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Flow Cytometry for Rapid Detection of Salmonella spp. in Seed Sprouts

Byron Brehm-Stecher (corresponding) , Bledar Bisha (2014)

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 Salmonella. 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 Enterobacteriaceae. This heavy background may present challenges to the detection of Salmonella, especially if this pathogen is present in relatively low numbers. We combined DNA-based fluorescence in situ hybridization (FISH) with flow cytometry (FCM) for the rapid molecular detection of Salmonella enterica 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 S. 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 S. 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 Salmonella in this challenging food matrix and provides industry with a useful tool for compliance with sprout production standards proposed in the Food Safety Modernization Act (FSMA).

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DOI:: 10.14293/S2199-1006.1.SOR-LIFE.AJ19WR.v1.RYFPCC

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Bisha and Brehm-Stecher reported the combined DNA-based fluorescence in situ hybridization (FISH) with flow cytometry (FCM) method to detect Salmonella in seed sprouts samples. Quick and sensitive method to detect Salmonella is important in terms of food safety concern, this manuscript is of scientific merit and I recommend to be acceptable for publication. However, I do have some suggestions:

The authors put too many introductory materials in the Discussion section. The first 5 paragraphs of the Discussion should be removed to the Introduction section or omit some of them. They should discuss the results presented in the manuscript more.

P2, Bacterial strains and culture conditions: spell out Escherichia coli ATCC 25922

P4, Pre-analytical sample preparation: Individual samples (1.3 ml) were aliquoted into in 1.5 ml polypropylene microcentrifuge tubes.., please remove “in”.

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Flow Cytometry for Rapid Detection of Salmonella spp. in Seed Sprouts

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