Abstract:
A plating media for identifying  Yersinia pestis  bacteria having nutrients for promoting growth of  Yersinia pestis  thereby producing beta-glucosidase, carbohydrate that is incapable of reacting with  Yersinia pestis,  a pH indicator dye, a chromogenic substrate that reacts to beta-glucosidase to form precipitate, and an agent to solidify the mixture, whereby a microorganism which ferments the carbohydrate but does not produce beta-glucosidase will produce colonies of the color determined by the pH indicator dye,  Yersinia pestis  and other microorganisms that do not ferment the carbohydrate but produce beta-glucosidase activate the substrate to color their colonies with the color of precipitate released by the substrate, and other bacteria which ferment the carbohydrate and produce beta-glucosidase produce colonies of the color that results from mixing the colors described above.

Description:
[0001]     The present invention relates to processes for detecting the presence of  Yersinia pestis  bacteria in foods, water, humans and animals. It also relates to plating media for the identification and enumeration of bacteria, particularly  Yersinia pestis  bacteria.  
       BACKGROUND OF THE INVENTION  
       [0002]      Yersinia pestis  is the causative bacteria for the plague or “black death” in man. Plague is one of the oldest known diseases of mankind dating back to A.D. 1348, and it is often fatal. During the 14 th  century, an epidemic of the plague in Europe caused 25 million deaths. Plague is known to be carried by rodents and spread among humans by fleas that have become infected through contact with infected rodents.  
         [0003]     Since 1900, the Unites States has been substantially free of the plague until recent outbreaks among infants. These outbreaks are believed to be caused by  Yersinia pestis  bacteria present in powdered infant formula.  Yersinia pestis  is substantially dormant at temperatures below 37 degrees Celsius and can survive for long periods in stored powdered infant formula. Further, once a human being or animal is inoculated with the  Yersinia pestis  bacteria, plague develops rapidly. Accordingly, there is a pressing need for means to rapidly identify  Yersinia pestis  bacteria in foods, water, humans and animals. A plating media described in the paper Ber, et al., Development of an Improved Selective Agar Medium for Isolation of  Yersinia Pestis,  Applied and Environmental Microbiology, 60: 5787-5795 (2003), documents an effort to satisfy this need, but the plating medium of this article requires long incubation times and produces colonies of  Yersinia pestis  on the medium of the color of the medium, thus making the medium difficult to count and tending to produce many false positives.  
       SUMMARY OF THE INVENTION  
       [0004]     It is an object of the present invention to provide a plating medium in which  Yersinia pestis  bacteria will grow and form colonies on the surface of the medium that may be readily observed and enumerated. However, test samples containing  Yersinia pestis  generally include a plurality of other microorganisms, and it is desirable for plating media to suppress unwanted bacteria and to differentiate colonies of  Yersinia pestis  from those of other microorganisms. It is known to use various characteristics of bacterial colonies to differentiate the colonies produced on a plating medium by different microorganisms from a mixed sample, including the size, shape, presence of a dome, and the color of the colony. The media of the present invention differentiate  Yersinia pestis  colonies from the colonies of other bacteria on the basis of contrasting colors.  
         [0005]     Culture media containing agar, a carbohydrate and a pH indicator dye have long been used to observe colonies on the surface of the media. The metabolic activity of the microorganisms in the media produces acid which influences the indicator dye and causes the color of the colonies to change. More recently chromogenic substrates have been incorporated in agar medium for coloring colonies of bacteria that utilize the substrate.  
         [0006]     It is an object of the present invention to provide a plating medium for identifying and enumerating colonies of  Yersinia pestis  bacteria that are differentiated by color from the colonies of other microorganisms that are likely to be present in a test sample and cannot be suppressed. The inventor has found that  Yersinia pestis  will not ferment certain carbohydrates, but is positive with respect to certain substrates. Further, almost all other microorganisms that are likely to be present in a test sample for  Yersinia pestis  and cannot be suppressed are capable of fermenting a carbohydrate, including  Yersinia enterocolitica.  The inventor has provided a plating medium including only carbohydrates that are negative for  Yersinia pestis  and positive for other microorganisms that are likely to be in a test sample, and a substrate that is positive for  Yersinia pestis.  Since all of the bacteria which cannot be suppressed, other than  Yersinia pestis,  will not ferment the same carbohydrate, it is desirable to provide a plurality of carbohydrates in the plating media according to the present invention. However, a single carbohydrate may be used in the media of the present invention to differentiate against those bacteria which are positive with respect to that carbohydrate.  
         [0007]     The one or more carbohydrates and pH indicator dye, and the substrate of the present invention will display four distinctive colors in a fully processed plate. The first color is the background color of the medium. The second color is the color of colonies that metabolize the substrate but are negative with respect to all carbohydrates, this color being the color of the precipitate released by the substrate.  Yersinia pestis  bacteria produce colonies of this color. The third color is the color of colonies of unsuppressed bacteria that are positive with respect to one or more carbohydrates and negative with respect to the substrate. Thus the third color is the color produced by the indicator dye. The fourth color is the color of colonies of bacteria that are positive for both the substrate and a carbohydrate, and hence the fourth color is a blend of the colors of the precipitate from the substrate and the indicator dye.  
         [0008]     The invention will be more readily understood from the following detailed description of the invention, which has no drawing. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0009]     The plating media of the present invention contain nutrients to promote the growth of  Yersinia pestis,  especially protein. Media containing the following ingredients, either singly or in various mixtures, have been found to be suitable for promoting the growth of  Yersinia pestis:  tryptone, bacto-peptone, proteose-peptone, meat infusions including heart infusion broth, yeast extract, soytone, and ammonium ferrous sulfate hexahydrate, may be 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 nutrients which will support the growth of microorganisms that may be used in place of or in addition to the above named nutrients.  
         [0010]     Test samples can be expected to contain bacteria other than  Yersinia pestis  that may be inhibited or suppressed. Gram positive bacteria and fungi (yeasts) may be inhibited. Such undesired microorganisms may be inhibited by mixing one or more of the following ingredients into the medium: bile salts #3, sodium deoxycholate, irgasan, vancomycin, and cycloheximide. Also, other inhibitors of gram positive bacteria and molds can be employed.  
         [0011]     Sodium chloride may be added to the medium for osmolarity purposes.  
         [0012]     The detection system for  Yersinia pestis  of the present invention utilizes a solid plating medium. Preferably agar is added to the other ingredients of the medium to provide the desired mixture for a solid medium. The detection system also uses a pH indicator dye to indicate changes in the pH of the medium.  
         [0013]     Media for detecting  Yersinia pestis  according to the present invention contain at least one and preferably a plurality of carbohydrates, none of which are fermented by the  Yersinia pestis  bacteria. Suitable carbohydrates are lactose, rhamnose, sucrose, cellobiose, adonitol, inositol, dulcitol, fucose, melezitose, and raffinose.  
         [0014]     Closely related bacteria that produce acid from a carbohydrate include the coliforms, such as  Escherichia, Klebisella  and  Enterobacter.  Most  Citrobacter  species and  Yersinia enterocolitica  produce acid from sucrose, and Salmonella will ferment dulcitol. Most species in the genis  Serritia  produce acid from adonitol, whereas,  Providencia  ferments inositol. All four of the above mentioned carbohydrates are not utilized by  Yersinia pestis.  Further as presently known, the above-identified bacteria do not significantly produce acid from the carbohydrates referred to with respect to other bacteria.  
         [0015]     A medium according to the present invention also contains one or more substrates that react to the enzyme beta-glucosidase. Growth of microorganisms in a medium according to the present invention, specifically including  Yersinia pestis,  can result in production of beta-glucosidase, and the media of the present invention include one or more substrates that react with the beta-glucosidase enzyme. Suitable substrates are 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside, and 3-indoxyl-beta-D-glucopyranoside, which produce an insoluble precipitate that contrasts with the color of the medium and is the second color (with a dark or indigo blue color). In media according to the hereinafter described examples, the presence of colonies of microorganisms that produce beta-glucosidase in the medium and do not produce acids from the carbohydrates, such as  Yersinia pestis,  are dark or indigo blue and sharply contrast with the color of the plating medium.  
         [0016]     While  Yersinia pestis  is lactose, dulcitol, adonitol, inositol, sucrose, rhamnose and cellobiose negative, other bacteria that are likely to be present in a sample are positive for one or more of these carbohydrates. Microbial fermenting of lactose, dulcitol, adonitol, inositol sucrose rhamnose or cellobiose changes the pH of the medium, thus producing colonies influenced by the indicator dye, a third color. In the following examples this third color is yellow. Hence, the presence of a yellow colony on the surface of an incubated plating medium of the examples is an indication of the presence of a microorganism other than  Yersinia pestis.  Colonies produced by a microorganism which is both a lactose, dulcitol, adonitol, inositol, sucrose, rhamnose or cellobiose fermenter and beta-glucosidase positive are of a fourth color that is a blend of the second and third colors, namely, green in the following examples.  
         [0017]     3-indoxyl-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside are chromogens that react to the presence of beta-glucosidase. Beta-glucosidase is an enzyme produced by  Yersinia pestis  and other enterics, and this enzyme reacts with 3-indoxyl-beta-D-glucopyranoside, or 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside to produce an insoluble indigo blue precipitate. Other chromogens may be used in place of, or in combination with, 3-indoxyl-beta-D-glucopyranoside or 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside. Two such other substrates are 6-chloro-3-indoxyl-beta-D-glucopyronoside and 5-bromo-6-chloro-3-indoxyl-beta-D-glucopyranoside which respond to beta-glucosidase by producing a salmon and majenta precipitate, respectively, in the plating medium that may be observed and counted.  
         [0018]     It is desirable to include an ingredient in the medium to enhance the production the production of the beta-glucosidase enzyme. In the following examples of media according to the present invention, isopropyl-beta-D-thioglucopyranoside or 3-O-methyl-beta-D-glucopyranoside is added to the plating medium for this purpose.  
       EXAMPLE I  
       [0019]     The ingredients for an exemplary embodiment of the plating media of the present invention and the amounts thereof are set forth in the following Table I.  
                                             TABLE I                                   MATERIAL   MEASUREMENT                                        Tryptone   5.0   grams/liter           Bacto-peptone   10.0   grams/liter           Beef (meat) extract   5.0   grams/liter           Dulcitol   8.0   grams/liter           Lactose   8.0   grams/liter           Sucrose   8.0   grams/liter           Sodium chloride   5.0   grams/liter           Inositol   8.0   grams/liter           Cycloheximide   0.100   grams/liter           5-bromo-4-chloro-3-indoxyl-beta-D-   0.200   grams/liter           glucopyranoside           Phenol red   0.1   grams/liter           Bile salts #3   1.0   grams/liter           3-indoxyl-beta-D-glucopyranoside   0.200   grams/liter           Isopropyl-beta-D-thioglucopyranoside   0.100   grams/liter           Agar   15.0   grams/liter           Vancomycin   8.0   milligrams/liter                      
 
         [0020]     Except for vancomycin, the ingredients are mixed in any order, the pH adjusted to 6.7 to 6.9, boiled, and the mixture is permitted to cool to 50 degrees Celsius. Thereafter, sterile vancomycin is added aseptically to the other ingredients. The composition is then poured into plates, thereafter permitted to dry for 48 to 72 hours in the dark, and then it is ready to be used. Storage time of poured plates is as much as 90 days at 2 to 8 degrees Celsius.  
       EXAMPLE II  
       [0021]     The ingredients and the amounts thereof of a second example of the present invention are set forth in the following Table II.  
                                             TABLE II                                   Supplier   Grams/liter                                        Chemical                   Heart Infusion   Difco   25.00           Broth           Yeast Extract   Difco   6.00           Soytone   Difco   3.13           Ammonium ferrous   Sigma   0.50           sulfate hexahydrate   (F3754)           Sucrose   Anyplace   8.00           Rhamnose   Anyplace   8.00           Cellobiose   Anyplace   8.00           Lactose   Anyplace   8.00           5-Bromo-4-Chloro-   Biosynth AG   0.20           3-Indoxyl-β-D-   or Inalco           Gluopyranoside           3-O-Methyl-D-   Biosynth AG   0.15           glucopyranose           Brom cresol purple   Anyplace   0.02           Agar   Difco   15.00           Supplements           provided by the           user           Irgasan       0.001           DP 300 (Dissolve           in 95% ethanol)           Vancomycin   Sigma   0.010           hydrochloride   V 2002                      
 
         [0022]     Except for irgasan and vancomycin hydrochloride, the ingredients are mixed in any order, the pH adjusted to 7.2 to 7.40, boiled and thereafter the mixture is permitted to cool to 50 degrees Celsius. Thereafter, sterile irgasan and vancomycin hydrochloride are added aseptically to the other ingredients, and the final pH is adjusted to 7.1 to 7.3. The composition is then poured into plates and permitted to dry for 48 to 72 hours in the dark, and then it is ready to be used. Storage time of poured plates is as much as 60 days at 2 to 8 degrees Celsius.  
         [0023]     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 at a temperature of 28 to 30 degrees Celsius for a period of time to permit growth of the microorganisms in the test sample to observable colonies. If powdered baby formula is to be examined for the presence of  Yersinia pestis,  it is preferable to mix a small quantity of the powdered baby formula in a nutrient broth, to allow the mixture to incubate for a period of time and to thereafter inoculate the mass of plating media with the liquid mixture. The inventor has found that with the plating media of the examples described above, a period of 48 hours of incubation is sufficient time for  Yersinia pestis  colonies to grow into colonies that are readily observable with the naked eye. It is believed that the abundant growth of microorganisms in the plating medium of Example I is due to the nutrients provided by the tryptone, bacto-peptone, and meat extract, and the abundant growth of microorganisms in the plating medium of Example II is due to the nutrients provided by the heart infusion broth, yeast extract, soytone and ammonium ferrous sulfate hexahydrate. Finally, the surface of the plating media is assayed and the presence and number of blue with black, or indigo blue, colonies recorded, and these colonies are identified as  Yersinia pestis.  Also, the presence of white or yellow to green colored colonies is noted as an indication of microorganisms other than  Yersinia pestis.    
         [0024]     Table III tabulates the growth of a number of microorganisms that are likely to be in a sample to be examined for  Yersinia pestis  after incubation for a period of 48 hours at 28 to 30 degrees Celsius in the plating media of Example II.  
                       TABLE III                           Growth on                 Yersinia pestis             Chromogenic       Bacterial strain   Plating Medium   Colony Color                     Yersinia pestis     +   B         Aeromonas hydrophila     +   Y/G         Aeromonas veronii     +   G         Aeromonoas sobria     +   G         Citrobacter freundii     +   Y         Edwardsiella tarda     +   Y         Enterobacter aerogenes     —         Enterobacter cloacae     —       Generic  Escherichia coli     —         Hafnia alvei  (2 strains)   —         Klebsiella pneumoniae     +   W         Lactobacillus     +   W         Listeria monocytogenes     —         Listeria seeligeri     —         Morganella morganii     +   W         Proteus mirabilis     —         Providencia rettgeri     +   B/G         Pseudomonas aeruginosa     +   W         Salmonella  spp. (4 strains)   —         Shigella boydii     —         Shigella flexneri     —         Shigella sonnei     —         Vibrio cholerae     —         Vibrio parahaemolyticus     —         Vibrio vulnificus     —         Yersinia enterocolitica  (8   +   Y       strains)         Yersinia enterocolitica  1A   +   G/Y         Yersinia aldovae     +   B/G         Yersinia fredricksenii     +   G         Yersinia intermedia     +   G         Yersinia kristensenii     +   Y/G         Yersinia pseudotuberculosis  (6   +   G/Y       strains)         Yersinia pseudotuberculosis  (7   +   G       strains)                 Key:            W = White;            Y = Yellow;            G = Green; and            B = Blue.            + = Growth and            — = no growth.             
 
 It is to be noted that no special equipment is required to observe the incubated surface of the plating medium. The time required observing the number and presence of blue with black colonies is far less than required when other similarly colored colonies are present. Subsequent confirmation of blue with black colonies uses known tests. Also, there are no ingredients in the plating medium that are especially costly. Hence, an assay of a test sample may be made at modest cost. 
 
         [0025]     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.