Note: Descriptions are shown in the official language in which they were submitted.
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2012287 ~
Field of Invention
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This invention relates to a method and apparatus for
detecting E._coli. More particularly this invention relates
to the rapid detection of E. coli in clinical specimens,
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food and water samples.
Cross Reference to Related Applications
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This application is related to copending United States
Patent Application Serial Nos. 080,731 and 039,435 filed 3
August 1987 and 17 April 1987 respectively in the names of
Wolfe et al. and Ley et al. respectively, which applications
are presently pending.
Background of Invention and Prior Art
Escherichia coli is the most common gram negative
bacterium isolated in clinical laboratories and accounts for
between 70 and 95',~ of all urinary tract infections. E. coli
is also considered to be a primary indicator of the presence
of human or animal sewage in public water supplies and an
indication of contamination of food supplies. Rapid,
sensitive and specific identification of E. coli is,
therefore, of considerable importance in a clinical, water
testing or food quality control laboratory. It has been
dislcovered (Kilian, Acta Pathol. Microbiol. Scand. Sect.
B84: 245-251) that E. coli is one of very few bacteria that
produce the enzyme ~-glucuronidase, and hence measurement of
~-glucuronidase activity provides a quantitative measure for
the presence of E. coli. Tests to measure ~-glucuronidase
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presently include the liberation of yellow P-nitrophenol
following hydrolysis of P-nitrophenyl- ~-D-glucuronide and
fluorogenic recognition of methyl umbelliferone following
hydrolysis of 4-methyl-umbelliferyl- ~ -D-glucuronide (MUG).
Both of these tests are relatively time consuming and rather
difficult to read. If a solid support (agar) is used, the
liberated yellow colour (P-NP) diffuses to distinguish
against the background agar into which it diffuses and in
the case of MUG, not only is special fluorescence equipment
required but eye strain on the technicians who use the
equipment routinely, is extreme.
In the related applications referred to above there is
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described a process for the synthesis of indoxyl-~-D-
glucuronide (IBDG), a compound which others had predicted
but had been unable to produce, and the properties of which
were largely unknown. Ley et al. have demonstrated that at
levels of about 800 mg/l IBDG, visual enumeration of E. coli
colonies in environmental samples, such as drinking water,
i9 readily achieved. The ~-glucuronidase of the E. coli
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hydrolyses the IBDG and the indoxyl portion thereof is
rapidly oxidized to indigo which is readily visible as a
deep blue coloration, which is quantitatively indicative of ~-
the number of colonies of E. coli present in the sample.
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Object of Invention ;
An object of the present invention is to extend the use
of IBDG as a diagnostic tool for the rapid identification ;~
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201Z287
and enumeration of E. coli in biological samples, such as
urine. E. coli is a common urinary pathogen and testing
therefore has, heretofore, been a relatively time consuming
(24 hours plus) and laborious procedure which is subject to
a relatively high percentage of false positive results.
Another object of the invention is to provide an
apparatus which incorporates a substrate of IBDG for the
rapid detection of E. coli in biological samples.
Brief Statement of Invention
Thus, by one aspect of this invention there is provided ;~
a method for enumerating E. coli in a biological sample
comprising culturing said sample in a medium containing
indoxyl-~-D-glucuronide or a salt thereof so as to produce
an indigo blue colouring indicative of glucuronidase enzyme
activity.
By another aspect of this invention there is provided a -
diagnostic article for the colorimetric assay of E. coli in
a biologically derived specimen comprising an absorbent
surface upon which is absorbed indoxyl- ~D-Glucuronide.
Detailed Description of Preferred Embodiments
I MacConkey agar (BBL Microbiology Systems) was
supplemented with 0.8 g of IBDC per liter (MAC-IBDG). Urine
specimens from patients in the Hotel Dieu Hospital,
Kingston, Ontario, Canada, were used to inoculate MAC-IBDG
plates with a O.Ol-ml calibrated loop. Deep blue colonies ~ -
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produced after 18 h at 35C were scored as positive for E.
coli. The colonies were distinct, as diffusion of indigo
did not occur. the results of the direct screening of urine
samples are shown in Table 1. No false-positive reactions
were noted. (API 20E) [Analytab Products] and Fox panels
[Beckman Instru~ents, Inc.] were used to identify the
isolates.) Of 152 gram-negative organisms screened, 99 were
E. coli. Eighty-three of these were positive for IBDG
hydrolysis. Of the 16 false-negative strains (10.5%), 6
were slow (> 18 h~ hydrolyzers. The sensitivity and
specificity of the MAC-IBDG plate identification directly
from urine were 88.8 and 100%, respectively.
A further 198 organisms isolated from multiple body
sites were inoculated onto MAC-IBDG plates with a Cathra
replicator. Organisms were identified by replica plating
biochemical agents and by the API 20E. A total of 76 E.
coli and 122 Enterobacteriaceae and Pseudomonas strains were
tested (Table 1). No false-positive reactions were seen.
Of the eight false-negative strains (4.0%), one was E. coli
0157:H7. the sensitivlty and specificity of this test were
90.4 and 100~, respectively.
Incorporation of IBDG into agar provides an
inexpensive, stable, and direct visualization of E. coli as
dark blue colonies. There were no false-positive reaotlons
for 350 clinical isolates tested. The test proved useful
for both direct screening of urine and replica plate
technology. False-negative reactions do not represent a
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problem in these clinical settings, as all IBDG-negative ~ ~ '
organisms are routinely identified if present in sufficient
numbers. Although many Shigella and Salmonella species are
known to be ~-glucuronidase positive, these organisms are
rarely found in urine and could be excluded by spot indole
and o-nitrophenyl-~-D-galactopyranoside tests. It was of
interest to note that E. coli 0157:H7 did not hydrolyze
IBDG, as noted by others with E. coli 0157:H7 in MUG agar.
TABLE 1. ~-Glucuronidase activity on NAC-I8DG plates
OrganismNo. testedNo. IBDG :
(n - 350) positive :-
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E. coli
Cathra replicator 76 68
Calibrated loop99 83
Pseudomonas aeruginosa 39 0
Pseudomonas maltophilia 5 0
Pseudomonas cepacia 1 0
Providencia rettgeri 4 0
Providencia stuartii 1 0
Proteus mirabilis 5 0
Proteus vulgaris 1 0
Proteus sp. 1 0
Alcaligenes spp. 2 0
Enterobacter cloacae 1 0
Enterobacter aerogenes 21 0
Klebsiella pneumoniae 23 0
Klebsiella oxytoca 14 0
Serratia spp. 6 0
Serratia marcescens 1 0
Hafnia alvei 8 0
Acinetobacter spp. 2 0
Morganella morganii 5 0
Moraxella sp. 1 0
Salmonella sp. 1 0
Citrobacter spp. 4 o
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A number of investigators have noted the difficulty of
incorporating MUG into agar. Diffusion of fluorescence
occurs rapidly and plates have to be read within 12 to 16 h
even with the addition of plate dividers. Similarly, p-
nitrophenyl-~-D-glucuronide agar also results in extensive
diffusion of the product from the colony. In contrast, IBDG
plates are stable, and no diffusion occurs, as the indigo
dye is insoluble. In addition, indigo production does not
alter the viability of the colonies, so colonies may be
picked directly for further sensitivity testing.
Rapid tests for E. coli identification rely on the
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identification of ~-glucuronidase from colonies following -
isolation in pure cultures. The advantage of the MAC-IBDG ;~
system is the immediate visualization and detection of E.
coli on primary plates. The IBnG substrate is also ideal
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for Dip Slides, for automated detection in panels, and for
Dip-Sticks in conjunction with urine screening to detect
leukocyte esterase. ~ ;
It is also contemplated within the scope of this inven-
tion that IBDG may be incorporated with an E. coli nutrient ~;~
in a water soluble substrate to provide a simple home test -
kit for water quality studies. It is also contemplated that
IBDG may be incorporated into a thin film substrate of the -
Petrifilm type sold by the 3M COmpdny.
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