METHOD FOR ISOLATION AND IDENTIFICATION OF ESCHERICHIA COLI 0157:H7 AND PLATING MEDIA FOR SAID PROCESS

Abstract: A solid plating medium for the presumptive detection of Escherichia coli 0157:H7 which selectively promotes growth of Escherichia coli, inhibits growth of gram positive microorganims, inhibits growth of Proteus species, inhibits the growth of Escherichia coli other than Escherichia coli 0157:H7, wherein said plating medium includes agar, a carbohydrate medium which is not fermented by Escherichia coli 0157:H7 but is fermented by other microorganisms to include other strains of Escherichia coli and results in a first color when fermented, a chromogenic beta-galactosidase substrate which produces a second color when fermented by beta-galactosidase, and a pH indicator dye which changes said medium to a third color.

Description: METHOD FOR ISOLATION AND IDENTIFICATION OF ESCHERICHIA COLI 0157 :H7 AND PLATING MEDIA FOR SAID PROCESS The present invention relates to a process for isolating Escherichia coli 0157 :H7 from other strains of Escherichia coli and other microorganisms, and to a solid plating media suitable for use in that process. BACKGROUND OF THE INVENTION 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. Pathoαenesis, 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 microoranisms 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. United States Patent 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 analyse 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 CA. 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 Shigell . STATEMENT OF INVENTION It is an object of the present invention to provide a method of isolating and presumptively identifying Escherichia coli 0157 :H7 which utilizes solid plating media and is more reliable than plating media methods known to the art, that is, makes a positive presumptive identification of Escherichia coli 0157 :H7 and reduces the percentage of false positive determinations from that of prior art plating media methods. It is a further object of the present invention to provide a method of isolating and presumptively identifying Escherichia coli 0157 :H7 which utilizes a solid plating medium and achieves its results in a significantly shorter time than processes of the prior ar . It is a further object of the present invention to provide a method of isolating and presumptively identifying Escherichia coli 0157 :H7 which utilizes a solid plating medium and achieves its results at a significantly lower cost than processes of the prior art. It is also an object of the present invention to provide plating media adapted for use in the methods described above. The present invention comprises a solid plating medium which utilizes three mechanisms to produce an indication of the presence of Escherichia coli 0157 :H7 in a test sample. First, the plating medium contains one or more carbohydrates to provide growth of microorganisms and which are not fermented by Escherichia coli 0157 :H7 but may be fermented by other microorganisms including other strains of Escherichia coli. If a microorganism is present which ferments the carbohydrate, the medium is selected to change to a first color and indicates the presence of a microorganism other than Escherichia coli 0157 :H7. Second, the plating medium contains a chromogen responsive to the presence of beta-galactosidase, and third, the plating medium contains ingredients for restricting the growth of microorganisms other than Escherichia coli 0157:H7. More specifically, a solid plating medium for the presumptive identification of Escherichia coli 0157 :H7 according to the present invention comprises: (1) an ingredient for selectively promoting the growth of Escherichia coli cells under incubation which is not fermented by Escherichia coli 0157 :H7 but is fermented by other microorganisms including other strains of Escherichia coli, and -if fermented the growth ingredient changes to a first color; (2) an ingredient for inhibiting the growth of gram positive microorganisms under incubation; (3) an ingredient for inhibiting the growth of Proteus sp. under incubation; (4) an ingredient for inhibiting the growth of strains of Escherichia coli other than Escherichia coli-0157:H7 under incubation; (5) a chromogenic substrate capable of forming a second color upon reacting to beta galactosidase; and (6) a pH indicator dye which changes to a third color when the pH of the medium changes . The method of presumptive identification of Escherichia coli 0157 :H7 according to the present invention comprises inoculating a mass of the plating medium described above with the sample under test, thereafter incubating the mass of inoculated plating medium at a temperature between 30 degrees and 40 degrees Celsius for a sufficient period to produce microorganism colonies in the mass of plating medium, and thereafter examining the surface of the mass of plating medium for colonies of the second color. Emergence of the first or third color indicates the presence of microorganisms other than Escherichia coli 0157 :H7. The invention will be more readily understood from the following detailed description of the invention, which contains no drawing. DETAILED DESCRIPTION OF THE INVENTION The inventors' preferred detection system for Escherichia coli 0157 :H7 utilizes solid plating media containing sorbitol, indoxyl-beta-D-galactopyranoside and tellurite. Growth of most microorganisms in sorbitol, including Escherichia coli, results in production of beta-galactosidase, which in turn produces an enzyme which reacts with indoxyl-beta-D-galactopyranoside to produce an insoluble percipitate with a blue color. Hence, the presence of colonies of microorganisms in the medium are clearly visible and clearly defined. The presence of tellurite in the medium suppresses the growth of most microorganisms other than Escherichia coli 0157 :H7 and a few others which are suppressed by other means to be described hereinafter. Hence, the observance of blue with black percipitate colonies on the surface of the plating medium is a direct indication of Escherichia coli 0157 :H7 and a presumptive identification. While Escherichia coli 0157 :H7 is sorbitol negative, 80.3% of Escherichia coli, and most other enteric microorganisms, are sorbitol positive. Sorbitol reacts to the presence of sorbitol positive microorganisms to produce white to yellow colonies . Hence, the presence of a white to yellow colony on the surface of an incubated plating medium of the present invention, is an indication of the presence of a microorganism other than Escherichia coli 0157 :H7, thus making a blue colony indication nondeterminative of Escherichia coli 0157:H7. Sorbitol is, in general terms, the carbohydrate ingredient of the plating medium. Other carbohydrates which are not fermented by Escherichia coli 0157 :H7 are salicin, inositol and adonitol, and these compositions and sorbitol, or a mixture thereof, have been found suitable for the carbohydrate ingredient of the plating media. Modified buffered peptone water consisting of peptone, NaCl, Na2HPθ4, KH2 O4, casamino acids, yeast extract and lactose has also been found satisfactory. Alternative carbohydrates at concentrations up to 10.0 grams per liter are mannitol, dulcitol, d-sorbitol, I-arabinose, I-rhamnose, maltone, d-xylose, trehalose, d-mannose and melibiose. Indoxyl-beta-D-galactopyranoside is a chromogen which determines the presence of beta-galactosidase. Beta- galactosidase is an enzyme produced by Escherichia coli and other coliforms, and this enzyme reacts with indoxyl-beta-D- galactopyranoside to produce an insoluble indigo blue percipitate. Other chromogens may be used in place of, or in combination with, indoxyl-beta-D- galactopyranoside, such as 5-bromo-4-chloro-3-indoxyl- beta-D-galactopyranoside . In the preferred embodiment, isopropyl-beta-D- thiogalactopranoside is also added to the plating medium. This ingredient enhances the production of the beta-galactosidase enzyme. In the preferred embodiment of the present invention, other inhibitors which will not inhibit the growth of Escherichia coli 0157 :H7 are employed in addition to tellurite. An inhibitor for gram positive bacteria is also utilized, and in the preferred composition it is bile salts #3. Other inhibitors of gram positive bacteria can also be employed. The medium of the preferred embodiment also contain a growth inhibitor for Proteus sp, namely, sodium novobiocin. Other inhibitors for Proteus s . could also be employed. The media of the present invention also contain nutrients to promote the growth of microorganisms, especially protein. In the preferred embodiment, a mixture of tryptone, bacto-peptone and proteose-peptone is used, but it is to be understood that each of these ingredients can be separately used or used in combination with other nutrients. Further, there are many other nutrient which will support the growth of microorganisms which may be used in place of the above named nutrients . The preferred embodiment also uses a pH indicator dye to permit ready determination of the pH of the medium. The pH of the medium should be adjusted to 6.6 to 6.8. Sodium chloride is also added to the medium for osmolarity purposes. There are some Escherichia coli in addition to Escherichia coli 0157 :H7, and some other microorganisms, which are not sorbitol fermenters and are beta-glucuronidase active. The medium described above is not responsive to such microorganisms, but is more specific to Escherichia coli 0157 :H7. Optionally, a beta-glucuronidase chromogen may be admixed with the medium to produce a response to beta-glucuronidase. A preferred beta-glucuronidase chromogen is 6-chloro-3-indoxyl-beta-D-glucuronide which responds to beta-glucuronidase by producing a salmon precipitate in the plating medium which may be observed and counted. The preferred embodiment of a plating medium contains the ingredients in the proportions set forth in the following Table I. TABLE I MATERIAL MEASUREMENT Tryptone 5.0 grams/liter Bacto-peptone 10.0 grams/liter Proteose-peptone 3.0 grams/liter Sorbitol 12.0 grams/liter Salicin 10.0 grams/liter Inositol 10.0 grams/liter Adonitol 8.0 grams/liter Sodium chloride 5.0 grams/liter TABLE I (Continued) MATERIAL MEASUREMENT Phenol red 0.1 grams/liter Bile salts #3 1.25 grams/liter indoxyl -beta-D-galactopyranoside 0.120 grams/liter 5 -bromo-4-chloro-3 -indoxyl -beta-D-galactopyranoside 0.120 grams/liter isopropyl-beta-D-thiogalactopyranoside 0.100 grams/liter Agar 15 grams/liter Novobiocin 10 milligrams per liter Potassium tellurite 0.5 to 2.0 milligrams/liter OPTIONAL 6-chloro-3 -indoxyl-beta-D-glucuronide 0.120 grams/liter Except for novobiocin and potassium tellurite, the ingredients are mixed in any order, the pH adjusted to 6.6 to 6.8, boiled to sterilize the mixture, and the mixture is permitted to cool to room temperature. Thereafter sterile novobiocin and sterile potassium tellurite at room temperature are added aeseptically to the other ingredients. The composition is then poured onto plates and permitted to dry for 48 to 72 hours, and it is then ready to be used. The process of the present invention requires a plate or mass of the plating medium to be inoculated with the test sample, and the inoculated mass is then incubated for a period of time to permit growth of the microorganisms in the test sample to observable colonies. The inventor has found that with the preferred plating medium described above, a period of 24 hours of incubation is sufficient time for Escherichia coli 0157 :H7 colonies present in raw hamburger to grow into colonies which are readily observable with a naked eye. It is believed that the rapid growth of microorganisms in the preferred plating medium is due to the abundance of nutrients provided by the tryptone, bacto-peptone, proteose-peptone, sorbitol salicin, inositol and adonitol. The surface of the plating medium mass is then assayed and the presence and number of blue with black percipitate colonies recorded. Also, the presence of white or salmon colored colonies is noted as an indication of microorganisms other than Escherichia coli 0157:H7. It is to be noted that no special equipment is required to observe the incubated mass of plating medium, and the time required to note the number and presence of blue with black percipitate colonies is far less than required when other colonies are present. Also, there are no ingredients in the plating medium that are especially costly. Hence, an assay of a test sample may be made at reduced cost from assays with prior plating media, and it may be made in the same day that the test sample was taken. The following Table II sets forth examples of use of the plating medium set forth in Table I, without the optional substrate, by the process described above, the test sample containing the microorganism shown in the left column and the observed colonial description being set forth in the right column. TABLE II # of Bacterial species Strains Colonial Morphology Escherichia coli 0157 :H7 26 Domed to raised colony; Typical strains 1.5 -2.5 mm in diameter. Dark blue to black color. No ring around colony. Escherichia coli 0157 :H7 1 Domed to raised colony; Beta-glucuronidase 2.0 mm in diameter. positive Dark blue to black color. No ring around colony. Bacterial species Strains Colonial Morphology Escherichia coli 0157 :H7 1 Domed to raised colony; Sorbitol positive 2.0 mm in diameter. Blue to turquoise color. No ring around colony. TABLE II (Continued) # of Escherichia her annii 2 Domed; pinpoint to <1 mm in diameter; clear to light blue color Escherichia coli 3 strains no growth. Pinpoint to 2 mm in diameter; clear to green color. Salmonella spp. Minimal growth; <1 to 1 mm in diameter; clear/ white to yellow color Pseudomonas cepacia Domed; 1 to 2 mm in diameter. Clear color. Pseudomonas aeruginosa Domed; pinpoint to <1 mm in diameter. Clear color. Providencia stuartii Domed; <1 mm in diameter. Clear to white color. Pseudomonas picketti Klebsiella spp Enterobacter spp . Proteus spp . Morganella sp . 16 No growth for all strains Citrobacter spp . Acinetobacter calcoaceticus Providencia alcalifaciens Yersinia enterocolitica Those skilled in the art will devise other methods of utilizing the plating media of the present invention, and other plating media than those specifically described in the foregoing specification within the scope of the present invention. It is therefore intended that the scope of the present invention be not limited by the foregoing specification, but rather only by the appended claims .

Claims: The invention claimed is: 1. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising a mixture of (1) an ingredient which selectively promotes growth of Escherichia coli cells under incubation, (2) an ingredient which inhibits growth of gram positive microorganisms under incubation, (3) an ingredient that inhibits growth of Proteus sp. under incubation, (4) an ingredient which inhibits the growth of strains of Escherichia coli other than Escherichia coli 0157 :H7 under incubation, (5) a carbohydrate medium that under incubation is not fermented by Escherichia coli 0157 :H7 but which is fermented by other microorganisms including other strains of Escherichia coli. said carbohydrate medium forming colonies of a first color when fermented by a microorganism, (6) a chromogenic beta-galactosidase substrate which forms colonies of a second color which contrasts with the first color upon reacting to beta-galactosidase, (7) a pH indicator dye which changes the plating medium to a third color which contrasts with the first and second colors when the pH of the medium changes, and (8) a mass of agar sufficient to solidify the mixture . 2. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 in combination with an ingredient which provides nutrients to facilitate the growth of microorganisms. 3. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 2 wherein the ingredient which provides nutrients to facilitate the growth of microorganisms comprises one or more members of the group tryptone, bacto-peptone, and proteose-peptone. 4. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 in combination with isopropyl-beta-D-thiogalactopyranoside . 5. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 wherein the carbohydrate medium that is not fermented by Escherichia coli 0157 :H7 but is fermented by other strains of Escherichia coli comprises one or more members of the group consisting of sorbitol, salicin, inositol, and adonitol. 6. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 wherein the ingredient which inhibits growth of gram positive microorganisms comprises bile salts. 7 An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 wherein the chromogenic beta galactosidase substrate which forms a second color upon reacting to beta-galactosidase comprises a member selected from the group consisting of indoxyl-beta-D-galactopyranoside and 5-bromo-4-chloro-3- indoxyl -beta-D-galactopyranoside . 8 An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 wherein the ingredient which inhibits the growth of Proteus sp . consists essentially of sodium novobiocin. 9. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 1 wherein the ingredient which inhibits the growth of Escherichia coli strains other than Escherichia coli 0157 :H7 consists essentially of potassium tellurite. 10. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprises a mixture of (1) an ingredient selectively promoting the growth of Escherichia coli cells under incubation consisting essentially of one or more members of the group tryptone, bacto-peptone, and proteose-peptone, (2) an ingredient inhibiting the growth of gram positive microorganisms consisting essentially of bile salts, (3) an ingredient inhibiting the growth of Proteus sp. consisting essentially of sodium novobiocin, (4) an ingredient inhibiting the growth of strains of Escherichia coli other than Escherichia coli 0157 :H7 consisting essentially of potassium tellurite, (5) a carbohydrate medium that is not fermented by Escherichia coli 0157 :H7 but which is fermented by other strains of Escherichia coli consisting essentially of one or more members of the group sorbitol, salicin, inositol, and adonitol, said carbohydrate medium forming colonies of a first color to microorganisms which ferment the medium, (6) a chromogenic beta-galactosidase substrate which forms a second color upon reacting to beta glucuronidase consisting essentially of one or more members of the group indoxyl-beta-D- galactopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D- galactopyranoside, (7) a pH indicator dye which changes to a third color when pH of the medium changes consists essentially of phenol red, the mixture having a pH of 6.6 to 6.8 before being exposed to a test sample, and (8) a mass of agar sufficient to solidify the mixture. 11. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 10 and sodium chloride admixed therein. 12. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 10 and 6-chloro-3- indoxyl-beta-D-glucuronide mixed therein. 13. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising a mixture of 5.0 grams per liter tryptone, 10.0 grams per liter bacto-peptone, 3.0 grams per liter proteose-peptone, 1.25 grams per liter bile salts #3, 10 milligrams per liter of sodium novobiocin, 0.5 to 2.0 milligrams per liter potassium tellurite, 12.0 grams per liter sorbitol, 10.0 grams per liter salicin, 10.0 grams per liter inositol, 15.0 grams per liter agar, 8.0 grams per liter adonitol, 120 milligrams per liter indoxyl-beta-D- galactopyranoside, 120 milligrams per liter 5-bromo-4-chloro-3-indoxyl-beta-D- galactopyranoside, 100 milligrams per liter isopropyl-beta-D- thiogalactopyranoside, 0.100 gram per liter phenol red, and 5.0 grams per liter sodium chloride, the mixture having a pH of 6.6 to 6.8 before being exposed to a test sample. 14. An isolation plating medium adapted for use in processes for the presumptive detection of Escherichia coli 0157 :H7 comprising the mixture of claim 13 including 120 milligrams per liter 6 -chloro-3- indoxyl -beta-D- glucuronide . 15. The method of detecting the presence of Escherichia coli 0157 :H7 in a test sample comprising the steps of inoculating a solid plating medium with said test sample, wherein said plating medium comprises a mixture of (1) an ingredient which selectively promotes growth of Escherichia coli cells, (2) an ingredient which inhibits growth of gram positive microorganisms, (3) an ingredient which inhibits growth of Proteus sp . , (4) an ingredient which inhibits growth of strains of Escherichia coli other than Escherichia coli 0157 :H7, (5) a carbohydrate medium that is not fermented by Escherichia coli 0157 :H7 but is fermented by other strains of Escherichia coli, said carbohydrate medium producing colonies of a first color upon fermentation, (6) a chromogenic beta galactosidase substrate capable of forming a second color which contrasts with the first color upon reacting t© beta-galactosidase, (7) a pH indicator dye which changes the color of the plating medium to a third contrasting color when the pH of the medium changes, and (8) a mass of agar sufficient to solidify the mixture, thereafter incubating said plating medium for a sufficient period to obtain colonies of microorganisms producing one or more of said colors, and examining the surface of said plating medium for colonies of said first, second and third colors. 16. The method of detecting the presence of Escherichia coli 0157 :H7 in a test sample comprising the steps of inoculating a solid plating medium with said test sample, wherein said plating medium comprises a mixture of 5.0 grams per liter tryptone, 10.0 grams per liter bacto-peptone, 3.0 grams per liter proteose-peptone, 1.25 grams per liter bile salts #3, 10 milligrams per liter of sodium novobiocin, 0.5 to 2.0 milligrams per liter potassium tellurite, 12.0 grams per liter sorbitol, 10.0 grams per liter salicin, 10.0 grams per liter inositol, 8.0 grams per liter adonitol, ' 120 milligrams per liter indoxyl-beta-D- galactopyranoside, 120 milligrams per liter 5-bromo-4-chloro-3-indoxyl- beta-D-galactopyranoside, 100 milligrams per liter isopropyl-beta-D-thiogalactopyranoside, 0.10 grams per liter phenol red, 5.0 grams per liter sodium chloride, and 15 grams per liter of agar, the mixture having a pH of 6.6 to 6.8 before being exposed to a test sample, then incubating said plating medium for at least 24 hours to obtain colonies of microorganisms generating one or more of said colors, and then examining the surface of said plating medium for colonies of colors differing from the color of the remainder of the plating medium.

Inventors: RESTAINO LAWRENCE, RESTAINO, LAWRENCE
Applicants: R & F LAB INC, R & F LABORATORIES, INC.
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1