PONE-D-20-08668

Spectrum of antibiotic resistant bacteria and fungi isolated from chronically infected wounds in a rural district hospital in Ghana

PLOS ONE

Dear Dr dekker,

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.

I have received the reviews of your manuscript. While your paper addresses an important question, the reviewers stated several concerns about your study and did not recommend publication in its present form.  All reviewers voice a number of concerns regarding the rational of why Ghana rural localization was chosen for such observation and how data analysis were done.  These comments need to be addressed carefully. In addition there were numerous issues identified where additional experimentation and documentation is needed.  Please see reviewers’ insightful comments below. Personally, at a more detailed level, I find the manuscript could benefit from strengthening the rationale of the study (see specific comments below).

Specific comments:

1. It is customary to abbreviate the name of the genus after the first time it is used, for examples, Staphylococcus aureus should be S. aureus after first mention in the paper (Ref:  International Code of Nomenclature of Bacteria: Bacteriological Code, 1990 Revision).
2. It may be valuable to have brief description of the different stages of wounds to give readers a general idea since this was mentioned in the results.
3. Line 59 – 61:  Move this sentence down as third paragraph and expand to explain what other fungal pathogens could cause problem.
4. Line 72 – 73:  The rationale of the study needs to be strengthen further here.
5. Line 87 – 90:  Need approval number.
6. Line 229 – 234:  This begs the question on how to access whether there are biofilm formation on the open wound.
7. Line 253 – 255:  This sentence is awkward, please rephrase for clarity.
8. Line 335 – 340:  Reference 9&10 need space in between for format consistency.

We would appreciate receiving your revised manuscript by Jun 19 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information

[Note: HTML markup is below. Please do not edit.]

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

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: Title: Spectrum of antibiotic resistant bacteria and fungi isolated from chronically infected

wounds in a rural district hospital in Ghana

The manuscript entitled “Spectrum of antibiotic resistant bacteria and fungi isolated from chronically infected wounds in a rural district hospital in Ghana “ described a pathogen distribution in chronic wounds in rural Ghana. It seems that information matched to the international patterns with a predominance of Pseudomonas aeruginosa and Staphylococcus aureus. Moreover, very high resistance rates were also observed suggestion the use or not of certain antimicrobials. Data here reported are important but major revisions are necessary before manuscript acceptance. Furthermore, some suggestions were here providing in order to improve the manuscript quality.

Suggestions:

1. Its is not clear why Ghana rural localization was chosen for such observation. For me it is obvious, specially because I could observe some Ghanaian authors. However, at introduction authors are invited to better explain spot location, adding such information in a World context.

2. In line 99, authors described that take 6 hours to transport the sample. I assume that collection was in a not easy place. But is this not a limitation? Authors could at least describe the possible contaminations occurring at this time.

3. In line 113, authors are invited to better describe the MALDI biotyper analyses including the number of spectra used in each identification and the number of technical and biological replicates utilized.

4. Please provide the list of antibiotics used for susceptibility tests. This could be added as supplementary material.

5. Please provide the primers sequence used at fungal identification.

6. There is some problem in Figure 1. The same occurs in figure 2, inserted in main text. Nevertheless at the end of main text is everything ok.

7. Table 2. Its is amazing that all strains were 100% resistant to CXM. This is not impossible but unusual. Authors are invited to clearly discussed. Moreover, is also remarkable that Klebsiella show so high rates of resistance. This is also an important data.

8. Why did authors did not test the fungal susceptibility to antifungals? Such data could also be important in order to determine the difficult in the region.

Reviewer #2: The objective of the study is to determine the epidemiology of microbial pathogens in chroncially infected wounds to support therapeutic choices for physician.

The aetiology of the chronic wounds in Table 1 is not welly defined (does cut means surgical incision or trauma?). Suggest for author to classify the types of wound by diabetic, arterial, infectious, surgical, trauma, venous and pressure ulcers.

Authors have performed logistic regression for wound size, traditional medicine and multiple bacterial infections. The p-value for the regression analysis are not stated in the manuscript.

Suggest to look into

1. Association of antibiotic usage and antibiotic resistance.

2. Association of types of wound and antibiotic resistance.

In row 211, authors have mentioned that "In 23(22%) wound, fungal DNA was amplified by broad range PCR". Does it means only 23 wounds were amplified for fungal detection instead of 105 wounds? Please justify.

Reviewer #3: Dear Editor,

Thank you for having chosen me as Referee for this paper.

I was pleased to read this work related to chronic infected wound in Ghana.

The authors provide detailed information on the epidemiology of microbial pathogens in chronically infected wounds in rural Ghana with the aim to support therapeutic choices for physicians. For the study were enrolled 105 patients with 207 microbial isolates. Isolated strains were characterized by a high resistant rate also against carbapenem. They concluded that for a correct management of these infections it is important to perform microbiological diagnostic approach including susceptibility testing.

The work is well structured and the topic, the antibiotic resistance of pathogen microrganisms isolated from chronically infected wounds in undeveloped countries such as Ghana, is of significant importance and fits within the scope of Plos one.

The results justify the conclusion.

All sections are presented with adequate clarity.

I suggest to emphasize the poor situation in Africa and in rural Ghana in the Introduction section.

In my opinion, the overall content of the paper is significant and the manuscript could be recommended for publication in “Plos one” after minor revision.

I have suggested some corrections and formulated some suggestions that might help the Authors in improving the manuscript.

Minor revision

In all parts of the manuscript, I recommend pointing the name of the bacteria except for the first time

• Ethics Statement and Ethical considerations (lines 86-90): Please, include the approval number of Ethical Committee

• Please, change “flora” with “microbiota” in all parts of the MS

• Page 7, lines 122-123, please insert “ATCC” before the number of the strains

• Page 7, line 150. Please, change 47 with 49

• Page 7 lines 160-163, Were the antimicrobial patterns of strains isolated from patients previously treated with antibiotics different from those of strains isolated from patients that used herbal treatments? Please insert a comment in Discussion

• Page 7, line 160. Please, add the space (n=11;10%)

• Page 12, line 228. Could be useful insert a comment regarding the following reference:

Synergistic interactions of Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro wound model. DeLeon S, Clinton A, Fowler H, Everett J, Horswill AR, Rumbaugh KP. Infect Immun. 2014 Nov;82(11):4718-28. doi: 10.1128/IAI.02198-14. Epub 2014 Aug 25

• Page 12, lines 235-237 Please the sentence is not clear, please rephrase it

• Page 12, line 238. Please, add uppercase letter

• Page 12, line 239 delete full stop and insert comma

• Page 13, lines 255-260. Please, rephrase the sentences

• Page 13, line 259 delete full stop and insert comma

• Page 16, line 305, change number 3 with 1

• Figure 1 it is not clear, please, re-write the legend or modify the figure

**********

6. 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: No

Reviewer #3: Yes: Luigina Cellini, Dept of Pharmacy, "G. d'Annunzio" University, Chieti, Italy

Mara Di Giulio, Associate Professor, Dept of Pharmacy, "G. d'Annunzio" University, Chieti, Italy

Silvia Di Lodovico, PhD, "G. d'Annunzio" University, Chieti, Italy

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Spectrum of antibiotic resistant bacteria and fungi isolated from chronically infected wounds in a rural district hospital in Ghana

PONE-D-20-08668R1

Dear Dr. dekker,

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,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

**********

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

**********

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 was improved been acceptable in the present form. In that view I have no additional comments

Reviewer #3: This is a much improved version of the previously submitted manuscript.

The present revision is suitable for publication on Plos One

**********

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: Yes: Octavio Luiz Franco

Reviewer #3: Yes: Luigina Cellini