Effects of an Eco-Friendly Sanitizing Wash on Spinach Leaf Bacterial Community Structure and Diversity
Round 1
Reviewer 1 Report
The manuscript written by Tenzin provides an brief insight into microbiota of spinach leaves after treatment with several disinfectants.
In general, the manuscript is not polished and needs some editorial work. I recommend either rejection or major revision. Please find my major and minor remarks below.
Major remarks:
- The manuscript and supplemental materials are disconnected, and it seems like the authors did not submit correct supplemental files.
- The authors excluded taxa with lower abundance from the analyses. This makes me question the diversity indices and other results.
- There are too many figures and tables for provided data. Most of them do not add any value to the manuscript.
- Relative abundances are important. However, it would be interesting to see absolute abundance - the effect of disinfectants is diminished - are they effective or not?
- The figures are in different styles and with different color/pattern schemes, which makes the manuscript even less consistent (other inconsistencies described below)
- I am missing the analysis of controls (disinfectants alone, with no spinach). This is the only reason why the diversity would be higher in spinach treated in PAA than in tap water.
- The authors used a commericially available disinfectant which is not in a pure form. At this point we do not know if the diversity changed because of PAA, H2O2 or something else.
- I am not sure if the amount of data is sufficient for a serious publication as it does not answer some relevant questions. For readers (and consumers) it would be more interesting to see if these disinfectants are efficient in reducing pathogen load, if there are persisters present after disinfection, if normal microbiota is able to "protect" the contaminants from disinfectants etc. I am aware that there was a lot of work invested into this manuscript. However, at this point, it seems like an analysis of controls, without experimental groups.
- I have some serious concerns about DNA isolation. The authors used peptone water, and not a buffer with EDTA.
Minor remarks:
L45-46: This statement is very vague. Are we talking about core microbiota? I would assume that microbiota is different in different environments
L52: what about the source of water, fertilizer, etc? was any research done on the source of these pathogens?
L58 and thorughout the manuscript: please be consistent with terminology. Microbiome and microbial communities/microbiota are not the same. Microbiome is just a collection of genetic material that is not necessarily from living organisms. So, microbiome cannot be (strictly speaking) responsible for anything. But microbiota can be.
L59-60: quotation marks not needed.
L61-62: only peptides? what about outer secondary metabolites?
L64: Bacterial population. was assessed? has been assessed? is generally assessed? unclear.
L67: DGGE is the abbreviation. Since this is the only place in the manuscript where you use DGGE or T-RFLP, there is no need to abbreviate these methods.
L68: usually, people analyze 16S rRNA gene, not the RNA itself.
Bacterial communities, as the community is not identical on every leaf of every spinach in the world.
L70 and the whole paragraph.: Bacterial communities. Analyses.
L77-8: this sentence can be easily contracted.
L79: not true. It has low reactivity compared to chlorine (Banach, J. L., Sampers, I., Van Haute, S., and van der Fels-Klerx, H. J. (2015). Effect of disinfectants on preventing the cross-contamination of pathogens in fresh produce washing water. Inl. J. Environ. Res. Public Health 12, 8658–8677. doi: 10.3390/ijerph120808658)
L82: define EO.
L83: which oxidants? PAA?
L92 and throughout (again): bacteriaL community.
L102: define ECAS
L98-105:
L108: grown in soil, hydroponically?
L109: define Ecas4 Australia
L110: this is a brand. double distilled? deionized? define.
L113a: 50 ppm of what? PPA? H2O2? Mixture?
L113b. define ORP
L116 - What country is Hanna from?
L117: "specific" not needed since you specify them in the same sentence.
L119-120: what is the difference between different ORPs, except the values?
L119: see my comment for L113a.
L122: space between 25 and g
L122: homogenized how? was the peptone sterile or not? Why did you homogenize them in peptone, and not e.g. TE buffer?
L127-132: Since the homogenization method is not mentioned, I am curious if you were able to get DNA from biofilms and all viable bacteria. This part is not very clear.
L133: PCR acronym explained above.
L133-134: you would not loose a lot of space if you pasted two primer sequences here or put them to supplemental.
L163: is V3-V4 enough information for identification to the species level?
L168: post hoc italic?
L197: phylum and family OR phyla and families. Just be consistent.
L202-3: please clarify- is this average or total?
L203: uncharacterized phyla - candidate phyla or bad sequences or unique? Candidate phyla are important as well.
L208: why did you exclude these sequences form further analyses? They contribute to the biodiveristy.
Table 1: please define the abundance, the sample etc. This table caption is not very informative.
L215: Table S1 not attached.
Figure 1: This figure is not informative enough (therefor not conclusive), since you present compiled data per treatment or per day post treatment. I would like to see how the bacterial communities change in every single type of disinfectant post treatment.
L233: Table S1 not attached.
251: 50 ppm or 52 +- 2?
Table 2: not intuitive, as the days do not follow logically. Please re-think the SE presentation. it is slightly confusing.
Figure 2: In such shape and form, it figure is not suitable for publication. Please make sure that everything is readable, that the fonts are consistent, that the colors are consistent, legend positions and forms are consistent. For provided information, this figure could be presented as 2 panels x 2 panels, occupying half of a page.
Table 3 caption: please define all acronyms and unify the number of decimal numbers.
Table 4: explain all acronyms
Figure 3: panels are not uniform. In panel A, you have ASVs on x-axis, in panel B, you have taxa. Unify. This could easily be one panel figure.
Author Response
We would like to thank you for your comments, which prompted us to review and improve the form of our manuscript. Kindly find the following responses to your remarks.
Major remarks:
1. The manuscript and supplemental materials are disconnected, and it seems like the authors did not submit correct supplemental files.
We are sorry for the problems encountered by the Reviewer; however, we are prone to consider that it was an isolated case since the other two reviewers raised no objections
2. The authors excluded taxa with lower abundance from the analyses. This makes me question the diversity indices and other results.
Looking at the core microbiota in other studies, phyla with an abundance percentage <0.1% were removed from the analyses; we decided to follow the same procedure.
3. There are too many figures and tables for the provided data. Most of them do not add any value to the manuscript.
We are sorry to disagree with the Reviewer. Figure 1 shows the relative abundance at phyla levels for treatment types and for day 0, 5 and 10 (sampling days) after storage respectively. Figure 2 shows the bacteria community differentiation for all treatment types and sampling days. Figure 3 presents the relative abundance ratios of assembled sequence variants and taxa level families that are different in different sampling days.
Table 1 present total phyla abundance and percentage abundances, and table 2 present the data on species richness and evenness for all the samples.
Table 3 and 4 present PERMANOVA and ANOVA test results. Table 5 presents Analysis of the composition of microbiomes test results and relative abundances that significantly contributed to the differentiation of microbiome composition for sampling days.
5. Relative abundances are important. However, it would be interesting to see absolute abundance - the effect of disinfectants is diminished - are they effective or not?
The method to establish an absolute abundance of microbiome composition so far is not available and moreover to quantify the absolute abundance of the microbiome may not be possible for vegetables that are produced at metric tonnes every day.
6. The figures are in different styles and with different colour/pattern schemes, which makes the manuscript even less consistent (other inconsistencies described below)
Figures have now been made consistent throughout the manuscript.
7. I am missing the analysis of controls (disinfectants alone, with no spinach). This is the only reason why the diversity would be higher in spinach treated in PAA than in tap water.
I don’t understand the comment: is the Reviewer suggesting that the sanitisers are useless and, in addition, may represent a source of microbial contamination?
8. The authors used a commercially available disinfectant, which is not in a pure form. At this point, we do not know if the diversity changed because of PAA, H2O2 or something else.
Peroxyacetic acid is synthesised upon treatment of acetic acid with hydrogen peroxide:
H2O2 + CH3COOH ⇌ CH3COOOH + H2O
As indicated by the double arrow, the reaction products are in equilibrium with the reagents, which means that all compounds are simultaneously present; in other words, it is not possible to isolate PAA in a pure form. It is a disinfectant commonly used in the processing of vegetables and, in our study, it was used as a comparison for the effectiveness of EO water.
9. I am not sure if the amount of data is sufficient for a serious publication, as it does not answer some relevant questions. For readers (and consumers) it would be more interesting to see if these disinfectants are efficient in reducing pathogen load if there are persisters present after disinfection if normal microbiota is able to "protect" the contaminants from disinfectants etc. I am aware that there was a lot of work invested in this manuscript. However, at this point, it seems like an analysis of controls, without experimental groups.
As commented by the Reviewer, the data presented in this manuscript were obtained in the frame of a more in-depth investigation, which studied the effectiveness of the various sanitizers in reducing the microbial load on spinach leaves. Since it was not possible to report all the data within a single paper, we presented the results on effectiveness in another paper (Ogunniyi et al., “A pH-neutral electrolyzed oxidizing water significantly reduces microbial contamination of fresh spinach leaves”. International Journal of Food Microbiology, under review.)
10. I have some serious concerns about DNA isolation. The authors used peptone water, and not a buffer with EDTA.
The DNA was extracted and purified as per the Qiagen QIAamp DNA Mini Kit (Cat. #51304).
Minor remarks:
L45-46: This statement is very vague. Are we talking about core microbiota? I would assume that microbiota is different in different environments
Response: This statement (now L44-45) is now revised to make it clearer as
“The bacterial communities associated with edible leafy vegetables are less diversified than those of farm soil and coastal seawater habitats.”
L52: what about the source of water, fertilizer, etc? was any research done on the source of these pathogens?
Response: Our interest has been focused on the influence of sanitizer washing on the microbiome profile of minimally processed spinach; the actual sources of the pathogens have not been taken into account.
L58 and throughout the manuscript: please be consistent with terminology. Microbiome and microbial communities/microbiota are not the same. The microbiome is just a collection of genetic material that is not necessarily from living organisms. So, the microbiome cannot be (strictly speaking) responsible for anything. But microbiota can be.
Response: Microbiome and microbial communities/microbiota terminologies are used as per the context of the sentence and following the published literature norms.
L59-60: quotation marks not needed.
Response: as suggested, quotation marks were removed
L61-62: only peptides? what about outer secondary metabolites?
Response: We agree that secondary metabolites are intrinsic plant factors that could influence the microbiota composition. We have included this information
L64: Bacterial population. was assessed? has been assessed? is generally assessed? unclear.
Response: the sentence was modified and changed to ‘is generally assessed’
L67: DGGE is the abbreviation. Since this is the only place in the manuscript where you use DGGE or T-RFLP, there is no need to abbreviate these methods.
Response: we removed abbreviations used only once from the text
L68: usually, people analyze the 16S rRNA gene, not the RNA itself.
Response: changed to 16S rRNA gene
L70 and the whole paragraph: Bacterial communities. Analyses.
Response: There is no problem using the term bacterial community in these contexts.
L77-8: this sentence can be easily contracted.
Response: We revised/shortened the sentence as follows: “For leafy vegetable processing, chlorine- or peroxyacetic acid (PAA)-based sanitizers are commonly used.’
L79: not true. It has low reactivity compared to chlorine (Banach, J. L., Sampers, I., Van Haute, S., and van der Fels-Klerx, H. J. (2015). Effect of disinfectants on preventing the cross-contamination of pathogens in fresh produce washing water. Intl. J. Environ. Res. Public Health 12, 8658–8677. doi: 10.3390/ijerph120808658)
Response: we changed the statement as follows “Chlorine is used for its effectiveness and low cost, whereas PAA for its activity over a wide pH range and limited reaction with the organic matter”
L82: define EO.
Response: this abbreviation is addressed throughout the manuscript.
L83: which oxidants? PAA?
Response: we changed the statement as follows “Izumi [28] reported that neutral EO water containing 50 mg/L of free available chlorine (FAC)…”
L92 and throughout (again): bacterial community.
Response: As responded in item L70 above
L102: define ECAS
Response: ECAS was removed and all references made to EO water (the two terms are interchangeably used in literature)
L108: grown in soil, hydroponically?
Response: grown in soil (the statement was modified)
L109: define Ecas4 Australia
Response: Ecas4 Australia has been changed to Ecas4 Australia Pty Ltd
L110: this is a brand. double-distilled? deionized? define.
Response: Milli-Q Academic A10, Millipore has been added in parenthesis. Milli-Q water is a deionised/demineralised water and passed through a filter to remove all life forms.
L113a: 50 ppm of what? PPA? H2O2? Mixture?
Response: PAA concentration was measured for the PAA-based sanitiser
L113b. define ORP
Response: all acronyms were defined
L116 - What country is Hanna from?
Response: Hanna Instruments has a network of over 40 subsidiaries in 32 countries around the world. Hanna Instruments is headquartered in Woonsocket, Rhode Island and has manufacturing sites in Mauritius, Singapore, Hungary and Romania.
L117: "specific" not needed since you specify them in the same sentence.
Response: the word specific here is used as an adjective
L119-120: what is the difference between different ORPs, except the values?
Response: the ORP value reflects the antimicrobial activity of the solution; the higher the ORP value, the more effective is the solution
L119: see my comment for L113a.
Response: PAA concentration was measured
L122: space between 25 and g
Response: done
L122: homogenized how? was the peptone sterile or not? Why did you homogenize them in peptone, and not e.g. TE buffer?
Response: Peptone water was sterile, and 0.1% peptone water is a minimal media used for suspension for microbial enumeration procedures.
L127-132: Since the homogenization method is not mentioned, I am curious if you were able to get DNA from biofilms and all viable bacteria. This part is not very clear.
Response: The sentence was modified as follows: “Samples (3 × 25 g) from each treatment were homogenized in 225 mL of sterile 0.1% peptone water for 60 s in a stomacher (BA 6021 Stomacher, Seward Ltd, UK) immediately after treatment (day 0) and stored at -20 °C.”
For DNA extraction and purification, DNA extraction kit from Qiagen QIAamp DNA Mini Kit (Cat. #51304) was used.
L133: PCR acronym explained above.
Response: PCR full form removed
L133-134: you would not lose a lot of space if you pasted two primer sequences here or put them to supplemental.
Response: Reverse and forward primer details are as follows:
Forward: 5'CGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse: 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC
is available at Klindworth A, Pruesse E, Schweer T, Peplles J, Quast C, et al. (2013).
L163: is V3-V4 enough information for identification to the species level?
Response: V3-V4 hypervariable region allows for reliable identification of bacterial at genera level and allows for more in-depth characterization of the microbial composition.
L168: post hoc italic?
Response: post hoc changed to italics
L197: phylum and family OR phyla and families. Just be consistent.
Response: Changed to phyla and families
L202-3: please clarify- is this average or total?
Response: It is the total number of ASVs (we added the information to the text)
L203: uncharacterized phyla - candidate phyla or bad sequences or unique? Candidate phyla are important as well.
Response: At the phyla level, unassigned phyla in a widely studied vegetable like spinach are almost always artefacts that do not exist in nature.
L208: why did you exclude these sequences from further analyses? They contribute to the biodiversity.
Response: Looking at the core microbiota in other studies, phyla with an abundance percentage <0.1% were removed from the analyses; we decided to follow the same procedure.
Table 1: please define the abundance, the sample etc. This table caption is not very informative.
Response: We added the percentage abundance of phyla
L215: Table S1 not attached.
Response: Sorry, attached
Figure 1: This figure is not informative enough (therefore not conclusive) since you present compiled data per treatment or per day post-treatment. I would like to see how bacterial communities change in every single type of disinfectant post-treatment.
Response: Diversity changes in the bacterial community after disinfectant treatments are described in detail in section 3.2, 3.3 and 3.4
L233: Table S1 not attached.
Response: Sorry, attached
251: 50 ppm or 52 +- 2?
Response: “50 ppm” was our target value; as discussed in section 2.1 (L119-120), a minimum of variability was found, which however did not alter the nominal value
Table 2: not intuitive, as the days do not follow logically. Please re-think the SE presentation. it is slightly confusing.
Response: SE has been removed from the Table, and the sequence of days corrected
Figure 2: In such shape and form, it figure is not suitable for publication. Please make sure that everything is readable, that the fonts are consistent, that the colours are consistent, legend positions and forms are consistent. For provided information, this figure could be presented as 2 panels x 2 panels, occupying half of a page.
Response: A new figure 3 is attached in the manuscript
Table 3 caption: please define all acronyms and unify the number of decimal numbers.
Response: Acronyms expanded, and decimal numbers made consistent.
Table 4: explain all acronyms
Response: Acronyms expanded
Figure 3: panels are not uniform. In panel A, you have ASVs on the x-axis, in panel B, you have taxa. Unify. This could easily be one panel figure.
Response: The analysis of differentially abundant taxa among the types of sanitization and days (0, 5 and 10) post-treatment, at ASV and family level were analysed and presented as ASVs and taxonomic level family.
Reviewer 2 Report
This manuscript aims to assess the microbiome of spinach leaves following washing with a number of different sanitisers. While there are quite a few published studies of the microbiome of spinach/other cut vegetables after these treatments and refrigeration, it would appear that this is the first to do a head-to-head comparison of the between the effects electrolysed oxidising water and PAA.
The study is carefully conducted and well written, however I have some minor comments.
- All abbreviations should be in full at first use eg ORP
- Please check the correct email address for author 3.
- If as stated " Ecas4 played no role in the study design, data collection and interpretation, writing and decision to submit the article for publication." The role of the author whose attribution is ECAS Pty Ltd needs to be clearly described and to be consistent with the criteria for inclusion as an author.
Author Response
We would like to thank you for reviewing our manuscript and kindly find the following responses to your remarks.
1. All abbreviations should be in full at first use eg ORP
Response: All done
2. Please check the correct email address for author 3.
Response: Done
3. If as stated "Ecas4 played no role in the study design, data collection and interpretation, writing and decision to submit the article for publication", the role of the author whose attribution is ECAS Pty Ltd needs to be clearly described and to be consistent with the criteria for inclusion as an author.
Response: Dr. Sergio Ferro reviewed on the manuscript drafts however had no role in data acquisition and in the decision to write the article
Reviewer 3 Report
1- LINE 204 AND 205, WHY IT HAS DECIDED ON WASH DAY 0 AND DAY 5? PLEASE JUSTIFY.
2-LINE 221, THERE IS ANOTHER WASH AFTER DAY 10, AND 4 DEGREE HAS BEEN MENTIONED. IS THAT THE STORAGE CONDITION BETWEEN DAY 5-10? WHY THAT TEMP HAS BEEN DECIDED? PLEASE JUSTIFY.
3-
Author Response
We would like to thank you for reviewing our manuscript and for your time. Kindly find the following responses.
- LINE 204 AND 205, WHY IT HAS DECIDED ON WASH DAY 0 AND DAY 5? PLEASE JUSTIFY.
Response: Spinach leaves, washed and stored at 4 ºC, usually exhibit up to 10 days of shelf life due to the growth of spoilage and psychotropic microorganisms above the acceptable level. Moreover, some sanitisers are known to adversely affect the shelf life of spinach, therefore day 0, day 5 and day 10 were chosen.
- LINE 221, THERE IS ANOTHER WASH AFTER DAY 10, AND 4 DEGREE HAS BEEN MENTIONED. IS THAT THE STORAGE CONDITION BETWEEN DAY 5-10? WHY THAT TEMP HAS BEEN DECIDED? PLEASE JUSTIFY.
Response: The treatment of samples is described in section 2.1 (Lines 118-125): the spinach leaves were washed on day 0, with either tap water or a sanitizing solution; no further washing was done after day 10.
4 ºC is the temperature at which retails shops store ready-to-eat spinach leaves
Round 2
Reviewer 1 Report
The authors addressed most of my comments satisfactory, however, not all of them. There are still few major concerns that need to be addressed before I can make a final decision.
1.Supplemental material: this issue has not been fully resolved.
Authors: "We are sorry for the problems encountered by the Reviewer; however, we are prone to consider that it was an isolated case since the other two reviewers raised no objections"
Reviewer: Even if it was an isolated event, I would recommend the authors to double check the submitted files. The odds of the reviewers not checking supplemental files are higher than only one reviewer receiving incorrect files. For the second review, I received 3 files: the corrected manuscript, two supplemental tables in a .txt format, and Unpublished material entitled "A pH-neutral electrolyzed oxidizing water significantly reduces microbial contamination of fresh spinach leaves", which looks like another manuscript that has not been quoted in the submitted manuscript. And in my previous review, when I referred to disconencted files, I was referring to this manuscript that is currently in review (if I understood your comments correctly).
Files I have not received:
Figure S1: Visualization of Shannon and Inverse-Simpson diversity (alpha-diversity) and Chao and ACE richness metrics of all samples.
Appendix 1 (mentioned in L189-190): "The fully reproducible code for statistical analysis is available as a supplementary document."
2. Authors: "Looking at the core microbiota in other studies, phyla with an abundance percentage <0.1% were removed from the analyses; we decided to follow the same procedure."
Reviewer: Stronger justification requested. "Other people did it as well" without additional explanation/justification and references for data discrimination is not a reason strong enough.
3. Authors:" I don’t understand the comment: is the Reviewer suggesting that the sanitisers are useless and, in addition, may represent a source of microbial contamination?"
Reviewer: I am concerned about your DNA isolation method. You used peptone water, which is not DNA free, instead of any buffer that would "protect" the DNA integrity, e.g. TE buffer. At this point, the DNA content in pure peptone water is not known. We don't know how peptone was prepared by the manufacturer. Are there any remains of the bacterial DNA? that's why it would be necessary to include some sort of negative control to make sure that your methods provide you with reliable data.
4. Authors: "The DNA was extracted and purified as per the Qiagen QIAamp DNA Mini Kit (Cat. #51304)."
Reviewer: I checked the manual, and the manufacturer does not recommend homogenization in peptone water.
5. Authors: "Diversity changes in the bacterial community after disinfectant treatments are described in detail in section 3.2, 3.3 and 3.4"
Reviewer: I would recommend the authors to provide visual data. Table 2 provides diversity indices for all samples. However, relative abundance for every day and every treatment as a figure would be greatly appreciated.
Minor remarks
L62 and throughout: please use "bacterial population" (bacterial, as an adjective, and not "bacteria population")
L82-3: since this is the first time you mention these bacteria, please spell out the genus name.
L89: reduce population size? bacterial size? reduce what? clarification needed.
L106-7 and throughout: 4 ± 1 °C (please use non-breaking space between value and unit, so that °C does not jump to the next row)
Table 2: In better form, however, please make sure that the font size and format are ok. In this version of the manuscript, certain fields are shifted, words broken in the middle etc. Make sure it is in a publishable form.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Round 3
Reviewer 1 Report
The authors addressed all of my concerns. I endorse this manuscript for publication
