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

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The manuscript requires a number of corrections and also some additional experiments
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    Rated 3 of 5.
Level of importance:
    Rated 3 of 5.
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    Rated 2 of 5.
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    Rated 3 of 5.
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    Rated 4 of 5.
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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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    Review information

    10.14293/S2199-1006.1.SOR-LIFE.AJ19WR.v1.RTLGOJ

    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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    Review text

    This is an interesting manuscript where the number of the Salmonella spp that are present in seed sprouts samples is analyzed. The manuscript requires a number of corrections and also some additional experiments (as suggested below). The authors should also prove that the proposal technique has a high sensitive for the Salmonella detection, and that the subpopulations detected by the probes used in the flow cytometry assay can be well separate (the noise shown in the pictures should be removed or the signal should be less than those of the population classified as Salmonella). In addition, they should demonstrate that the bacilli appearing in the SEM photographs are also Salmonella. Finally, they should propose a way to apply this technique to the socio-economic sector, because unlike what the author’s opinion, I do not think that it is a cheaper and easy to implement technique.



    Major comments



    1- In the end of INTRODUCTION section the author mentioned that “our study has direct relevance to public health and safety.” However, there is not a method to ensure non contamination of the seeds. Therefore, it is not a preventive measure during germination but only prevents the market lead to contaminated sprouts.



    2 In the “Cultural and molecular characterization of endogenous sprout microbiota” section: The authors should explain better the procedure used to perform the isolation of microorganisms from the seeds (this was by washing or making a homogenate of these seeds in 0.1% peptone water).



    3- In the same section: when the author mentioned that “Total nucleic acids were isolated from…..”. They should specify if the total DNA was extracted from individual colonies or from the sample that was deposited on selective medium plates?



    4- In the “Time course hybridization of Salmonella ser. Typhimurium in alfalfa sprouts” section and throughout the manuscript, delete “ser”



    5- In the footnote of figure 1: The last sentence should go in results or discussion, it is wrong to put it in figure legend. Moreover, and in accordance to those below mentioned, this asseveration does not apply for the subgroup IIIa.



    6- In Panel A and B of figure 2: the Authors should mention what is the difference in the preparation of both pictures to justify the observed differences, such as the presence of biofilm in one of them but not in the other.



    7- In the “Effects of physical and chemical pre-analytical sample preparation” section, the Denhardt’s solution: was the effect of this treatment confirmed by SEM?



    8- The way to mention for the first time each microorganism should be normalized. For example: the first time that Salmonella Typhimurium was mentioned, the authors should added (S. Typhimurium), and then only mention as S. Typhimurium.



    9- In the footnote of figure 5: a- Delete the sentence “As indicated by the graph, near –maximal staining intensity was achieved after 15 min hybridization”, because it is mentioned in results section. b- The sentence “These data suggest that while maximal staining may require longer hybridization, effective detection of Salmonella may be accomplished at earlier hybridization timepoints.” should be deleted from figure legend and be added to the results or conclusion sections.



    10- Figure 6: Although the authors made numerous efforts to increase the sensitivity of the test flow cytometry to detect Salmonella outbreaks, graphs of these tests show that there is still a lot of noise in the graphs and do not show that the group identified as Salmonella actually corresponds to this type of pathogen.



    11- In DISCUSSION section: As I above mentioned, despite to the author´s justification, in the flow cytometry graphs the probes used detected much noise, being Salmonella the smallest group represented. For this reason I do not see a significant increased in the sensitivity of this microorganism detection.



    12- In DISCUSSION section the authors mentioned that Sal3 and Salm-63 probes are targeting different region of ribosome corresponding to E. coli rRNA. However, in the table and references was mentioned that all these probes are designed specifically for Salmonella, but not for E. coli detection. This point should be clarified by the authors.



    13- In DISCUSSION section: “We expect that this probe would bind to the rRNA of subroup IIIa strains and modify the localized rRNA accessibility for Salm-63, potentially yielding a more intense signal for strains belonging to this subgroup”. These assays should be performed by authors, not only be proposed, in order to give more sense to this manuscript and convincingly demonstrate that the here proposed method has advantages over those currently used.



    14- In CONCLUSIONS section: “Concentration of our samples with TTF and centrifugation enabled us to reliably detect S. Typhimurium in alfalfa sprouts at levels as low as 103 CFU ml/1 sprout wash (104 CFU g/1 sprouts).” This indicates that the developed method is not a very sensitive procedure forSalmonella detection, because the samples should be concentrated.

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