Antibiofilm Efficacy of Quercetin against Vibrio parahaemolyticus Biofilm on Food-Contact Surfaces in the Food Industry

Round 1

Reviewer 1 Report (Previous Reviewer 4)

This manuscript showed that quercetin suppressed motility and biofilm formation of Vibrio parahaemolyticus. However, I have a concern as follows in this manuscript.

1.       In the results section

I wonder QC concentration used in this study suppressed growth of V.p.

Please show MIC results, such as changes in OD600 or colony numbers.

Author Response

Thank you dear respected reviewer for evaluating our manuscript to improve it.

Comments and Suggestions for Authors

This manuscript showed that quercetin suppressed motility and biofilm formation of Vibrio parahaemolyticus. However, I have a concern as follows in this manuscript.

1. In the results section

I wonder QC concentration used in this study suppressed growth of V.p.

Response: We are thankful to the respected reviewer for the initial evaluation of our manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript. Certain amounts of quercetin did not affect the growth of bacteria. Please check the reference below:

https://0-www-sciencedirect-com.brum.beds.ac.uk/science/article/pii/S0956713522001578

Please show MIC results, such as changes in OD600 or colony numbers.

Response: We are thankful to the respected reviewer for the initial evaluation of our manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript. Corrected in the manuscript Line 267-282 and we have added also MIC results in Figure 1.

Thank you very much respected reviewer for your valuable comments on improving our manuscript.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

The manuscript entitled "Antibiofilm efficacy of quercetin against Vibrio parahaemolyticus biofilm on food contact surfaces in the food industry" aims to evaluate the efficacy of Quercetin as a food additive in reducing V. parahaemolyticus biofilm formation on steel coupon (SS) and hand gloves (HG) as well as testing its antimicrobial activities.

The manuscript presents an interesting topic dealt with great methodological complexity. However, it still has several problems related to the form and presentation of some results.

For this reason, the manuscript should not be accepted in the present form but revised and resubmitted.

The introduction should be implemented with a focus on why this research was done.

below are some comments and suggestions for the authors.

Line 158 why did the authors choose the initial 10 5 CFU bacterial suspension? Is there a reference or previous work?

Line 184 should further detail be added, i.e. how did the authors get the swimming / swarming percentage?

Line 251 space between V. parahaemolyticus and ATCC ...

line 252 perhaps to make the manuscript more readable the authors should use only one mode of expression of the quercetin concentrations used, that is ½, 1 / 4,1 / 8 of MIC or 110, 55,27,5 ug / ml ...

line 258 "Quercetin reduced the motility of V. parahaemolyticus by 18 and 79% respectively" in figure 1 it seems that the percentage of reduction is greater than 18% when treated with 1/8 MIC please check figure and data .

Line 288 the name of the bacteria must be written in italics.

Line 311 lacked comments on the results of the genetic analysis.

Line 335 "quercetin disrupts cell-to-cell connections" is very unlikely that this sentence is only recognized by scanning electron micrographs. Is there any other analysis to support this sentence? Why are 1/8 MIC figs not shown?

Author Response

Thank you dear respected reviewer for evaluating our manuscript to improve it.

Comments and Suggestions for Authors

The manuscript entitled "Antibiofilm efficacy of quercetin against Vibrio parahaemolyticus biofilm on food contact surfaces in the food industry" aims to evaluate the efficacy of Quercetin as a food additive in reducing V. parahaemolyticus biofilm formation on steel coupon (SS) and hand gloves (HG) as well as testing its antimicrobial activities.

The manuscript presents an interesting topic dealt with great methodological complexity. However, it still has several problems related to the form and presentation of some results.

For this reason, the manuscript should not be accepted in the present form but revised and resubmitted.

Response: We are thankful to the respected editor and reviewer for the initial evaluation of our manuscript. We also acknowledge the efforts made for processing of the manuscript within suitable time period. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

The introduction should be implemented with a focus on why this research was done.

Response: Corrected in the manuscript Line 125-152 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

below are some comments and suggestions for the authors.

Line 158 why did the authors choose the initial 10 5 CFU bacterial suspension? Is there a reference or previous work?

Response: Corrected in the manuscript Line 163-175 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

Line 184 should further detail be added, i.e. how did the authors get the swimming / swarming percentage?

Response: Corrected in the manuscript Line 207-209 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

Line 251 space between V. parahaemolyticus and ATCC ...

Response: Corrected in the manuscript Line 275 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

line 252 perhaps to make the manuscript more readable the authors should use only one mode of expression of the quercetin concentrations used, that is ½, 1 / 4,1 / 8 of MIC or 110, 55,27,5 ug / ml ...

Response: Corrected in the manuscript Line 276-277 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

line 258 "Quercetin reduced the motility of V. parahaemolyticus by 18 and 79% respectively" in figure 1 it seems that the percentage of reduction is greater than 18% when treated with 1/8 MIC please check figure and data .

Response: Corrected in the manuscript Line 288 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

Line 288 the name of the bacteria must be written in italics.

Response: Corrected in the manuscript Line 319 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

Line 311 lacked comments on the results of the genetic analysis.

Response: Corrected in the manuscript Line 342-353 and throughout the manuscript. We appreciate the comments and made appropriate corrections throughout the revised manuscript.

Line 335 "quercetin disrupts cell-to-cell connections" is very unlikely that this sentence is only recognized by scanning electron micrographs. Is there any other analysis to support this sentence? Why are 1/8 MIC figs not shown?

Response: We have performed only FE-SEM for visual analysis for cell-to cell connections. FE-SEM images show cell-to-cell connections (Figure 6). 1/8MIC did not show difference and representative figure to compare with control and quercetin (1/2 MIC). We appreciate the comments and made appropriate corrections throughout the revised manuscript. For more details Please check the links below:

https://0-www-sciencedirect-com.brum.beds.ac.uk/science/article/pii/S0956713521009348

https://0-www-mdpi-com.brum.beds.ac.uk/2076-3921/11/9/1733/htm

Thank you very much respected reviewer for your valuable comments to improve our manuscript.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (Previous Reviewer 2)

The authors improve the manuscript. However, the text should be better in some parts, and language/style editing below some points/and suggestions. The manuscript should be accepted after minor revisions.

Line 38 please include reference  doi: 10.1146/annurev.mi.49.100195.003431.

Line 49 please insert references such as , https://0-doi-org.brum.beds.ac.uk/10.1016/j.ijfoodmicro.2014.07.007

Line63-63 and lines 72-72 give similar information that should be rewritten

Line 86-88 the sentence seems redundant please rewrite. Moreover “In V. parahaemolyticus strains, several biofilm-associated genes (vp0950, 88 vp0952, and vp0962) have been reported” appear very short.

Line 117 please include macroalgal extracts in a recent work  https://0-doi-org.brum.beds.ac.uk/10.1016/j.aquaculture.2021.737729

Line 145 “biofilm eradication” suggest replacing with “antibiofilm activity”

Line 177 may be its more simple “dilute it serial dilutions until it contained” replace to “reach the…”

Line 203 “were conducted” replace with “were performed”

243 one extra “(” please remove

In line 347 the figure should be improved maybe with the more evident arrows. 

Author Response

Thank you respected reviewer for evaluating our manuscript to improve it.

Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In the study of Roy et al., the effect of quercetin on Vibrio parahaemolyticus biofilm was investigated. The study is very interesting, well written and complete. However, the introduction is too long. The authors should consider shorten the introduction, particularly the information regarding the biofilms. my biggest concern is with the bacterial suspension dilutions. The authors add 100 μL of quercetin serially diluted to TSB and 100 μ L of bacterial suspension (105 log CFU/mL). However, this addition will cause the bacterial concentration to drop by half which will lead to the MIC assessment being incorrect as the concentration of the bacterial suspension is lower than described in the susceptibility testing guidelines. And same for the biofilm formation assay in which 100 µL of bacterial suspension (105 log CFU/mL) are added to 10 mL TSB.

Line 38-39: The sentence is confusing. Something is missing.

Line 46: at risk of contamination with…

Line 60: The reference is missing.

Line 75: including seawater AND marine organisms 

Line 79: “according to reports”. However, only one study is cited. Please add more references.

Line 259: The first 3 sentences of the discussion are quite odd.

Reviewer 2 Report

The paper “Eradication efficacy of quercetin against Vibrio parahaemolyticus biofilm on food processing appliances” evaluated the effectiveness of quercetin as a food additive in reducing V. parahaemolyticus biofilm on stainless-steel coupons and hand gloves (HG). The paper appears to provide a lot of data, but it is not well organized and appears to be lacking in results declared in the abstract such as scanning electron microscopy and the cell to cell interaction.

The manuscript needs to be revised by the authors. I believe that the paper in its current form is insufficient for publication in this journal.

The title “eradication” and the aims of the abstract “prevent biofilm formation” seem did not match because eradication is usually referred to as preformed biofilm whereas prevention is related to measures before biofilm formation.

The introduction appears insufficient and inconsistent with the theme of the paper, with no mention of the relationship between motility and biofilm formation and no explanation of the genetic analysis performed. Why did the authors choose these genes? how does their expression affect biofilm formation?

The methods are exposed in a less clear manner. More detail should be added

The results are interesting however the figures need to be improved and no sufficient comments are reported.

Some other minor observations:

In line 39 the sentence is not clear please rephrase.

Line 43 “the main causes” should be “the factors that play a pivotal role in the infections are…”

Line 51 please avoid acronyms “CDC” also for the institutions

Line 143 the species “ V. pahaemolyticus” should be in italics

Line 149 Please insert information on Vibrio CHROMagar

Line 150 “The MIC of quercetin was 220 μg/mL” this is a result shoed be moved in the result section

Line 158 What is the final concentration of quercitin in each agar plate?

Line 167-174 the methodology is less clear please rewrite

Line 211 the y axis in figure 1 is not clear in the bracket there are mm should be a percentage if I understand correctly also the some in Fig 2

Line 249 any comments are presented after figure 5 

Reviewer 3 Report

This manuscript describes the effectiveness of quercetin in reducing Vibrio parahaemolyticus biofilm formation. Quercetin is already known to have antibacterial properties against both Gram-positive and Gram-negative bacteria. Authors should describe what novelty of this work is compared to previous research results.

1.      In Abstract, the result of field emission scanning electron microscopy was presented. But, there were no experimental methods and results in manuscript.

2.      In Table 1, if citations were made, please describe references.  

3.      Lines 143, 285, 320. The name of microorganisms should be shown in italics. Please change it throughout the entire manuscript.

4.      Only one strain, Vibrio parahaemolyticus ATCC27969, was used in this study. It seems that it is necessary to evaluate for other strains of V. parahaemolyticus.  

5.      Lines 259-262. Please delete sentences.

Reviewer 4 Report

This manuscript showed that quercetin suppressed motility and biofilm formation of Vibrio parahaemolyticus. However, I have some concern as follows in this manuscript.

1. The title is misleading.  

There is no data that show food processing application.

2. The manuscript is very descriptive and lacks a clear mechanistic insight.

2-1. For example, authors mentioned ROS in introduction and discussion section, however, there is no data related that.

2-2. Authors did not show why and how vp0952 and vp0962 as virulence related genes. These genes are related to the biofilm formation. There are many other genes involved virulence of V. parahaemolyticus, such as TDH, TRH, T3SS1, and T3SS2.

3. Results

Please show MIC results, such as changes in OD600or colony numbers.

4. V. parahaemolyticus can disinfected by pure water. Why authors use quercetin. Because this application is difficult to use than pure water.