Escherichia coli 0157:H7 has been recognized as an important human pathogen. Studies have shown that it is principally transmitted through food, Escherichia coli 0157:H7: Epidemiology, Pathogenesis, and Methods for Detection in Food, Nisha V. Padhye and Michael P. Doyle--Journal of Food Protection, Vol. 55, No. 7, Pages 555-565 (July 1992). There is thus a need for a rapid diagnostic test for the presence of Escherichia coli 0157:H7 in food in order to prevent the spread of Escherichia coli 0157:H7 through the food supply.
Pradhye and Doyle, supra, survey methods of detection of Escherichia coli 0157:H7. A stable characteristic of Escherichia coli 0157:H7 is that it will not ferment sorbitol within 24 hours whereas other strains of Escherichia coli will produce fermentation in sorbitol under incubation temperatures within 24 hours, and this characteristic has been used in processes for the isolation of Escherichia coli 0157:H7 from other enterics. Since there are microorganisms other than Escherichia coli 0157:H7 that do not ferment sorbitol, including some strains of Escherichia coli, this characteristic is not sufficiently specific to serve as an identifying test for Escherichia coli 0157:H7.
Anita J. Okrend, Bonnie E. Rose and Charles P. Lattuada describe an improved plating medium in Use of 5-Bromo-4-Chloro-3-Indoxyl-Beta-D-Glucuronide in MacConkey Sorbitol Agar in the Isolation of Escherichia coli 0157:H7 from Ground Beef, Journal of Food Protection, Vol. 53, No.11, Pages 941-943 (November 1990). This article describes a plating medium in which 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide acid cyclohexylammonium salt was dissolved in ethanol and the solution added to MacConkey Sorbitol Agar. Since approximately 97% of all Escherichia coli are beta-glucuronidase positive, but Escherichia coli 0157:H7 is beta-glucuronidase negative, this medium responds to the presence of Escherichia coli 0157:H7 by isolating white colonies rather than isolating blue colonies resulting from beta-glucuronidase positive microorganisms.
The process of isolating and detecting the presence of Escherichia coli 0157:H7 in a test sample by means of the processes described above, requires inoculation of the plating medium with the test sample, incubating the inoculated plating medium for a period of time, usually over night, and examining the surface of the plating medium to locate colonies of microorganisms in the incubated plating medium. Identification of Escherichia coli 0157:H7 is determined by the shape of the colony, size of the colony and color of the colony in the plating medium.
The color of the colony in the plating medium is a characteristic of the particular medium. U.S. Pat. No. 5,464,755 of Barry Bochner entitled Microbiological Medium and Method of Assay; for Bacteria describes a plating medium adapted to produce colonies in three different colors. The examination of incubated plating media under a microscope is an exacting and time consuming task, and in the plating methods of the prior art, a positive response results in a presumptive identification of Escherichia coli 0157:H7 which must be verified by other testing methods. In short, an identification of Escherichia coli 0157:H7 cannot be made by prior art methods in less than about one day and at substantial expense due to the labor required to analyze the plating medium and the cost of the plating materials.
In an article published in the Journal of Microbiology--Volume 39 (1993) at pages 133-158, by P. M. Zadik, P. A. Chapman and C. A. Siddons, entitled Use of Tellurite for the Selection of Verocytotoxigenic Escherichia coli 0157:H7, experiments are described in which plating media containing mixtures of MacConkey sorbitol and potassium tellurite are subjected to mixed cultures of microorganisms. It was found that such plating media can be effective to reduce the growth of other strains of Escherichia coli than Escherichia coli 0157:H7 without materially affecting the growth of Escherichia coli 0157:H7. Further, such plating media were found to suppress the growth of other important enteric microorganisms, excepting Shigella.
USPTO Backgrounds
USPTO Backgrounds