Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters

Authors: Jenkins, Michael; Endale, Dinku; Fisher, Dwight

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2007
Publication Date: 6/1/2008
Citation: Jenkins, M., Endale, D.M., Fisher, D.S. 2008. Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters. Journal of Applied Microbiology. 104:1562-1568.

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 Salmonella. Because small numbers of Salmonella can cause disease, and because the indicator of fecal contamination, E. coli, may not indicate its presence, a method is needed to detect and enumerate dilute concentrations of viable Salmonella. The capacity to quantify small numbers of Salmonella in environmental water samples would expand out present understanding of its fate and movement across various landscapes. To resolve this problem three proven technologies were combined: 1) a filtration method for filtering large volumes of environmental water, 2) a standard most probable number (MPN) dilution scheme for counting bacteria with prescribed growth media, and 3) a genetic method for confirming the identity of Salmonella. The efficiency of the filtration system was tested in the lab. The entire method was tested on surface waters from a watershed with animal agriculture. This method has determined the concentration of Salmonella in 20 liter samples taken from the inflow and outflow streams of a pond in an agricultural watershed as low as 0.1 Salmonella cells/liter and identified substantial fluxes of Salmonella when the fecal indicator bacterium E. coli was not detected. The sensitivity of this method will enhance our understanding of the fate and transport of Salmonella in agricultural watersheds and because it has the potential for developing culture collections it may prove helpful in identifying the actual environmental sources of this pathogen.

Technical Abstract: Aim: To better understand and manage the fate and transport of Salmonella in agricultural watersheds we developed a culture-based, five tube-four dilution most probable number (MPN) method for enumerating dilute densities of Salmonella in environmental waters. Methods: The MPN method was a combination of a filtration technique for large sample volumes of environmental water, standard selective media for Salmonella, and a TaqMan confirmation step. Results: This method has determined the density of Salmonella in 20 l samples of inflow and outflow streams of pond water as low as 0.1 MPN l-1, and a low 95% confidence level of plus or minus 0.015 MPN l-1. Salmonella densities ranged from not detectable to 0.55 MPN l-1 for inflow samples and from not detectable to 3.4 MPN l-1 for outflow samples. Salmonella densities of inflow samples were associated with densities of E. coli and fecal enterococci that indicated stream contamination with feces, and with non-detectable outflow densities of the fecal indicator bacteria. The MPN methodology was extended to flux determinations by integrating with volumetric measurements of inflow (mean flux of 2.5 l s-1) and outflow (mean flux of 5.6 l s-1). Fluxes of Salmonella ranged from 100 to greater than 104 MPN h-1. Conclusion: This is a culture-based method that can detect small numbers of Salmonella in environmental waters containing animal agriculture and wildlife. Impact: Applying this method to environmental waters will improve our understanding of the transport and fate of Salmonella in agricultural watersheds, and can be the basis of valuable collections of environmental Salmonella.