Culture-dependent enumeration methods failed to simultaneously detect disinfectant-injured and genetically modified Escherichia coli in drinking water

Underestimation of Escherichia coli in drinking water, an indicator microorganism of sanitary risk, may result in potential risks of waterborne diseases. However, the detection of disinfectant-injured or genetically modified (GM) E. coli has been largely overlooked so far. To evaluate the accuracy of culture-dependent enumeration with regard to disinfectant-injured and GM E. coli, chlorine-or ozone-injured wild-type (WT) and GM E. coli were prepared and characterized. Then, water samples contaminated with these E. coli strains were assayed by four widely used methods, including lactose tryptose broth-based multiple-tube fermentation (MTF), mendo- based membrane filtration method (MFM), an enzyme substrate test (EST) known as Colilert, and Petrifilm-based testing slip method (TSM). It was found that MTF was the most effective method to detect disinfectant-injured WT E. coli (with 76.9% trials detecting all these bacteria), while this method could not effectively detect GM E. coli (with uninjured bacteria undetectable and a maximal detection rate of 21.5% for the injured). The EST was the only method which enabled considerable enumeration of uninjured GM E. coli, with a detection rate of over 93%. However, the detection rate declined to lower than 45.4% once the GM E. coli was injured by disinfectants. The MFM was invalid for both disinfectant-injured and GM E. coli. This is the first study to report the failure of these commonly used enumeration methods to simultaneously detect disinfectant-injured and GM E. coli. Thus, it highlights the urgent requirement for the development of a more accurate and versatile enumeration method which allows the detection of disinfectant-injured and GM E. coli on the assessment of microbial quality of drinking water.