Understanding the cause of false negative beta-D-glucuronidase reactions in culture media containing fermentable carbohydrate.

AIMS: To explain the basis for false negative beta-glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. METHODS AND RESULTS: Escherichia coli strains were assessed for their reactions in culture media containing a beta-d-glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of beta-D-glucuronidase at pH 5.0 and pH 7.2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme beta-D-glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose-containing media expressed virtually no beta-D-glucuronidase activity at pH 5.0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. CONCLUSIONS: E. coli that failed to produce green colonies on MLGA produced lower levels of beta-D-glucuronidase than did strains that formed green colonies, the difference being greater at pH 5.0 than pH 7.2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme beta-D-glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7.2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).

False-negative beta-D-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia coli from water.

AIMS: Testing for beta-D-glucuronidase activity has become the basis of many methods for the detection of Escherichia coli in both food and water. Used in combination with tests for the presence of beta-D-glucuronidase, these tests offer a simple method for simultaneously detecting coliforms and E. coli. The purpose of this study was to determine the effectiveness of several different procedures in detecting beta-D-glucuronidase activity and hence in detecting E. coli. METHODS AND RESULTS: The ability of membrane lactose glucuronide agar (MLGA), Colilert-18, MI agar, Colitag and Chromocult agar to detect beta-D-glucuronidase activity was tested with over 1000 isolates of E. coli recovered from naturally contaminated water samples. Four of the media gave very similar results but MLGA failed to detect glucuronidase activity in 15.6% of the cultures tested. CONCLUSIONS: MLGA had very poor sensitivity for the detection of beta-D-glucuronidase activity in strains of E. coli isolated from naturally contaminated water. This is probably because of the fact that beta-D-glucuronidase activity is pH-sensitive and that acid is formed by E. coli during fermentation of lactose in MLGA. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of E. coli in drinking water is the primary test used to establish faecal contamination. The poor sensitivity of MLGA in detecting beta-D-glucuronidase activity suggests that this medium and others containing high concentrations of fermentable carbohydrate should not be used for the detection of E. coli.