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Title: Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Author
item Jenkins, Michael
item Endale, Dinku
item Fisher, Dwight
item GAY, P - University Of Florida

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/28/2008
Publication Date: 2/1/2009
Citation: Jenkins, M., Endale, D.M., Fisher, D.S., Gay, P.A. 2009. Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters. Journal of Applied Microbiology. 106:572-579.

Interpretive Summary: Watersheds with animal agriculture have the potential to adversely impact recreational waters and threaten public health by contaminating surface waters with fecal pathogens such as Escherichia coli O157:H7. Because small numbers of E. coli O157:H7 can cause disease and commonly used indicators of fecal contamination may not indicate the presence of this pathogen, scientists and public health officials need a method to detect dilute concentrations of the pathogen in order to understand its viability and movement in the environment. Scientists at the USDA-ARS J. Phil Campbell Sr. Natural Resource Conservation Center developed a method combining three technologies to detect low numbers of E. coli O157:H7 and tested the method with samples from an agricultural watershed. The method combined filtering large volumes of environmental water, a technique to count bacteria using a laboratory growth media with dilutions and immunological assay, and a genetic method for confirming the identity of E. coli O157:H7. Their technique proved very sensitive to detecting concentrations of E. coli O157:H7 from samples taken from the inflow and outflow streams of a pond and within the pond in an agricultural watershed, even when standard indicator organisms of fecal contamination could not be detected. The sensitivity of this method will enhance our understanding of the fate and transport of E. coli O157:H7 in agricultural watersheds. Additionally, because it has potential for developing culture collections, it may prove helpful in identifying sources of this pathogen in the environment. This method of quantifying concentrations of E. coli O157:H7 in environmental waters is a tool that federal, state, and local agencies concerned with food and water protection can use to better manage contamination risks, and can be used in efforts to prevent E. coli O157:H7 contamination of fresh produce.

Technical Abstract: To better understand and manage the fate and transport of Escherichia coli O157:H7 in agricultural watersheds we developed a culture-based, five tube-multiple dilution most probable number (MPN) method for enumerating dilute densities of E. coli O157:H7 in environmental waters. The MPN method was a combination of a filtration technique for large sample volumes of environmental water, standard selective media, biochemical and immunological tests for E. coli O157:H7, and a TaqMan confirmation step. This method has determined the density of E. coli O157:H7 in 20 L samples of pond-inflow and -outflow streams and within pond water as low as 0.1 MPN L-1; and with a 95% confidence level as low as 0.015 MPN L-1. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN L-1 for pond inflow samples, from not detectable to 0.9 MPN L-1 for pond outflow samples, and from not detectable to 18 MPN L-1 for within pond samples. Not all E. coli O157:H7 densities of pond inflow samples were associated with densities of E. coli and fecal enterococci that indicated stream contamination with feces. Escherichia coli O157:H7 densities within pond and pond outflow were associated with densities of fecal indicators that were either not detectable or below the criterion indicative of fecal contamination. The MPN methodology was extended to flux determinations by integrating with volumetric measurements of pond inflow (mean flux of 2.0 L s-1) and outflow (mean flux of 2.8 L s-1). Fluxes of E. coli O157:H7 ranged from less than 55 to greater than 104 MPN h-1. This is a culture-based method that can detect small numbers of viable/culturable E. coli O157:H7 in environmental waters of watersheds containing animal agriculture and wildlife. Applying this method to environmental waters will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of valuable collections of environmental E. coli O157:H7. It may be used as a tool to better manage contamination risks, and can be used in efforts to prevent E. coli O157:H7 contamination of field-grown, fresh produce.