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Project ID: ICA4-CT-2001-10014
Finanziato nell'ambito di: FP5-INCO 2
Paese: Italy

Development of a simple and inexpensive phenotypic test for rapid Escherichia coli identification during fieldwork

Presumptive identification of E. coli isolates based on the colony morphology on McConkey agar can be affected by poor specificity, especially when carried out by inexperienced personnel. Moreover, approximately 10% of E. coli isolates are lactose-negative, and these isolates are going to be missed if only lactose-positive colonies are considered.

To facilitate the process of selection of E. coli isolates grown onto the screening plates, a simple and inexpensive phenotypic test for rapid identification of E. coli, suitable for field work, was developed. The test is based on the detection of the enzyme Beta-D-glucuronidase using the substrate 4-methylumbelliferyl-Beta-D-glucuronide (MUG) which, upon hydrolysis by the enzyme, yields the fluorogenic compound 4-methylumbelliferone. Production of this enzyme is considered to be diagnostic for E. coli: 94 to 97% of E. coli strains are reported to produce this enzyme, while other bacteria that produce this enzyme do not grow on media selective for coliforms (McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354).

The test is carried out as follows: 10 microL of MUG 80 microg/mL, dissolved in 0.05 M Sorensen s phosphate buffer (pH 7.5), is dispensed in a well of a microtiter plate. A small amount of the colony to be tested is taken with a sterile toothpick and suspended in the MUG solution. After 2 hours of incubation at 37°C the plate is observed under UV light (a portable battery-operated Wood�s lamp is suitable). An intense blue fluorescence indicates a positive reaction, while no or a weak fluorescence indicates a negative reaction. In each test positive and negative controls are always inserted as references.

The test was validated using 250 isolates from the pilot study. It allowed correct identification of 91% of E. coli isolates (in agreement with the data from the literature that report that Beta-D-glucuronidase production occurs in approximately 90% of E. coli isolates - McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354), with no false positives.

Compared to the MUG-tests based on the use of culture media supplemented with MUG, this test is considerably less expensive (the cost is approximately 0.002 Euro per test) and is feasible for routine application in fieldwork in low-resources settings such as those participating in this project. Moreover, the use of microtiter plates and small volumes facilitates a quick and simple analysis of a high number of strains.
The major usefulness of a similar test, which allows rapid, cheap and specific confirmation of E. coli isolates, consists in reducing the rate of false positives derived from collection of lactose fermenters. Moreover, since production of Beta-D-glucuronidase is not related to production of Beta-galactosidase, the test allows reliable identification of E. coli isolates among lactose nonfermenting colonies, thus avoiding the systematic missing of similar isolates if lactose-positivity is considered for screening purposes.

Due to these features, it was decided to use this test during the ARS-Baseline activities in Bolivia and Peru, as a supporting tool for interpretation of results of the rapid screening method. During the first days of the ARS-Baseline activities, the MUG-test was carried out for several coliform and atypical colonies, and this approach allowed to considerably improve the ability of the local personnel to classify isolates grown on MacConkey agar plates. Although this training was rather time-consuming, due to the necessity of a two-step reading of plates (selection of the colonies to test with MUG, followed by observation of the colonies knowing the results of the MUG-test), it resulted very helpful to increase the ability of the local personnel to differentiate E. coli colonies from the usual mucoid, lactose-fermenting, MUG-negative colonies of Klebsiella spp.. The MUG-test was also used to recognise the morphology of lactose-nonfermenting E. coli colonies. After a first period of training, the MUG-test was used whenever doubts persisted.

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Dipartimento di Biologia Molecolare - Sezione di Microbiologia
Viale Bracci
53100 Siena
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