Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-08-22
2004-07-20
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S968000, C435S039000, C435S038000, C435S040000, C435S029000, C435S973000
Reexamination Certificate
active
06764832
ABSTRACT:
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
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.
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.
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)
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 negatives 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.
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).
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
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.
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.
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.
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.
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.
The invention will be more readily understood from the following detailed description, which contains no drawings.
REFERENCES:
patent: 3870601 (1975-03-01), Warren et al.
patent: 5726031 (1998-03-01), Roth et al.
patent: 6350588 (2002-02-01), Roth et al.
Burmeister Marshall A.
Leary Louise N.
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