Protocol for the recovery and detection of Escherichia coli in environmental water samples

- Rapid protocol (less than 75 min) for E. coli detection in environmental waters developed.
- Optimization of sample preparation for best performance including E. coli recovery and extraction of GUS marker enzyme.
- Evaluation of this protocol against the standard method and a literature rapid method.

To achieve active management of bathing areas and to reduce risk associated with the presence of fecal pollution, tests capable of rapid on-site assessment of microbiological water quality are required. A protocol for the recovery and detection of fecal pollution indicator bacteria, E. coli, using β-glucuronidase (GUS) activity was developed. The developed protocol involves two main steps: sample preparation and GUS activity measurement. In the sample preparation step, syringe filters were used with a dual purpose, for the recovery and pre-concentration of E. coli from the water matrix and as μL reactors for bacteria lysis and GUS extraction. Subsequently, GUS activity was measured using a continuous fluorometric method developed previously. The optimum GUS recovery conditions for the sample preparation step were found to be 100 μL PELB (supplemented with 1 mg mL−1 lysozyme and 20 mM DTT) at 37 °C for 30 min. The protocol was evaluated on environmental samples (fresh and seawater) against an establish GUS assay method (Coliplage®). GUS activities corresponding to samples containing as low as 26 MPN E. coli 100 mL−1 were detected for the seawater sample and as low as 110 MPN E. coli 100 mL−1 for the freshwater samples. By comparison with the Coliplage® method, this protocol offered an improvement in the measured GUS activities of 3.1 fold for freshwater samples and 4.1 fold for seawater samples. Furthermore, the protocol developed here, has a time-to-result of 75 min, and successfully addresses the requirement for tests capable of rapid assessment of microbiological water quality.

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