CA2012287A1 - Rapid detection of e.coli - Google Patents
Rapid detection of e.coliInfo
- Publication number
- CA2012287A1 CA2012287A1 CA 2012287 CA2012287A CA2012287A1 CA 2012287 A1 CA2012287 A1 CA 2012287A1 CA 2012287 CA2012287 CA 2012287 CA 2012287 A CA2012287 A CA 2012287A CA 2012287 A1 CA2012287 A1 CA 2012287A1
- Authority
- CA
- Canada
- Prior art keywords
- coli
- ibdg
- agar
- glucuronide
- indoxyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Abstract
Abstract of Disclosure A method and apparatus for the rapid visual detection and enumeration of E. coli in a biological sample, in which indoxyl- .beta.-D-Glucuronide and a nutrient for E. coli is absorbed onto a substrate surface, such as a dip slide, dip-stick or thin film.
Description
~ ` :
2012287 ~
Field of Invention ;
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, -food and water samples.
Cross Reference to Related Applications ..
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 ;~0122~7 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 .: - - ': ' ' 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 , , ~ :":
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.
.. .. . .
Object of Invention ;
An object of the present invention is to extend the use of IBDG as a diagnostic tool for the rapid identification ;~
2012287 ~
Field of Invention ;
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, -food and water samples.
Cross Reference to Related Applications ..
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 ;~0122~7 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 .: - - ': ' ' 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 , , ~ :":
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.
.. .. . .
Object of Invention ;
An object of the present invention is to extend the use of IBDG as a diagnostic tool for the rapid identification ;~
2 -~
.,: ' . :, :, 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 ~ -~ ' "':
.,: ' . :, :, 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 ~ -~ ' "':
3 ~ ~ ~
;~0~2Z87 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 :, ;~, '~', ' '',:
:
20~2Z87 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 :-.,. ~ .
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 20~Z87 ',.,'.' ~.,;',':.
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 ~: i:: :.:
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 . .
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.
6 ~ ;
-'" ~''
;~0~2Z87 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 :, ;~, '~', ' '',:
:
20~2Z87 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 :-.,. ~ .
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 20~Z87 ',.,'.' ~.,;',':.
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 ~: i:: :.:
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 . .
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.
6 ~ ;
-'" ~''
Claims (7)
1. A method for enumerating E. coli in a biological sample comprising culturing said sample in a medium containing indoxyl-.beta.-D-glucuronide or a salt thereof so as to produce an indigo blue colouring indicative of glucuronidase enzyme activity.
2. A method as claimed in claim 1 wherein said biological sample is urine.
3. A method as claimed in claim 1 wherein said medium comprises agar.
4. A method as claimed in claim 3 wherein said agar is MacConkey's Agar.
5. A diagnostic article for the colorimetric assay of E. coli in a biologically derived specimen comprising an absorbent surface upon which is absorbed indoxyl-.beta.-D-Glucuronide.
6. A diagnostic article as claimed in claim 5 wherein said surface includes a nutrient for E. coli.
7. A diagnostic article as claimed in claim 6 wherein said surface is selected from a film surface, a dip slide and a dip-stick.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US32476189A | 1989-03-17 | 1989-03-17 | |
| US324,761 | 1989-03-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2012287A1 true CA2012287A1 (en) | 1990-09-17 |
Family
ID=23264991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2012287 Abandoned CA2012287A1 (en) | 1989-03-17 | 1990-03-15 | Rapid detection of e.coli |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2012287A1 (en) |
-
1990
- 1990-03-15 CA CA 2012287 patent/CA2012287A1/en not_active Abandoned
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |