Development and validation of a FISH-based method for the detection and quantification of E. coli and coliform bacteria in water samples

Abstract

Monitoring of microbiological contaminants in water supplies requires fast and sensitive methods for the specific detection of indicator organisms or pathogens. We developed a protocol for the simultaneous detection of E. coli and coliform bacteria based on the Fluorescence in situ Hybridization (FISH) technology. This protocol consists of two approaches. The first allows the direct detection of single E. coli and coliform bacterial cells on the filter membranes. The second approach includes incubation of the filter membranes on a nutrient agar plate and subsequent detection of the grown micro-colonies. Both approaches were validated using drinking water samples spiked with pure cultures and naturally contaminated water samples. The effects of heat, chlorine and UV disinfection were also investigated. The micro-colony approach yielded very good results for all samples and conditions tested, and thus can be thoroughly recommended for usage as an alternative method to detect E. coli and coliform bacteria in water samples. However, during this study, some limitations became visible for the single cell approach. The method cannot be applied for water samples which have been disinfected by UV irradiation. In addition, our results indicated that green fluorescent dyes are not suitable to be used with chlorine disinfected samples.

Biological Stability of Drinking Water: Controlling Factors, Methods, and Challenges

“…In the Netherlands, the presence of Aeromonas is also included in legislation and often tested as an indicator for biological stability ( Waterleidingbesluit, 2001 ). Specific detection is traditionally performed by cultivation on selective media, and more recently with molecular-based techniques such as qPCR or with the use of specific antibodies ( Rompré et al, 2002 ; Schets et al, 2002 ; Tallon et al, 2005 ; Hügler et al, 2011 ; Douterelo et al, 2014 ). Besides specific detection, cultivation is also used worldwide in drinking water monitoring for HPC methods ( Allen et al, 2004 ; Pepper et al, 2004 ; Uhl and Schaule, 2004 ; Batté et al, 2006 ).…”

Development of Fluorescence In Situ Hybridization as a Rapid, Accurate Method for Detecting Coliforms in Water Samples

“…For example, Garcia-Armisen et al [19] and Baudart et al [20] used an improved FISH method to detect viable E. coli in river water, wastewater, seawater, and freshwater samples. In addition, Ootsubo et al [40] used FISH with probe D following cultivation for enumeration of Enterobacteriaceae in food and environmental water samples, and the experimental time was within 7 h. Hügler et al [41] also devel-oped and validated a FISH-based method for the detection and quantification of E. coli and coliform bacteria in water samples. Their protocol consisted of two approaches: direct detection of single E. coli and coliform bacteria on filter membranes, and incubation of the filter membranes on nutrient agar plates with subsequent detection of microcolonies.…”

A Preliminary Study on Detection of Serogenic E.coli in Fishes to Conduct a Microbial Assessment of Coastal Water

“…A bacterial species associated with infection via ingestion of edible products of marine origin is Escherichia coli. The occurrence of this bacterium in food is directly related to fecal contamination and unhygienic conditions during the handling process (16). There are numerous potential causes for E. coli contamination which can include the quality of ice during storage to the unhygienic handling processes (11).…”

“…To prevent the colonization of the intestine with pathogenic bacteria, E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for 3 days, but its numbers decline slowly afterward (16).…”