ARS | Publication request: Most probable number methods for enumerating salmonella and E. coli 0157:H7 in environmental waters

Submitted to: USDA-CSREES National Water Quality Conference
Publication Type: Abstract Only
Publication Acceptance Date: December 4, 2007
Publication Date: February 3, 2008
Citation: Jenkins, M., Fisher, D.S., Endale, D.M., Lowrance, R.R., Newton, L., Gay, P., Vellidis, G. 2008. Most probable number methods for enumerating salmonella and E. coli 0157:H7 in environmental waters. Proceedings of the 2008 USDA-CSREES National Water Quality Conference, February 3-7, 2008, Sparks, Nevada. CD-ROM.

Technical Abstract: Background Most agricultural animals such as beef and dairy cattle, swine, and poultry are a source of Salmonella and E. coli0157H:7. Watersheds with animal agriculture can adversely impact recreational waters and threaten public. To understand better and manage the fate and transport of these pathogens in agricultural watersheds a most probable number (MPN) method for each of them was developed to determine their concentrations in environmental waters. Methods Each MPN method begins with a filtration step. As much 20 l of water is filtered through a FALP 293 mm membrane with a 1 µm pore size. The filtered material is scrubbed off the filter with a brush in PBS, and consolidated by centrifugation. The pellet is resuspended in PBS and used to inoculate tubes of Tetrathionate for Salmonella and Laural Tryptose Broth (LTB) for E. coli0157:H7. The tubes are arranged in a 5-tube, four dilution scheme. Positive tetrathionate tubes are inoculated onto Brilliant Green agar plates; presumptive Salmonella isolates are streaked for purity on Beef Heart Infusion (BHI) agar plates. Fresh colonies are stabbed into slants of Lysine Iron Agar and Triple Sugar Iron agar. If both slants are positive for Salmonella, confirmation is made with a TaqMan assay using primers and probe for the junction between the virulence genes SipB and SipC. Positive LTB tubes are spread on Sorbitol MacConkey agar plates for isolating colonies. Suspected E. coli0157:H7 colonies are inoculated into tubes of LTB-MUG and checked for gas production and non-fluorescence. Corresponding colonies are tested for glutamate decarboxylase (GAD) activity and latex agglutination. If positive for both GAD and agglutination, confirmation is made with a TaqMan assay using primers and probe for the virulence gene eaeA. MPN of cells is calculated with FDA’s preferred MPN method. Results and Conclusions These MPN methods have determined the densities of Salmonella and E. coli0157:H7 as low as 0.1 MPN l-1 of waters impacted by animal agriculture. They can be used as tools for better understanding fluxes of these pathogens in watersheds impacted by animal agriculture.