Flow cytometry for rapid detection of Salmonella spp. in seed sprouts

Abstract

<p>Seed sprouts (alfalfa, mung bean, radish, etc.) have been implicated in several recent national and international outbreaks of salmonellosis. Conditions used for sprouting are also conducive to the growth of <em>Salmonella</em>. As a result, this pathogen can quickly grow to very high cell densities during sprouting without any detectable organoleptic impact. Seed sprouts typically also support heavy growth (~108 CFU g−1) of a heterogeneous microbiota consisting of various bacterial, yeast, and mold species, often dominated by non-pathogenic members of the family <em>Enterobacteriaceae</em>. This heavy background may present challenges to the detection of <em>Salmonella</em>, especially if this pathogen is present in relatively low numbers. We combined DNA-based fluorescence <em>in situ</em> hybridization (FISH) with flow cytometry (FCM) for the rapid molecular detection of <em>Salmonella enterica</em> ser. Typhimurium in artificially contaminated alfalfa and other seed sprouts. Components of the assay included a set of cooperatively binding probes, a chemical blocking treatment intended to reduce non-specific background, and sample concentration via tangential flow filtration (TFF). We were able to detect <em>S</em>. Typhimurium in sprout wash at levels as low as 103 CFU ml−1 sprout wash (104 CFU g−1 sprouts) against high microbial backgrounds (~108 CFU g−1 sprouts). Hybridization times were typically 30 min, with additional washing, but we ultimately found that <em>S</em>. Typhimurium could be readily detected using hybridization times as short as 2 min, without a wash step. These results clearly demonstrate the potential of combined DNA-FISH and FCM for rapid detection of <em>Salmonella</em> in this challenging food matrix and provide industry with a useful tool for compliance with sprout production standards proposed in the Food Safety Modernization Act (FSMA).</p>