Submitted to: Journal of Applied Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2008
Publication Date: February 2, 2009
Citation: Jenkins, M., Endale, D.M., Fisher, D.S. 2009. A most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli 0157:H7 in surface waters [abstract]. Journal of Applied Microbiology. 106:572-579. Technical Abstract: Background Beef and dairy cattle are a source of pathogenic Escherichia coli 0157:H7. Agricultural watersheds with beef or dairy cattle can thus adversely impact surface waters and threaten public health as exemplified by the occurrence of spinach contaminated with E. coli 0157:H7 that was marketed throughout the US. To understand better and manage the fate and transport of this pathogen in agricultural watersheds a most probable number (MPN) method for enumerating E. coli 0157:H7 in environmental waters was developed. Methods The first step is to filter 20 L of environmental water through a FALP 293 mm membrane filter with a 1-µm-pore-size, and centrifuge material removed from filter. The pellet is resuspended in 5 mL of PBS, and 1-mL aliquots are used to inoculate the first five tubes containing 9 mL of laural tryptose broth (LTB) followed by three 10-fold dilutions to complete a five-tube, four by 10-fold dilution scheme. Positive LTB tubes are inoculated onto sorbitol MacConkey (SMAC) plates for isolating colonies. Three colorless colonies are transferred to SMAC plates and streaked for purity. Tubes of LTB-MUG with durham tubes for collecting gas are inoculated with these isolates. Isolates from non-fluorescing tubes with gas production are then tested for glutamate decarboxylase (GAD) activity and latex agglutination. If isolates are both GAD and agglutination positive, confirmation is made by real time-PCR with primers and probe for the virulence gene eaeA. MPN and 95% confidence limits of E. coli 0157:H7 cells are calculated by the number of positive tubes with all determinations having improbability ratios >0.0001. Results This method has determined the density of E. coli 0157:H7 in 20-L samples of surface water from a watershed with cattle and wildlife to be as low as 1 cell/L. Conclusion This represents a culture-based method with a real-time PCR confirmation step that can be considered a tool for quantifying dilute concentrations and fluxes of E. coli 0157:H7 in surface waters of watersheds impacted by animal agriculture, and developing collections of environmental isolates of E. coli 0157:H7 for forensic purposes.