Wednesday, November 15, 2006 - 1:45 PM
237-1

A Most Probable Number Method for Enumerating Escherichia coli 0157:H7 in Environmental Waters.

Michael Jenkins, USDA-ARS, 1420 Experiment Station Rd., Watkinsville, GA 30677-2373

Beef and dairy cattle are a source of pathogenic Escherichia coli 0157:H7.  Agricultural watersheds with beef or dairy cattle can thus adversely impact recreational waters and threaten public health especially at the expanding interface of urban and agricultural lands.  To understand better and manage the fate and transport of this zoonotic pathogen in agricultural watersheds a most probable number (MPN) method for enumerating E. coli 0157:H7 in environmental waters was developed.  The first step is to filter, for example, 20 l of environmental water through a FALP 293 mm membrane filter with a 1 µm pore size, and centrifuge material removed from filter.  Centrifuged pellet is resuspended in PBS and used to inoculate tubes of laural tryptose broth (LTB) arranged in a 5-tube four or more dilution scheme.  Positive LTB tubes are inoculated onto sorbitol MacConkey  (SMAC) plates for isolating colonies.  Two to three colorless colonies are transferred to SMAC plates and streaked for isolation.  Tubes of LTB-MUG with durham tubes for collecting gas are inoculated with these isolates.  Non-fluorescing tubes with gas production are putative E. coli 0157:H7.  Corresponding isolates are confirmed by testing for glutamate decarboxylase activity and latex agglutination.  Further confirmation can be made by PCR with primers for the virulence gene eaeA.  MPN of cells is calculated using Microsoft Excel spread sheet and its Solver.  This method has determined the density of E. coli 0157:H7 in a 20 l sample of pond water from a watershed with cattle to be 0.01 cells/100 ml.  This represents a culture-based method to which real-time PCR methods can be compared.

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