfalfa and brassicaceous vegetables, it is best to incubate samples with Colitag 3.0 at 44.5 deg C, in order to eliminate competition from endogenous coliforms. We propose to extend these studies to other fruits and vegetables that are eaten raw. For example salad ingredients, tree fruits, and berries. 2) Use of Colitag principles for detection of pathogens such as O157:H7 and Salmonella. For O157, we propose to experiment with factors such as incubation temperature (O157 is too temperature-sensitive to grow at the usual fecal coliform incubation temperature of 44.5 deg C. Yet it is slightly more thermotolerant than is most of the competing microflora.) We also propose to modify a solid medium we devised, mX medium, in order to have a more economical alternative for isolation and confirmation of positive O157 cultures. For Salmonella, we propose to explore the use of slightly acidic media such as Colitag 3.0 for the initial enrichment step. We also propose to explore use of TMAO for both growth enhancement and self-neutralization of acidic selection media. 3) Re-examination of classical methods. For much of the 20th century, sanitary microbiologists have used a tedious, two-step incubation for coliform counting. We have recently discovered that they could have done the job in half the time if they had realized two simple facts: A) The interfering bacteria that necessitated the second incubation were actually members of a single genus, Aeromonas. B) Aeromonas interference could be eliminated very simply by formulating media with dinitrophenol, a cheap, readily available chemical. Had microbiologists known these two simple facts, they could have done 20th century coliform testing in half the time with half the trouble. We believe that the traditional use of lactose broth (LB) for the initial enrichment step of Salmonella isolation procedures will turn out to be a similar story. We suspect that LB works by allowing incidental bacteria to produce acid, and that the acid is the critical selective agent. We will investigate this hypothesis in the course of developing acidic enrichment media for Salmonella.
<P>
PROGRESS: 2004/10 TO 2009/09<BR>
In the final project period, we made progress in two areas: 1) Clarification of how aquatic aeromonads interfere with coliform tests for fecal contamination in water; and 2) Further optimization of the Colitag medium we devised some years ago. Details follow: 1) Aeromonas project: Our intitial hypothesis was that aeromonads generated false-positive coliform tests and necessitated the development of the tedious coliform test that was used for most of the 20th Century. We tested the hypothesis by observing gas formation by a wide range of aquatic aeromonads in the Presumptive Coliform Test. We obtained this wide range of bacteria in four very different ways: 1) From local creek water: We selected ampicillin-resistant bacteria, many of which were aeromonads. Then we tested the aeromonads. 2) From urban runoff: We did traditional coliform tests, cultured positive tubes, and isolated coliforms and aeromonads. 3) From the American Type Culture Collection. We ordered over a dozen lactose-positive aquatic aeromonads. 4) From our historical collection. We tested all the aeromonads that we had isolated in 15 years of water testing. We found that NONE of our PURE CULTURES of aeromonads produced false-positive presumptive coliform tests. These results leave us with an alternative hypothesis, that MIXED CULTURES of aeromonads and non-coliform enterobacteriaceae can symbiotically give the false-positive tests. In addition, we explored methods for eliminating Aeromonas interference from our rapid, one-step Colitag coliform test. We found that the antibiotic cefsulodin will eliminate the false-positives without compromising the test's sensitivity to environmentally injured coliforms.2) Further optimization of Colitag. When Colitag moved from thelaboratory to the field, it got a great deal of feedback from commercial formulators and end users. A major complaint was the malodorous and hygroscopic nature of a key ingredient, TMAO. We found that nitrate or fumarate could partially replace TMAO, but that some TMAO was needed for the brilliant color and fluorescence that we expect of Colitag.
<BR><BR>
IMPACT: 2004/10 TO 2009/09<BR>
While the Aeromonas experiments did not solve the mystery of the false positives in the presumptive coliform test, they did provide an easily testable alternative hypothesis. The Colitag optimization experiments paved the way form making the commercial Colitag medium more acceptable to producers and users.