PONE-D-20-29463

EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

PLOS ONE

Dear Dr. Flores Clavo,

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.

The comments by both referees are included at the end of this letter and I would kindly suggest you follow them should you decide to resubmit your MS to PLOS ONE. Both referees concluded -separately- that your MS falls under the "Major revision" decision and I also agree with them completely. Despite the particular comments by each reviewer I would kindly suggest that a major revision of the written English is performed since this will also increase the impact of the MS.   ​

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We look forward to receiving your revised manuscript.

Kind regards,

Luis Angel Maldonado Manjarrez, Ph.D.

Academic Editor

PLOS ONE

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"At the research agency of Peru (Concytec-Fondecyt) financially supported this work within the framework of the 041-01 call with the 190-2018 contract; as well as the National Council for Scientific and Technological Development of Brazil (CNPq), it was the authors are grateful to the cooperation of the postgraduate programs in Genetics and Molecular Biology and Biosciences of the Biology Institute of the State University of Campinas. Also, Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - (Scholarship-code 001)."

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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: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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: No

Reviewer #2: No

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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: No

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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: This manuscript describes the isolation of actinobacterial strains from a saline lagoon in Northwest Peru. The authors identified a selected group of actinobacteria by 16S rRNA gene sequencing and subjected the isolates to tests in order to determine whether they produce any bio-active compounds with activity against bacteria and various cancer cell lines. The extract from one of the most promising strains was evaluated via molecular networking, allowing for the prediction of compounds produced by this strain.

It is clear that the manuscript represents a large body of work, which also resulted in a rather lengthy manuscript. The authors would need to look at ways in which the manuscript can be shortened so that it is more streamlined. In addition, the authors also need to make sure that the way of citing references in the main text follow the journal’s requirements.

Here are some additional comments/suggestions to the authors:

1. The manuscript will require language editing before it can be considered for publication.

2. Figure 1: the caption for image ‘d’ is not included in the figure legend.

3. Figure 2 can probably be presented as supplementary material.

4. Page 8, section 2.2.: The authors described a very specific isolation protocol. This obviously would limit the amount and type of actinobacteria that would be isolated. What was the rationale behind using this method and were other isolation methods explored, but yielded no isolates of interest? In addition, many researchers often base their isolation protocol on the observed physicochemical characteristics of the environment the sample was collected from. Were any physicochemical analyses performed (e.g. sediment pH, level of salinity, presence of K, P, N, C, etc.)?

In addition, in this section and throughout the manuscript, when indicating a % solution, please indicate whether it is v/v or w/v, e.g. isolates were preserved in 20% (v/v) glycerol…

5. Page 9, section 2.3.1: What was the final pH of the R2A-ASW broth used?

6. Page 9-10, section 2.32: It is mentioned that modifications were made to the DNA extraction method reported by Pospiech and Neuman (1995), but no details are provided. Please expand on the modifications effected.

Line 161: The PCR protocol also calls for a large amount of DNA to be added. Is there any particular reason for that?

Line 166: What % agarose gel was used for the analysis of the amplicons?

Lines 171-172: Which program was used to assemble to DNA sequences?

7. Page 11, line 186: What was the rationale behind the use of R2A broth for the production of bio-active compounds? Actinobacteria can produce numerous different compounds, a process that is greatly affected by the type of growth media used. Were different types of media tested, or did you solely focus on this medium?

8. Page 11, lines 196-197: Here mention is made that the crude extracts were also analysed by LC-MS. Is this the same analyses reported later on in the methodology section (in which case the authors need to refer to the section) or was this a separate analysis (in which case more details are required)?

9. Page 12, line 208: The wording of this sentence does not make sense – the ‘three crude bacterial extracts’ can’t be ‘tested as antimicrobial producers’. Best to say that they were tested for antimicrobial activity.

10. Page 12, lines 211-212: What is meant by ‘pre-coated microbial cultures’?

11. Page 12, lines 217-218: Define the abbreviations ‘MDRs’ and ‘UCI’ – these have not been defined previously.

12. Throughout the manuscript, make sure that ‘sp.’ is not written in italics, e.g. page 13, line 244 – the ‘sp.’ should not be in italics.

13. Page 13, lines 244-245: It is indicated that the B-81 extract was diluted 1 in 10 in methanol, but there is no indication as to the final concentration of the extract. Please indicate, e.g. 1mg/ml, 0.1mg/ml, etc.

14. Page 15, section 3.1: The use of phenotypic characteristics to group actinobacteria is notoriously unreliable. Genera within a family may have very similar morphological features and you stand a chance of ‘missing out’ potentially unique/’rare’ actinobacteria, e.g. members of Kitasatospora and Streptomyces share phenotypic features, similarly members of Pseudonocardia and Nonomuraea. Why not identify all 166 strains?

15. Page 17: The text provided in this section is basically a repeat of what is presented in Table 3. Only highlight the most interesting rather than repeating what is already presented.

16. Page 18, line 317 and 324: ‘Streptomyces’ should be written in italics.

17. Page 18, lines 321-325: Here the authors highlight the types of actinobacteria isolated from the different environments. Care should be taken here especially since the authors only made use of a single isolation approach, which will result in isolation bias. They therefore can’t compare what they detected during their isolations to that of others – they can simply report it and mention that these were isolated under the experimental conditions applied.

18. Figure 3: A phylogenetic tree containing only the three selected strains (and related streptomycetes) is presented in this figure. It seems a bit out of place since the importance of these three strains only becomes clear once the results for antimicrobial and anticancer tests have been reported. This makes it seem out of order. I would recommend amending the tree to include all the streptomycete strains identified in the study and once the bio-activity has been reported, reference can be made to this figure. This also means that Table 3 can be shifted to supplementary material.

19. Page 20, line 364: No evidence has been provided to support the classification of the isolates as ‘extremophilic’.

20. Page 22, line 395: Note the spelling of ‘doxorubicin’ – please correct.

21. Page 22, line 398: It is mentioned that the extracts from ‘three different media’ were selected. This is not reported in the methodology section – please amend.

22. Page 30, line 544: Note the spelling of ‘cholic’ – please correct.

23. Page 18, line 333: incomplete surname provided of the reference Cortés-Albayay.

24. Page 36, reference 40: Not cited in the main text.

25. Page 37, reference 42: Not cited in main text or incorrect spelling of lead author.

26. Page 37, reference 48: Not cited in main text or incorrect spelling of l

Reviewer #2: The article “EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU” describes the isolation and antimicrobial and anticancer capacity of strains isolated from saline environments. Although some of the results are of interest the quality of the manuscript is not enough, and a careful revision of data presentation and English should be taken into account before publication.

It is not clear at any point, why the three strains of Streptomyces have been selected for further analysis, it is said that they have better inhibition results, but it has not been shown any experiment with the other strains.

The authors should clarify that the sampling was in agreement with Nagoya protocol (check comment from line 110).

Lines 30-31: Reconsider this sentence, it has no many sense as it is.

Line 35: add “and /or”

Line 36: add “these bacteria” before “were identified”

Lines 36-38: I imagine these numbers refer to abundance of strains but it is not clear here.

Line 38: Actinobacteria should be in italics

Line 60: Here should be a final dot instead of semicolon.

Line 69: Italics

Line 77: give a reference.

Line 110: The Nagoya protocol was signed by Peru in 2014 so, at least the sampling of 2015 should be authorised by the goverment.

Line 112: it should be b, c and d, isn't it?

Table 1: before was indicated that sampling was in July, clarified it. Indeed if it was in december the Nagoya protocol should be applied.

Figure 2: correct “bacterial” and “extremophilic bacteria”. But indeed this figure can be omitted.

Line 126: it should be actinomycetes, not actinomyces, but indeed better if you use "Actinobacteria"

Line 144: this amount means 2.43% not 5%, verified your data.

Lines 276-279: These characteristics do not allow the classification into genus, you can group them into groups with similar characteristics, but not genus. This classification could be obtained after 16S rRNA gene analysis.

Line 286: based on what? which strains were included in those groups? this should be further explained.

Line 288: What are the other 116 strains isolated?

Page 292: delete strain, and put "isolates" and “samples” in plural

Line 303: correct to “were similar” or “presented higher similarities with”

Lines 300-317: it has no sense to give all the data here again if you give them in table 3, keep just the text or the table but not both. I recommend to keep the table.

Line 317: Streptomyces should be in italics; delete “of the”

Line 318: change “to” by “from”

Line 323: add a space before “has”

Lines 324 and 388: Streptomyces should be in italics

Line 328: correct the reference

Figure 3: I do not understand why the authors has selected only three of their strains to construct the phylogenetic tree, a good analysis of their strains should include all the ones for which the 16s rRNA gene has been obtained, independently of their ability to produce secondary metabolites. The 16S rRNA gene sequences should be deposited in public databases, and their accession numbers included in the tree.

Line 345: genus names should be in italics.

Line 353-356: this is not true, that is only true for the strain M-92, but not for the other two strains represented in the tree.

Line 379: check the reference

Line 401: delete “the”

Lines 405-407: these values are higher than 50, Shouldn’t they be considered as inactive according to line 394 reference? This should be further discussed by the authors.

Line 474: give details about the molecules marked as 1, 2 ,3, 4, and 5 in the figure.

Line 541: it should be a comma after the first “study”. Indeed one “study” should be replaced.

Line 542: correct “actinobacteria”

Line 550: delete the final dot after “potential”

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-29463R1

EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

PLOS ONE

Dear Dr. Flores Clavo,

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.

As you can notice from the comments of the two reviewers, one has suggested major and one has suggested minor revisions to your manuscript. However, I have decided to go for the "minor revisions" decision based on the first revision of your manuscript. Despite the latter, I would kindly appreciate if you would check on those comments of both reviewers and address all of their points accordingly as indicated in the previous paragraph. ​

Please submit your revised manuscript by Jul 11 2021 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: http://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,

Luis Angel Maldonado Manjarrez, Ph.D.

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.

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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 #3: (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 #3: Yes

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

Reviewer #1: Yes

Reviewer #3: N/A

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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 #3: 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 #3: 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: It is clear from the revised manuscript that the authors applied great effort to address the comments/recommendations/queries of the reviewers. However, with the editing and additions there are still some additional comments/recommendations for the revised manuscript:

1. Page 2-3, lines 54-55: ‘Three Streptomyces sp. strains’ sound cumbersome – consider changing the wording to ‘Three streptomycetes, strains MW562814,…’

2. Page 3, lines 59-60: To streamline the sentence, consider writing ‘… and the multi-drug resistant Acinetobacter baumannii AC-972.’

3. Page 3, line 61: ‘…in the R2A medium was determined using a…’

4. Page 4, lines 79-81: Consider merging the two sentences to provide more flow to the information provided, e.g., ‘…were reported from saline soils of the ephemeral salty lakes in Buryatiya [4], while Streptomyces, Nocardiopsis, and…’

5. Page 4, lines 81-83. The sentence on biological activities seems out of place – I would recommend removing it.

6. Page 4, line 89: ‘…to the production of enzymes, which can…’

7. Page 8, lines 147, 156, 159-160: It is conventional to refer to 16S rRNA (as per other sections in the manuscript).

8. Page 8, line 158: What is meant by ‘represented’? Did you mean that the sequences were submitted to the EZBioCloud 16S database ‘Identify’ service?

9. Page 9, line 162: ‘Mega’ should be written as ‘MEGA’

10. Throughout the manuscript: For any % solutions, please indicate whether they are v/v or w/v solutions.

11. Page 10, line 187: What is meant by ‘adjusted to a total volume of 200�l’? And is the ‘plate’ referred to a 96-well plate?

12. Page 13, lines 251-252: ‘…were used to group isolates into 42 filamentous and 8 non-filamentous bacterial groups’. Can’t use the information to ‘classify’, rather ‘group’, and since all the isolates were bacteria, best to keep it generic at this stage and use an inclusive term such as ‘bacteria’.

13. Table 2: The Family names should also be written in italics.

14. Page 14, line 283: Here is it indicated that MEGA7 was used, whereas in the methodology section it is indicated that MEGA6 was used – please correct.

15. Page 15, line 298: Remove ‘to’

16. Page 15, line 303: Replace ‘Actinobacteria’ with ‘actinobacterium’

17. Page 16, line 310: Replace ‘like’ (colloquial term) with ‘such as’ (more scientific).

18. Page 16, line 312: Did you mean to say that a member of the genus Pseudonocardia was also recovered during this study? In which case, the wording needs to change to reflect this: ‘…and plants, was also recovered in this study.’

19. Page 16, line 316 and throughout the manuscript: If there is no reference made to specific strains of a genus, then it is best to write out spp. as species. For example, here, it is best to write ‘…Streptomyces species as the most…’

20. Table 3: Replace ‘Isolates of Streptomyces sp.’ with ‘Streptomyces isolates’

21. Figure 3: It is recommended that the figure should be removed. The addition of all streptomycete isolates to Figure 2 negated the need for this figure.

22. Page 22: Be consistent in how the names of bioactive compounds are presented, e.g., shellmicin is written in both lower case and upper case in this section (see lines 430 and 436 vs 434 and 436).

23. Figure 4: How does the inset information (Streptomyces B-81 and Control) relate to the figure presented?

24. Page 25, line 463: What is meant by ‘rolling in the GNPS platform database’?

25. Page 25: Please see comment above about writing the names of bioactive compounds in lower case/upper case. Choose one and be consistent in its use.

26. Page 26, line 484: ‘gram-negative’ needs to be replaced with ‘Gram-negative’. ‘Gram’ refers to the surname of the scientist who designed the staining technique and should therefore always be written with a capital letter.

27. Page 28, line 519: Remove the additional full stop after ‘sp.’

28. Page 28, line 527: Remove ‘one’.

29. Even though it was attempted to highlight additional language editing required, it is recommended that the revised manuscript should be evaluated once again before resubmission.

Reviewer #3: The manuscript by Flores and colleagues deals with the isolation and application of actinobacteria recovered from lagoons in Peru. No doubt the MS is interesting but I feel some sections should be improved.

1. It does not seem to be fully details on the culture conditions, time, and morphology of the colonies as these were recovered from the isolation plates and subcultured for further analyses. I think this is important should other authors who read the MS may also be encouraged to perform or follow the protocols indicated on their MS. If this information is missing, then I do not see how other authors may follow or identify their cultures.

2. After sequencing the 16S of the isolates, the authors indicate a size of 1,206 nt but the 16S rDNA gene is nearly 1,500 nt. Is there any particular reason why the gene was only partially sequenced and not completed? I am not saying that the full gene should be sequenced to accept the ms but an explanation within the MS would be useful on why 300nt were missing (technical problems? primers used? sequencing reactions?).

3. Figure 3 indicates: Type strains within the genus Streptomyces and Pseudonocardia but the figure is not related nor include any Pseudonocardia, only Streptomyces!

4. In Figure 2 Streptomyces strain MW562807 falls in a subclade together with many other isolates. However, in that same tree MW562807 is also related to Streptomyces olivaceus and S. pactum. Have the authors checked if those two streptomycetes also show the same or similar biological activities to their isolate? Also since MW562807 is the most interesting strain, why the authors did not include on their study all related isolates to that strain acording to Figure 2? It would definitively be interesting to know if all the isolates within that subclade in Figure 2 do also show similar activities. Again, this is not meant to hold the MS any longer but a few lines within the conclusions or future work could be mention.

5. Conclusion. It is clear that the authors have a good set of interesting strains which have been analysed in the MS. There is the MW562805 and related isolates but, in my view, MS562814 also deserves more comments. That isolate is not related to any type strain and it seems to be a putative novel species as it is not related to any other streptomycete in their phylogenetic trees. If the authors are to continue with this kind of studies, which one they think deserve more attention? The MSW562805 subclade or the MS562814?

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Reviewer #1: No

Reviewer #3: No

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EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

PONE-D-20-29463R2

Dear Dr. Flores Clavo,

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.

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Despite the "acceptance" of your Manuscript, there are a few extra comments that should be addressed together with the technical requirements. A list of such minor comments which are merely mine follows:

1. The abstract should be shortened 3-5 lines. The chemical details can be easily avoided in the abstract.

2. Line 158, it says "on closest hits" and should be "on the closest hits"

3. Lines 277, 278, 281, 282, 284 285 and 286. All of these lines use the word "Streptomyces" but this paragraph is mainly devoted to Streptomyces hence it is redundant to re-write Streptomyces for each species and the corresponding comparisons. I suggest you simply use "S." which it is understood, due to the context of the paragraph, referring to Streptomyces.

4. Line 292. It currently says "has recovered" but I think it should be "was recovered".

5. Line 354. It says "Kitasospora" but it should be "Kitasatospora". Despite this typographical mistake I'm not quite sure if this outgroup would be the best because Kitasatospora belongs to the same family as Streptomyces. Perhaps a total different actinobacteria such as Micromonospora which is not related neither to Pseudonocardia nor Streptomyces would be better.

I strongly believe that these minor corrections should contribute to the improvement of the manuscript hence I kindly ask you to follow them together with the technical requirements of the editing process.  

Kind regards,

Luis Angel Maldonado Manjarrez, Ph.D.

Academic Editor

PLOS ONE