Abstract:
A solid growth plating medium in which Shigella organisms will grow and form colonies in the medium, and substantially, other microorganisms are inhibited or their colonies are differentiated from Shigella organisms. In one embodiment of the invention, colonies produced by Shigella appear with the color of the plating medium, usually a clear off-white color, that can be readily observed. In another embodiment, the fact that  Shigella boydii  and  Shigella sonnei  produce the enzyme alpha-galactosidase, but most  Shigella dysenteriae  and  Shigella flexneri  strains do not, is utilized with a chromogenic substrate to produce colonies of these microorganisms of a distinguishing color.

Description:
[0001]    The present invention relates to the presumptive identification of bacteria, and in particular to the presumptive identification of Shigella. The genus Shigella is classified in the family Enterobacteriaceae, and has four species, namely,  Shigella dysenteriae, Shigella flexneri, Shigella boydii  and  Shigella sonnei.  One embodiment of the present invention relates to plating media for identifying the genus Shigella, and a second embodiment of the present invention relates to plating media for simultaneously identifying  Shigella boydii  and  Shigella sonnei.    
         BACKGROUND OF THE INVENTION  
         [0002]    Shigella organisms produce ulcerative colon infections in humans called shigellosis or bacillary dysentery. The organisms are transmitted directly or through food or water contaminated with fecal matter. In order to combat the spread of shigellosis, public health professionals and health care professionals require a means for rapidly and quantitatively identifying the presence of Shigella organisms in samples containing a plurality of other organisms. Shigella is generally found in mixed samples containing organisms of other Enterobacteriaceae.  
           [0003]    Scientists have long searched for an effective means for isolating, detecting and identifying Shigella organisms. In 1987, the  Journal of Food Protection  (Smith, J. L.,  Shigella as a Foodbourne Pathogen,  volume 50, page 788) reviewed methods used for the isolation and detection of Shigella from suspect foods. Nonetheless, plating media developed prior to the present invention produce excessive false negative and false positive organism counts, and were difficult to read.  
           [0004]    The biochemical characteristics of Shigella, and its similarity to  Escherichia coli,  significantly increase the likelihood of false negative and false positive indications from a plating medium. Table I hereafter, published in the  Compendium of Methods for the Microbiological Examination of Foods,  Edited by Carl Vanderzant and Don F. Splittstoesser, 1992, American Public Health Association, page 429, makes it clear that Shigella does not have characteristics that are readily identified in a plating medium.  
                         TABLE 1                           Selected Biochemical Characteristics of the Genus Shigella       for Speciation            Medium   Reaction               KCN broth   negative       malonate broth   negative       tryptone broth (indole)   positive or negative ( S. dysenteriae  1,             S. flexneri  6 and  S. sonnei  are negative;             S. dysenteriae  2 is positive)       MR-VP medium   positive methyl red           negative Voges-Proskauer       citrate agar   negative       decarboxylase medium       with lysine   negative       with ornithine   negative       acetate differential agar   negative (some strains of  S. flexneri  4a are           positive)       indicator broth       with glucose   acid (some strains of  S. flexneri  6 and  S. boydii             13 and 14 produce acid and gas)       with adonitol, xylose,   no acid ( S. boydii  is variable on xylose)       cellobiose, dulcitol,       inositol, salicin       with lactose   no acid (some strains of  S. flexneri  2a and             S. boydii  9 produce acid,  S. sonnei  produces           acid after several days)       with sucrose   no acid ( S. sonnei  produces acid after several           days       with mannitol   acid ( S. dysenteriae  does not produce acid;           some strains of S. flexncri 4a and 6 do not           produce acid)       with raffinose   no acid ( S. flexneri  is variable;  S. sonnei             produces acid after several days)                  
 
           [0005]    From Table 1 it is clear that the different strains of Shigella do not react to conventional media in the same way, and that false positives and false negative are likely to occur. Paragraph 26.24 of the Compendium of Methods for the Microbiological Examination of Foods, supra, recommends the use of three media of different selectivity to assay a single sample in order to increase the accuracy of the media, which makes for a costly and cumbersome isolation procedure.  
           [0006]    Further, Shigella organisms do not react to many biochemical tests. They do not utilize KCN, malonate, citrate, or acetate as sole carbon sources. They produce acid from glucose and mannitol* but do not produce acid from sorbitol, adonitol, dulcitol, inositol, xylose*, cellobiose, lactose*, sucrose*, raffinose*, or salicin. Shigellae generally do not produce gas from sugar* (*see Table 1).  
           [0007]    The 1999 U.S. Pat. No. 5,871,944 of Russel G. Miller and Edward Mallinson entitled  Salmonellae Preferential Media  discloses a plating medium for identification of Salmonella and Shigella from a mixed bacterial sample with ingredients for preferential growth of these bacteria and a chromogenic substrate that reacts to the enzyme beta-galactosidase to differentiate other bacteria. Other bacteria that do not produce beta-galactosidase will not be differentiated from Salmonella or Shigella, resulting in false positives.  
         SUMMARY OF THE INVENTION  
         [0008]    It is an object of the present invention to provide a novel plating medium for isolating and quantitatively identifying Shigella organisms from a sample containing a plurality of different organisms; and a further object of the invention to provide a plating medium for making a combined identification of  Shigella boydii  and  Shigella sonnei  organisms from a mixed sample.  
           [0009]    It is also an object of this invention to provide such plating media for the isolation and identification of Shigella organisms that will produce fewer false positive and fewer false negative identifications than the plating media of the prior art.  
           [0010]    Further, it is an object of this invention to provide such a plating medium for the isolation and identification of Shigella organisms from a mixed sample that will produce a more readily readable plate with colonies of fewer colors than the plating media of the prior art to facilitate identification of Shigella organisms.  
           [0011]    For many public health and health care professionals,  Shigella boydii  and  Shigella sonnei  are the strains of Shigella of greatest interest. It is therefore a further object of the present invention to provide a plating medium for the combined isolation and quantitative identification of  Shigella boydii  and  Shigella sonnei  organisms.  
           [0012]    The objects of the invention are accomplished by providing a solid growth plating medium in which Shigella organisms will grow and form colonies in the medium. Substantially, other microorganisms are inhibited or their colonies are differentiated from Shigella organisms, i.e. transformed to a color distinguishing the Shigella colonies. Differentiation is accomplished by providing in that medium a plurality of biochemical ingredients that produce reactions with the other organisms of the sample to color the colonies of the other organisms a minimum of colors that contrast with the color of the Shigella colonies. In one embodiment of the invention, colonies produced by Shigella appear with the color of the plating medium, usually a clear off-white color, that can be readily observed. In another embodiment, the fact that  Shigella boydii  and  Shigella sonnei  produce the enzyme alpha-galactosidase, but most  Shigella dysenteriae  and  Shigella flexneri  strains do not, is utilized with a chromogenic substrate to produce colonies of these microorganisms of a distinguishing color.  
           [0013]    The invention will be more readily understood from the following detailed description, which contains no drawings. 
       
    
    
     DETAILED DESCRIPTION  
       [0014]    Mixed samples containing Shigellae often contain other bacteria of the Enterobacteriaceae and other bacteria. In both embodiment of the present invention, Shigellae form colonies in the plating medium of a particular color, and the other bacteria are subjected to reactions changing the color of these colonies to a different color.  
         [0015]    Before differentiation of the bacteria can occur, an inoculated plate is incubated to produce bacterial growth. Nutrients are provided in the plating medium to facilitate growth of Shigella, and the inventor has found casamino acids, dessicated beef extract, yeast extract, sodium pyruvate and soytone to be desirable nutrients. Agar functions to thicken the mixture, and it is used in sufficient quantity to solidify the mixture. Carbohydrates are also incorporated in the plating medium to provide for growth. The inventor has found carbohydrates of the group lactose, sucrose, salicin, 2-deoxy-D-ribose, adonitol, dulcitol, inositol, xylose and cellobiose to be particularly satisfactory.  
         [0016]    Some organisms can be prevented from colonizing the plating medium by incorporating inhibitors in the medium. If it is likely that they will be present in the sample, it is desirable to inhibit as many unwanted organisms as can reasonable be inhibited. One group of organisms that my readily be inhibited are gram-positive bacteria which are inhibited by bile salt #3 and other forms of bile salts. Other inhibitors that may be used in the media of this invention are tellurite to retard the growth of  Escherichia coli,  sodium novobiocin and cefixime to inhibit Proteus sp. and cefsulodin to suppress pseudomonas-like bacteria  
         [0017]    The plating media of the present invention differentiate the colonies of the microorganisms that cannot be inhibited from the Shigella colonies by use of a pH indicator dye and one or more chromogenic substrates. The chromogenic substrates react to an enzyme and produce a water insoluble precipitate of a particular color, and the precipitate is maintained in the region of the colony that produced it to color that colony with the color of the precipitate. Different chromogenic substrates react with different enzymes, but they can be selected to produce precipitate of essentially the same color. Hence, colonies of different bacteria, that react with different substrates, will produce colonies colored by precipitate of the same color, thus reducing the number of colors that the observer must note in order to count the plate.  
         [0018]    The inventor has found that the following chromogenic and fluorogenic substrates may be used in the media of the present invention: 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside, 6-chloro-3-indoxyl-alpha-D-galactopyranoside, 4-Methylumbelliferyl-alpha-D-galactopyranoside, 1-Naphthyl-alpha-D-galactopyranoside, 4-Nitrophenyl-alpha-D-galactopyranoside, 4-Methylumbelliferyl-alpha-D-glucopyranoside, 2-Naphthyl-alpha-D-glucopyranoside, 4-Nitrophenyl-alpha-D-glucopyranoside, 5-Bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside, 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside, 5-Bromo-6-chloro-3-indoxyl-beta-D-glucopyranoside, 3-indoxyl-beta-D-glucopyranoside, 6-chloro-3-indoxyl-beta-D-glucopyranoside, 5-Bromo-4-chloro-3-indoxyl-alpha-D-mannopyranoside, 2-(6-Bromonaphthyl)-alpha-D-mannopyranoside, 6-chloro-3-indoxyl-alpha-D-mannopyranoside, 4-Methylumbelliferyl-alpha-D-mannopyranoside, 4-Nitrophenyl-alpha-D-mannopyranoside, 4-Methylumbelliferyl-beta-D-mannopyranoside, 4-Nitrophenyl-beta-D-mannopyranoside, Methylumbelliferyl-beta-D-xylopyranoside, 2-Nitrophenyl-beta-D-xylopyranoside, 4-Nitrophenyl-beta-D-xylopyranoside, 5-Bromo-4-chloro-3-indoxyl-beta-D-fucopyranoside, 4-Methylumbelliferyl-beta-D-fucopyranoside, 4-Nitrophenyl-beta-D-fuicopyranoside, 4-Methylumbelliferyl-beta-D-glucopyranoside, 1-Naphthyl-beta-D-glucopyranoside, 2-Nitrophenyl-beta-D-glucopyranoside, 3-Nitrophenyl-beta-D-glucopyranoside, 4-Nitrophenyl-beta-D-glucopyranoside, 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-galactosaminide,, 6-chloro-3-indoxyl-N-acetyl-beta-D-galactosaminide, 4-Methylumbelliferyl-N-acetyl-beta-D-galctosaminide, and 4-Nitrophenyl-N-acetyl-beta-D-galactosaminide.  
         [0019]    The response of a chromogenic substrate to an enzyme may be inadequate to produce precipitate to fully color its colony. It is therefor desirable to add an enhancer to the medium to increase the production of precipitate. The inventor has found the following enhancers to be effective with the chromogenic substrates set forth above: 1-O-Methyl-alpha-D-galactopyranoside, 1-O-Methyl-beta-D-glucopyranoside, and 1-O-Methyl-alpha-D-mannopyranoside.  
         [0020]    Table 2 sets forth the ingredients of one particular plating medium according to the present invention. This plating medium is exemplary of one embodiment of the present invention, and will identify Shigella. All four strains of Shigella will form colonies in the plating medium of the color of the medium, an off-white solid.  
                                           TABLE 2                           Ingredients For General Shigella Medium                INGREDIENTS   GRAMS/LITER                            Casamino acids   14.37           13eef extract, dessicated   1.0           Yeast extract   6.0           Lactose   8.0           Soytone   5.0           Sodium chloride   5.0           Bile salts #3   0.6           Bile salts   0.2           Sucrose   8.0           Dulcitol   8.0           5-Bromo-4-chloro-3-indoxyl-beta-D-   0.2           gIucopyranoside           1-O-Methyl-beta-D-glucopyranoside   0.15           5-Bromo-4-chloro-3-indoxyl-beta-D-   0.2           fucopyranoside           5-Bromo-4-chloro-3-indoxyl-N-Acetyl-beta-D-   0.2           galactosaminide           Phenol red   0.08           Agar   15.0           Novobiocin   0.002           Cefsulodin   0.006                      
 
         [0021]    The ingredients, except for the last two, are mixed in any order, the pH is adjusted to 6.6 to 6.8, the ingredients are boiled to dissolve the agar, and the mixture is permitted to cool to room temperature. Thereafter, novobiocin and cefsulodin are added under sterile conditions. The composition is then poured into plates and permitted to dry for 48 to 72 hours, and it is then ready to be used. Storage time of poured plates is as much as 90 days at 2 to 8 degrees Celsius.  
         [0022]    Table 3 sets forth data taken with a batch of plating media containing the ingredients set forth in Table 2 from a mixed sample containing the named bacterial strains.  
                           TABLE 3                                   Organism   Colonial Morphology                             Shigella sonnei  (3 strains)   White to clear raised; 2-3 mm in               diameter           Shlgella  boydii     White to clear raised; 1 mm in               diameter           Shlgella  flexneri     White to clear raised; 2 mm in               diameter             Shigella dysenteriae     White to clear raised; 2 mm in               diameter             Escherichia coli     Yellow raised             Escherichia coli     Yellow-green raised             Klebseilla pneumoniae     Green raised             Enterobacter aerogenes     Green raised             E. coli  01 57:H7   Yellow raised             Salmonella derby     Yellow raised             Salmonella duesseldorf     Yellow raised                      
 
         [0023]    In some locations,  Shigella boydii  and  Shigella sonnei  have proven to be of particular interest to public health officials and health care workers. A second embodiment of the present invention set forth in Table 4 is effective in making a combination identification of  Shigella sonnei  and  Shigella boydii  from a mixed bacterial sample.  Shigella sonnei  and  Shigella boydii  produce the enzyme alpha-galactosidase, but most  Shigella dysenteria  and  Shigella flexneri  strains do not. The plating medium of Table 4 contains a substrate that is responsive to alpha-galactosidase, and hence produces a precipitate of a color to identify these strains of Shigella.  
                                           TABLE 4                           Ingredients for  Shigella sonnei  and  Shigella boydii  Medium                INGREDIENTS   GRAMS/LITER                            Casamino acids   14.5           Soytone   5.0           13eef extract, dessicated   1.0           Yeast extract   6.12           Sodium chloride   5.0           Lactose   8.0           Sucrose   8.0           Salicin   8.0           Dulcitol   8.0           Bile salts #3   0.6           5-Bromo-4-chloro-3-indoxyl-alpha-D-   0.2           galactopyranoside           1-O-Methyl-alpha-D-galactopyranoside   0.2           Bile salts   0.2           Phenol red   0.08           Agar   15.0           Novobiocin   0.002           Cefsulodin   0.006           Cefixime   0.00005                      
 
         [0024]    This medium may be prepared in the same manner as the medium of Table 2. In this medium, the chromogenic substrate, 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside, reacts with  Shigella sonnei  and  Shigella boydii  to add precipitate to these colonies, and to function as a differentiator with respect to most  Shigella flexneri  and  Shigella dysenteriae.  Accordingly, most  Shigella flexneri  and  Shigella dysenteriae  strains produce clear colonies in the medium, as shown in Table 5 which reports the test results produced by inoculating the medium of Table 4 with essentially the same sample used to produce the test results of Table 3.  
                           TABLE 5                                   Organism   Colonial Morphology                             Shigella sonnei     Blue center with clear ring; 2-3 mm in               diameter             Shigella boydii     Blue colony raised; 1 mm in               diameter             Shigella flexneri     Clear, raised colony, 2 mm. in               diameter             Shigella dysenteriae     Clear, raised colony, 2 mm. in               diameter             Escherichia coli     Yellow raised             Escherichia coli     Yellow-green raised             Klebseilla pneumoniae     Yellow raised             Enterobacter aerogenes     Yellow raised             E. coli  0157:H7   Yellow green raised             Salmonella derby     Yellow green raised             Salmonella duesseldorf     Yellow green raised           Enteroinvasive             Escherichia coli     Blue center with clear ring               1-3 mm. in diameter                      
 
         [0025]    The foregoing test results indicate that plating media according to the first embodiment of the present invention are capable of identifying Shigella bacteria from a mixed bacterial sample with accuracy, and further indicate that plating media according to the second embodiment of the present invention are capable of identifying combined  Shigella sonnei  and  Shigella boydii  from a mixed bacterial sample.  
         [0026]    In the first embodiment, a pH indicator dye (phenol red) and a plurality of chromogenic substrates (5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside, 5-Bromo-4-chloro-3-indoxyl-beta-D-fucopyranoside, and 5-Bromo-4-chloro-3-indoxyl -N-acetyl-beta-D-galactosaminide) are used to differentiate the colonies of Shigella from the colonies of other bacteria. Shigella does not produce enzymes that will react with the chromogenic substrates or acid to excite the pH indicator dye, and therefore clear Shigella colonies are formed on the surface of the medium. Those other bacteria that produce acid will cause the indicator dye to color these colonies with the color of the dye, namely yellow. Those other bacteria that produce an enzyme that reacts with any one or more of the chromogenic substrates will release precipitate into its colonies of the color of the substrate, namely dark blue, since all of the substrates release precipitate of this same dark blue color. Those other bacteria that produce acid and an enzyme that will react with one or more of the chromogenic substrates, will inject into its colonies both the dye of the pH indicator and the precipitate of the substrate, and the yellow dye will mix with the dark blue precipitate to color these colonies a shade of green.  
         [0027]    In the second embodiment, a pH indicator dye (phenol red) and one chromogenic substrates (5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside) are used to differentiate the colonies of  Shigella sonnei  and  Shigella boydii  from the colonies of other bacteria.  Shigella sonnei  and  Shigella boydii  produce the enzyme alpha-galactosidase, and this enzyme reacts with the chromogenic substrate 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside to release dark blue precipitate into its colonies. With some exceptions, other bacteria that produce the alpha- galactosidase enzyme, and hence release dark blue precipitate into its colonies, also produce acid and cause pH indicator dye to enter its colonies; thus producing a mixture of blue and yellow colors to achieve a shade of green. Other bacteria that do not produce an enzyme to react with the chromogen, but produce acid will have colonies colored by the the indicator dye, namely yellow; and other bacteria that neither produce an enzyme that reacts with the substrate nor acid will produce clear colonies in the medium.  
         [0028]    Those skilled in the art will devise many other applications for the present invention, and it is therefore intended that the scope of this invention be not limited by the foregoing disclosure, but only by the appended claims.