PONE-D-22-31146The impact of primary colonizers on the community composition of river biofilmPLOS ONE

Dear Dr. Marsh,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 10 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Robert P Smith

Academic Editor

PLOS ONE

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When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

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2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

   "Funding sources included the Michigan Department of Natural Resources State Wildlife Grants Program T-10-T-5 Study 237026 (https://www.michigan.gov/dnr/buy-and-apply/grants/aq-wl/wildlife562 hab), Center for Water Sciences Water Cube initiative at MSU (https://water.msu.edu/watercube/), and the Department of Microbiology and Molecular Genetics at MSU. In addition, we thank the Department of Microbiology and the College of Natural Science for partial funding including a Thesis Completion grant. We thank Dr. M. Bagdasarian (MSU) for providing Pseudomonas aeruginosa PA01."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

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Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Additional Editor Comments:

Good afternoon Dr. Marsh,

First, I would like to apologize for the length of time that it took to review your manuscript. As we discussed, it took quite a long time to find qualified reviewers. Nevertheless, I now have received two reviews.

Both reviewers appear to have found the manuscript to be technically sound. However, they have some suggestions about justification about experimental approaches, writing and the figures/figures legends that are worth addressing. I look forward to receiving your revised manuscript. If you have any questions, please do not hesitate to contact me.

Rob

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General

The authors investigated how the establishment of biofilms from river microbial communities were affected by pre-established biofilms by isolates from sturgeon eggs and vice versa. They conclude that certain taxa could act as probiotics if they are precolonized on sturgeon eggs and then exposed to possibly harmful taxa in the river communities. In general, I think this is a straightforward and interesting study, however there are several improvements that could be made to the writing and presentation of these data, and important methodology is missing.

The justification for using specific isolates should be highlighted more clearly. I do not see any informative on cell counts or growth phase at which these isolates were inoculated, and I’m not sure the absorbance values presented would allow for successful replication of these results. I There are other pieces of information missing from the methods section that I’ve highlighted below that need to be included.

The English is fine, but the scientific writing, especially in the results, could be improved.

The results could be tightened by removing statements that are not informative to the results. For example, Lines 265-266 don’t seem to add much and may be more appropriate for a legend, and Lines 284-288 are redundant of intro and methods. There is much more than can be cut.

The data presented in the results section and the figures/tables are not always clear. For example Table 1 and Figure 1 panel B and others like it. The legends need to be more way informative so the reader can understand them as well, but in general I think the presentation of data in some tables and figures should be re-considered (Table 1, Figure 1c, Figure 4 [way too small, no description of colors?], Figure 6, Figure 7 [too small of text]). In addition I think the results needs to be described more clearly with more informative information presented in the figures. I could not understand the results without spending time trying to figure out what the figures were presenting, and even then, the legends were missing necessary information.

I think the conclusion could be more strongly supported if there was a known egg pathogen to challenge the biofilm rather than inferring certain pathogens from the microbial community composition of the river. Certainly not all Aeromonas sp. are pathogenic to the eggs? However, I see the value in investigating as a microbial community as well because it is more representative of nature conditions.

Specific

Line 55 awkward sentence

Line 82: bd is not defined before this, I assume it is B. dendrobatidis

Line 83: suggestion: remove “in our labs”

Line 86: suggestion: alter language to avoid first person

Line 93: I think “with” is missing

Line 98. Starting with “our experimental strategy…” – consider removing this and everything after or shorten it.

The details of how isolates were identified need to be added. For example, you just say that partial 16S sequencing was used to identify. In addition, the justification for using these strains is lacking. For example, line124-125 states that these were representative of previous characterizations – is this something you can cite? How abundant are these taxa on the eggs? Are egg communities diverse or do they only harbor a few species? Following that at line 126, do you mean that these were of interest related to biofilms and resilience on eggs or just in general?

Llines 153 and on.. Can you provide more quantitative information about the overnight cultures inoculated? Cell count? Absorbance? Were they added at the same concentration?

Line 160 – avoid starting a sentence with a number.

Line 204 is awkward. Consider changing to “…500 cycle v2 reagent kit to generated 250 bp paired end reads.

Line 205 needs work to clarify.

Line 212-214 is awkward the way it is written. For example, the analysis of diversity of bacterial communities is redundant to all the following analyses listed.

Line 212: permanova

Can you provide the details of your mothur analysis? Trimming, filtering, chimera checks?

Isn’t is “t-test?”

Lines 228: “were” instead of was

The first paragraph of the results section doesn’t seem appropriate for the results, but perhaps intro or beginning of discussion.

Line 238: Is there data somewhere to show this or reference?

Is the sentence starting at line 239 needed? Also seems like the next two sentences are redundant of the methods.

Line 245: I don’t think stress is in an indicator of anything being different but rather an indicator of how well the data is represented in that 2 or 3D model.

Panel B in Figure 1 is not easy to interpret. The shaded boxes and non-shaded boxes need to be explained in the legend. According to the way it is now, one could interpret that T4, T8 etc are R values, because it is not clear here these are timepoints.

In figure 1 panel C what does Taxa S mean? Describe these things in the legend.

Line 267, what are 164 and 719? Rank abundance? You mention relative abundance right before this so this is a bit confusing.

Table one is not clear as is. The lettering is confusing and not intuitive.

Line 320 “described”

Lines 297 : an absorbance of 3.7 means nothing without any comparisons, which do not follow. What were the absorbances of the “low amounts” of biofilm? What is considered “low” and why?

The discussion could be streamlined and made me succinct.

Reviewer #2: Comments from the Reviewer of the Angoshtari et al. manuscript ‘The impact of primary colonizers on the community composition of river biofilm’ (PLoS ONE)

Angoshtari et al. present their research focussed on understanding how the first bacterial colonisers impact the subsequent recruitment and growth of other bacteria present in river water with the aim of better understanding how pathogens might be prevented from colonising sturgeon eggs and causing disease.

This is a very well-written manuscript and I have no serious concerns with it, other than to say it might be edited for length in paces, and that the print-quality of the figures was poor.

Comments

1. P. aeruginosa PA01 is not necessarily thought of as a model for river bacteria, and it should be made clear when it is first referred to (after the Methods) that P. aeruginosa strains are regularly recovered from fresh water systems.

2. Pg. 5, line 114. The species of sturgeon should be provided.

3. Pg. 6, line 127 and elsewhere. Whenever percentages are given, w/w, w/v or v/v should be stipulated in parentheses.

4. Pg. 8, line 175. It should be stated clearly that the type of biofilm being examined and measured in this work is a submerged solid surface – liquid (A-L) interface biofilm. Other types of biofilm are equally well-studies, including air – liquid surface interface biofilms that readily form in static microtitre dish assays.

5. Pg. 9, lines 201 & 203. 5’ and 3’ should be added to the primer sequences.

6. Pg. 10, line 234. Can ‘amounts’ be contextualised – were the biofilms thicker or more dense, or did they cover more of the microtitre well walls?

7. Pg. 10, line 238. Please indicate what type of biofilm was observed in these experiments. Is it possible that some wells produced A-L interface biofilms which were not registered by the crystal violet assay because the material was washed away?

8. Table 1. Could a contingency table / Chi Square analysis be conducted to see whether species abundance is dependent on abundance in river water and in biofilms?

9. Figure 2 does not specify what type of data are shown in the graph (means ± SE?).

10. Figure 3 A & B do not show means and errors.

11. Pg. 19, line 462. This statement is disingenuous especially after hydrophobicity is noted on the following page. Do the surfaces also differ in terms of microtopography which might aid recruitment from flowing water / protection from shear force, and flexibility which may also play a role in biofilm-formation and retention?

12. Pg. 20, line 488. Can any comment be made of whether, in the microtitre wells used for the biofilm assays, there was any evidence of A-L biofilm formation, incomplete surface colonisation or possible ripping of biofilms from the surface (which may all result from aging biofilms and/or over vigorous washing and staining)?

13. Pg. 21, line 511. Some references should be provided to contextualise biofilm formation by P. aeruginosa strains isolated from fresh water and/or tested in fresh water-based medium.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Andrew J. Spiers

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PONE-D-22-31146R1The impact of primary colonizers on the community composition of river biofilmPLOS ONE

Dear Dr. Marsh,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 17 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Robert P Smith

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Thank you for submitting your revised manuscript to PLoS One. I have now heard back from both previous reviewers. From their comments, it appears that you addressed the majority of their concerns from the first round of review. However, they recommended some stylistics edits and some minor edits that should help clarify a few areas of the manuscript. Both recommended a minor revision and I agree with both of the reviewers. I look forward to receiving your revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has improved, and I had a couple of follow ups that I think need to be addressed.

Table 1:

I was referring to the lettering scheme with panels “A and B” and then the lowercase lettering scheme a, b, c, d, e being confusing and not intuitive. I still think it is. And the legend is not appropriate. There is no acknowledgement of panels A and B and a, d, and e are just results.

I don’t think it’s necessary to have it with A and B and then a. b. c. d. and e. My suggestion is to just refer to A and B only in the legend. Since everything else is labeled already describing what it is, then just include the other information in the legend without the reference to any letter. Basically, eliminate the a,b,c,d,e

“Population” is also not the current term in the table or in the legend. These are OTUs representing unique taxa… But then you use phylotypes in some of the text but populations elsewhere. Is phylotype or OTU more appropriate here? Were you screening sequences again the database to generate phylotypes or clustering sequences for OTUs? This should be clear in the methods but it wasn't to me. Table 1 says OTUs. This is an inconsistency between population, OTU, and phylotypes is throughout the manuscript.

RE: response from authors in R1:

"The cell concentration of the inoculum is irrelevant to the amount of biofilm formed in 48 hours over a wide range of initial cell concentrations. We have determined this in studies with 500-600 freshwater isolates, including those that we used in this study. "

…

"This issue is addressed above. Briefly, what is important here is the biofilm formed after 48 hours. Healthy 16-hour cultures were used as the inoculum. Each strain has its own unique

growth rate and ability to form biofilm. The biomass of biofilm produced by each strain was

strain-dependent. Under our experimental conditions the biofilm amounts as measured by crystal violet were consistent for each strain at 48 hours, over a broad range of inoculum concentration. "

Okay, but I don’t see in the text or the snippet you added these specific comments that there was consistent biofilm with a range of inoculums. Is the evidence that inoculum size of differing ranges yields the same amount of biofilm at 48 hours something you can cite from previous work or is there data? Or personal comm?

In general I find there is a lot figure and now table explanation in the text rather than just describing the text. Addressing this could make the results more concise.

Line 419 refers to supplemental material – is this suppose to be to a specific figure?

Number of references at line 579/580 seem a bit excessive.

Reviewer #2: Comments from the Reviewer of the Angoshtari et al. revised manuscript ‘The impact of primary colonizers on the community composition of river biofilm’ (PLoS ONE)

Angoshtari et al. present a revised manuscript which deals well with the comments I had made earlier. There are some very minor comments that should be addressed:

1. Line 50. It is arguable that all extant aquatic metazoans were present for millions of years; perhaps this dramatic statement can be rewritten as ‘Aquatic metazoans …’.

2. Lines 56-57. This sentence seems repetitive and could be deleted.

3. Line 101. Correct ‘I’.

4. Pseudomonas aeruginosa PA01 was isolated from an Australian patient in 1954 and it is exceedingly unlikely that the exact same strain has been isolated since then – and certainly not from the Red Cedar River. The sentence in Lines 111 – 114 needs to be altered to say that ‘other strains of this species’ are routinely isolated from fresh water sources (the rest of the manuscript needs to be checked and corrected if necessary).

5. Pseudomonas aeruginosa PA01 is referred to at various points in the manuscript but there is inconsistency in naming – a consistent approach should be adopted, e.g., Pseudomonas aeruginosa PA01 and thereafter P. aeruginosa PA01.

6. P. aeruginosa PA01 is not necessarily thought of as a model for river bacteria, and it should be made clear when it is first referred to (after the Methods) that P. aeruginosa strains are regularly recovered from freshwater systems.

7. While a particular crystal violet assay was undertaken in this work, there are various forms of the assay, including washing steps and eluent. Perhaps referring to it as ‘the standard’ (Line 203 and possibly elsewhere) is inappropriate and it should be referred to as simply ‘the crystal violet assay’.

8. Figure legends and possibly elsewhere. The term ‘average’ should be avoided as there are three possibilities; please replace with ‘mean’ throughout.

9. Line 340. The end of the sentence ‘in the biofilm that traps crystal violet’ is not needed and arguably, is incorrect: crystal violet binds to the bacterial peptidoglycan layer (and probably more generally to proteins as it stains fingers too).

10. Line 478. It is not necessary to specify ‘Earth’s’ microbiome unless there are substantive reports that microbial biomes in orbit don’t form biofilms.

11. Line 518. Delete ‘+’ as 2000 is the critical value discussed here.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Andrew J Spiers

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

The impact of primary colonizers on the community composition of river biofilm

PONE-D-22-31146R2

Dear Dr. Marsh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Robert P Smith

Academic Editor

PLOS ONE

Additional Editor Comments: Thank you for addressing all of the minor comments raised by both reviewers.