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

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        Abstract

        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 (~10 8 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 10 3 CFU ml −1 sprout wash (10 4 CFU g −1 sprouts) against high microbial backgrounds (~10 8 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 provide industry with a useful tool for compliance with sprout production standards proposed in the Food Safety Modernization Act (FSMA).

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        Most cited references 50

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        A membrane-filter technique for the detection of complementary DNA.

         D Denhardt (1966)
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          Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens

           J Fahey,  Y. Zhang,  P Talalay (1997)
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            Dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in Helicobacter pylori-infected mice and humans.

            The isothiocyanate sulforaphane [SF; 1-isothiocyanato-4(R)-methylsulfinylbutane] is abundant in broccoli sprouts in the form of its glucosinolate precursor (glucoraphanin). SF is powerfully bactericidal against Helicobacter pylori infections, which are strongly associated with the worldwide pandemic of gastric cancer. Oral treatment with SF-rich broccoli sprouts of C57BL/6 female mice infected with H. pylori Sydney strain 1 and maintained on a high-salt (7.5% NaCl) diet reduced gastric bacterial colonization, attenuated mucosal expression of tumor necrosis factor-alpha and interleukin-1beta, mitigated corpus inflammation, and prevented expression of high salt-induced gastric corpus atrophy. This therapeutic effect was not observed in mice in which the nrf2 gene was deleted, strongly implicating the important role of Nrf2-dependent antioxidant and anti-inflammatory proteins in SF-dependent protection. Forty-eight H. pylori-infected patients were randomly assigned to feeding of broccoli sprouts (70 g/d; containing 420 micromol of SF precursor) for 8 weeks or to consumption of an equal weight of alfalfa sprouts (not containing SF) as placebo. Intervention with broccoli sprouts, but not with placebo, decreased the levels of urease measured by the urea breath test and H. pylori stool antigen (both biomarkers of H. pylori colonization) and serum pepsinogens I and II (biomarkers of gastric inflammation). Values recovered to their original levels 2 months after treatment was discontinued. Daily intake of sulforaphane-rich broccoli sprouts for 2 months reduces H. pylori colonization in mice and improves the sequelae of infection in infected mice and in humans. This treatment seems to enhance chemoprotection of the gastric mucosa against H. pylori-induced oxidative stress.
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              Author and article information

              Affiliations
              [1 ]Department of Animal Science, University of Wyoming, Laramie, WY, USA
              [2 ]Department of Food Science and Human Nutrition, Rapid Microbial Detection and Control Laboratory, Iowa State University, Ames, IA, USA
              Author notes
              [* ]Corresponding author's e-mail address: byron@ 123456iastate.edu
              Contributors
              (View ORCID Profile)
              Journal
              SOR-LIFE
              ScienceOpen Research
              ScienceOpen
              2199-1006
              31 December 2014
              : 0 (ID: 2b6c7758-f173-4f97-b71b-371fa127ede1 )
              : 0
              : 1-13
              2351:XE
              10.14293/S2199-1006.1.SOR-LIFE.AJ19WR.v1
              © 2014 B. Bisha and B.F. Brehm-Steccher.

              This work has been published open access under Creative Commons Attribution License CC BY 4.0 , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com .

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