Enzyme-based detection and differentiation of Escherichia coli and total coliforms in drinking water

The bacterial enzymes beta-glucuronidase and beta-galactosidase hydrolyze the synthetic substrates anthracene-beta-d-glucuronide and pyrene beta-d-galactopyranoside, respectively. The enzymes are produced by, and are therefore used to detect, Escherichia coli and total coliforms. Upon cleavage by these enzymes, the substrates release the soluble fluorescent molecules 2-hydroxyanthracene and 1-hydroxypyrene, which are then selectively partitioned in a partitioning element within a sample holder. Fluorescence is coupled into the wave-guide and is selectively detected by a spectrometer in a commercial instrument called Pathogen Detection SystemsRTM. The level of fluorescence produced is detected by the spectrometer and used to measure contamination with E. coli and coliforms in a presence or absence format. The impact of drinking water quality parameters such as colour, turbidity, pH, fluoride and aluminum concentrations on detection of these bacteria was investigated using the fluorescent substrates. The presence of fluoride had no significant negative effects on the fluorescence of the cleaved products and was found to enhance the fluorescence/enzymatic activity of the bacterial supernatant at 0.87 ppm. However, aluminum was found to enhance the fluorescence efficiency of the cleaved substrates. When aluminum is present during bacterial growth, a drastic reduction in enzyme activity at concentrations higher than 0.5 ppm was observed. Additionally, presence of aluminum was shown to reduce the rate of enzymatic hydrolysis of the substrates. Aluminum does not impact directly on the fluorescence of the cleaved products but rather on bacterial growth and on enzyme activities. The increase in the fluorescence of the cleaved products was observed when the pH of the medium was reduced, showing highest fluorescence at pH 5.5. It was also observed that pH affected on the levels of enzyme expression. Water physicochemical parameters such as turbidity and colour in field samples had no impact on the detection of E. coli and total coliforms. This method is able to detect 1 CFU/100 mL of these bacteria in about 17 h and less than 50 CFU/100 mL in about 12 h in field samples of recreational water.