Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis

Round 1

Reviewer 1 Report

The manuscript entitled: "Novel therapeutic strategies applied to Pseudomonas aeruginosa infections in cystic fibrosis"

The topic is interesting and worth to summarize recent developments.  However, I found the manuscript lacks in-depth analysis of work done in this area.  Many of the references are not recent, which may indicate ignorance of recent research been done.  The section on inorganic NP is brief and authors were only able to cite 2-3 references.  Some of the quoted sentences are not correct for example line 166, please check the liposomal encapsulation of doxorubicin as common example contrary to the quoted sentence on hydrophilic drugs encapsulation.  The used language requires extensive revision, there are several typos or grammar mistakes.  The clinical trials section is weak and does not fit the purpose of this review.  Polymeric NP section is fragmented and topics such as dendrimers vs polymeric materials seemed overlapped and often confused with biomolecules.  The authors should clarify the focus of this review and differentiate between different materials.  Some of the cited references are wrong or inappropriate to cite, for example, reference 8 which is a review article and the author should cite original research papers. Some sentences lack appropriate referencing such as lines 56, 306 and 359.  Acknowledgment section is wrong and needs to be revised, some of the references were not cited correctly Such as reference 19, ref 27 ref to fick's law in 1855 which not sure is needed for this type of review. Overall, this manuscript requires extensive revision, clarification of objectives and not suitable for publication in the current format. 

Author Response

Reviewer 1:

The manuscript entitled: "Novel therapeutic strategies applied to Pseudomonas aeruginosa infections in cystic fibrosis"

The topic is interesting and worth to summarize recent developments.  However, I found the manuscript lacks in-depth analysis of work done in this area.

We hope this updated version includes a better and more focused analysis of the advances in this area.

Many of the references are not recent, which may indicate ignorance of recent research been done. 

We have updated the reference list that hope fulfills the state of the art.

The section on inorganic NP is brief and authors were only able to cite 2-3 references. 

We have updated the reference list, please see track changes.

Some of the quoted sentences are not correct for example line 166, please check the liposomal encapsulation of doxorubicin as common example contrary to the quoted sentence on hydrophilic drugs encapsulation. 

We do not mention doxorubicin in the manuscript, we hope that our new version does not lead to confusion as the previous did.

The used language requires extensive revision, there are several typos or grammar mistakes. 

Two native English speakers have read and edit the manuscript in depth

The clinical trials section is weak and does not fit the purpose of this review. 

We would like to specially thank you this comment, we were unsure about this section and now we have moved it to supplementary material

Polymeric NP section is fragmented and topics such as dendrimers vs polymeric materials seemed overlapped and

often confused with biomolecules.  The authors should clarify the focus of this review and differentiate between different materials. 

We have changed and clarify this section and we hope is now more clear.

Some of the cited references are wrong or inappropriate to cite, for example, reference 8 which is a review article and the author should cite original research papers.

We have changed the references to mostly original articles, however some key ideas were taken from reviews and that is why we thought it was important to recognize the authors in their effort. We however, updated the references and increase the number of original citations, we hope is now better.

Some sentences lack appropriate referencing such as lines 56, 306 and 359. 

Thanks for this observation, we have now addressed this references.

Acknowledgment section is wrong and needs to be revised,

Thanks you for pointing this out, we forgot to update this section.

Some of the references were not cited correctly Such as reference 19, ref 27 ref to fick's law in 1855 which not sure is needed for this type of review.

We have updated this, please see the track changes

Overall, this manuscript requires extensive revision, clarification of objectives and not suitable for publication in the current format.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript reports on an overview of the therapeutic strategies for treatmenent (drug payload/delivery) of P. aeruginosa in the context of cystic fibrosis affected patients.

The topic is appropriate for the journal.

The title is adequate and correlate with the content of the article.

The abstract reports a consistent summary.

The work has a clear structure.

All sections are properly written and required for a complete understanding.

Nevertheless, there are major issues that requires to be addressed prior proceeding to acceptance for publications.

The manuscript should be carefully checked for English editing and redundant forms.

Examples:

Abstract, page 1, line 20, “an environment that promotes bacterial infections. Pseudomonas aeruginosa is a ubiquitous bacterium, which can be found in many different environments” (environment is very often repeated) Abstract, page 1, line 24 “When a CF lung is infected with P. aeruginosa, the decay of the patient is accelerated, but nowadays there is little that it can be done apart from controlling the infection with antibiotics, which are becoming less effective”. Please, provide to shorten the sentence in order to allow a more straightforward understanding by a broad public Abstract, page 1, line 28, “new hope”. Please, provide for a more technical or scientific synonym Introduction, page 2, line 43, “One of the most common opportunistic bacteria for patients with CF is Pseudomonas aeruginosa. P. aeruginosa is a gram-negative bacteria that is commonly found in soil and water…”.Please, try to rephrase to avoid the redundancy of “Pseudomonas aeruginosa”. It is clear strarting from the title that the review focuses on this specific type of bacteria. Introduction, page 2, line 58, “To ensure therapeutic dosage at the site of infection, multiple administrations of the drug are required each day, increasing not only the cost of treatment but also a burden for the patients as they will have to strictly administer the dosages recommended, affecting their personal life and schedules [6].”. Please, rephrase for a more convenient understanding. Introduction, page 2, line 64, “Presently”. Please, amend

Page 1, line 34, “Cystic Fibrosis (CF) is an inherited disease caused by a mutation of the cystic fibrosis transmembrane regulator (CFTR), leading to the generation of a dysfunctional protein impeding the chloride from reaching the cell surface [1]”. Please, be more specific: it is the first sentence of the manuscript.

Page 2, line 46: please provide for statistics for the disease impact on a worldwide basis, not just for US, and a reference of a timeframe of the collected data.

Page 2, line 73, “shuttles and vectors”. They are synonyms. Please, select just one of them.

Page 2, line 77, “Chen and coworkers tested a combination of minocycline and silver

dual-loaded polyphosphoester-based nanoparticles for the treatment of resistant P. aeruginosa strains. Authors used 4-epi-minocycline, a metabolite of minocycline, identified as an active antimicrobial against P. aeruginosa using a high-throughput screen. The antimicrobial activities of 4-epi-minocycline, minocycline, and silver acetate against clinical isolates of P. aeruginosa obtained from CF patients were evaluated in vitro. The results demonstrated that both silver and minocycline are potent antimicrobials alone and that the combination allows for a reduced dosage both therapeutics, while promoting a robust antimicrobial effect.”. References are severely lacking.

Page 2, line 84, “Furthermore, the proposed synergistic silver/minocycline combination can be coloaded into nanoparticles as a next-generation antibiotic to combat the threats presented by MDR pathogens [11].”. Please, amend “coloaded” with “co-loaded”, and “next generation antibiotics” with “next generation approaches”.

Page 2, line 95, “that is able to treat”. Please, amend with “feasible for treatment of”

Page 2, line 100, “leak” and “dissipation”. Please, amend with other words, more suitable for a scientific reader platform.

Page 2, line 104, “fusion interaction”. Please, amend with “fusion-interaction”.

Page 2, line 107, “liposomes were able to encapsulate 99% of the ciprofloxacin, allowing for once a day inhalation and a half-life of 10.5 hours [14]”. Please, rephrase. The core subject is not “the liposomes”, whereas the method developed.

Overall, it is strongly suggested to shorten sentences for a better understanding.

The conclusion section, although satisfactory, could be improved, specifically on sentence 385, page 11, “It is important to consider that these novel approaches are only at early stages and more research needs to be done in order to move towards the clinical trials phase.”. It reduces the impact of the overall concepts presented.

References should be uniformed (please, provide for journal abbreviations for all ref.)

Author Response

Reviewer 2:

The manuscript reports on an overview of the therapeutic strategies for treatment (drug payload/delivery) of P. aeruginosa in the context of cystic fibrosis affected patients.

The topic is appropriate for the journal.

The title is adequate and correlate with the content of the article.

The abstract reports a consistent summary.

The work has a clear structure.

All sections are properly written and required for a complete understanding.

We would like to thank the reviewer for the kind words that complement our work.

Nevertheless, there are major issues that require being addressed prior proceeding to acceptance for publications.

The manuscript should be carefully checked for English editing and redundant forms.

We have carefully read over and edited the manuscript

Examples:

Abstract, page 1, line 20, “an environment that promotes bacterial infections. Pseudomonas aeruginosa is a ubiquitous bacterium, which can be found in many different environments” (environment is very often repeated)

We like to thank the reviewer for pointing this out, we had now realized of how many redundancies the manuscript had and we have changed them.

Abstract, page 1, line 24 “When a CF lung is infected with P. aeruginosa, the decay of the patient is accelerated, but nowadays there is little that it can be done apart from controlling the infection with antibiotics, which are becoming less effective”. Please, provide to shorten the sentence in order to allow a more straightforward understanding by a broad public

We have changed it

Abstract, page 1, line 28, “new hope”. Please, provide for a more technical or scientific synonym Introduction,

We have changed this part of the abstract and used new prospects instead

page 2, line 43, “One of the most common opportunistic bacteria for patients with CF is Pseudomonas aeruginosa. P. aeruginosa is a gram-negative bacteria that is commonly found in soil and water…”.Please, try to rephrase to avoid the redundancy of “Pseudomonas aeruginosa”. It is clear starting from the title that the review focuses on this specific type of bacteria.

We have read this section and changed the sentences that contained redundancies.

Introduction, page 2, line 58, “To ensure therapeutic dosage at the site of infection, multiple administrations of the drug are required each day, increasing not only the cost of treatment but also a burden for the patients as they will have to strictly administer the dosages recommended, affecting their personal life and schedules [6].”. Please, rephrase for a more convenient understanding.

We have modified this sentence

Introduction, page 2, line 64, “Presently”. Please, amend

We have changed to currently

Page 1, line 34, “Cystic Fibrosis (CF) is an inherited disease caused by a mutation of the cystic fibrosis transmembrane regulator (CFTR), leading to the generation of a dysfunctional protein impeding the chloride from reaching the cell surface [1]”. Please, be more specific: it is the first sentence of the manuscript.

We have changed this sentence and hope is now more clear

Page 2, line 46: please provide for statistics for the disease impact on a worldwide basis, not just for US, and a reference of a timeframe of the collected data.

We have used CDC data for 2013 to obtain this information.

Page 2, line 73, “shuttles and vectors”. They are synonyms. Please, select just one of them.

We have removed shuttles

Page 2, line 77, “Chen and coworkers tested a combination of minocycline and silver

dual-loaded polyphosphoester-based nanoparticles for the treatment of resistant P. aeruginosa strains. Authors used 4-epi-minocycline, a metabolite of minocycline, identified as an active antimicrobial against P. aeruginosa using a high-throughput screen. The antimicrobial activities of 4-epi-minocycline, minocycline, and silver acetate against clinical isolates of P. aeruginosa obtained from CF patients were evaluated in vitro. The results demonstrated that both silver and minocycline are potent antimicrobials alone and that the combination allows for a reduced dosage both therapeutics, while promoting a robust antimicrobial effect.”. References are severely lacking.

This information was all from the same source (reference at the end), nevertheless, we have done some changes that hope avoid confusion

Page 2, line 84, “Furthermore, the proposed synergistic silver/minocycline combination can be coloaded into nanoparticles as a next-generation antibiotic to combat the threats presented by MDR pathogens [11].”. Please, amend “coloaded” with “co-loaded”, and “next generation antibiotics” with “next generation approaches”.

It has been replace by next generation approaches

Page 2, line 95, “that is able to treat”. Please, amend with “feasible for treatment of”

We have changed it

Page 2, line 100, “leak” and “dissipation”. Please, amend with other words, more suitable for a scientific reader platform.

It has been replace

Page 2, line 104, “fusion interaction”. Please, amend with “fusion-interaction”.

We have changed it

Page 2, line 107, “liposomes were able to encapsulate 99% of the ciprofloxacin, allowing for once a day inhalation and a half-life of 10.5 hours [14]”. Please, rephrase. The core subject is not “the liposomes”, whereas the method developed.

We have changed this

Overall, it is strongly suggested to shorten sentences for a better understanding.

Thanks for the comment, now two native English speakers have done an in depth editing of the manuscript.

The conclusion section, although satisfactory, could be improved, specifically on sentence 385, page 11, “It is important to consider that these novel approaches are only at early stages and more research needs to be done in order to move towards the clinical trials phase.”. It reduces the impact of the overall concepts presented.

We had changed this to avoid confusion

References should be uniformed (please, provide for journal abbreviations for all ref.)

Thanks for realizing of this mistake. We have gone throughout the entire manuscript and checked the abbreviations

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors,

I have the following suggestions:

Supplementary information:

Clinical Trials and Table 1. Summary of Clinical Trials

For Arikayce™/ liposomal amikacin.  What were the adverse events or effects? What were the laboratory abnormalities?

I recommend to include information on the causes of biofilm-associated tolerance and resistance against different classes of antibiotics, in the context of an enhanced effect of the nano approach (one paragraph). Suggested references:

Ciofu O1, Tolker-Nielsen T1. Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics. Front Microbiol. 2019 May 3;10:913. doi: 10.3389/fmicb.2019.00913. eCollection 2019.

Ciofu O, Rojo-Molinero E, Macià MD, Oliver A. Antibiotic treatment of biofilm infections. APMIS. 2017 Apr;125(4):304-319. doi: 10.1111/apm.12673. Review.

Mihai MM, Holban AM, Giurcaneanu C, Popa LG, Oanea RM, Lazar V, Chifiriuc MC1, Popa M, Popa MI. Microbial biofilms: impact on the pathogenesis of periodontitis, cystic fibrosis, chronic wounds and medical device-related infections. Curr Top Med Chem. 2015;15(16):1552-76.

Henriksen K1, Rørbo N2, Rybtke ML1, Martinet MG1, Tolker-Nielsen T1, Høiby N1,3, Middelboe M2, Ciofu O1.P. aeruginosa flow-cell biofilms are enhanced by repeated phage treatments but can be eradicated by phage-ciprofloxacin combination. Pathog Dis. 2019 Mar 1;77(2). pii: ftz011. doi: 10.1093/femspd/ftz011.

Corrections:

Incomplete phrase: While experiments examining the ability of phages to treat P. aeruginosa infection in lungs is lacking.

Misleading term: This solidifies the idea that phages. Suggestion of change:  Reinforces the idea

My best regards

Author Response

Reviewer 3:

Dear authors,

I have the following suggestions:

Thanks for this detail review it has significantly improved the manuscript. The references suggested were key for our message. Thanks

Supplementary information:

Clinical Trials and Table 1. Summary of Clinical Trials

For Arikayce™/ liposomal amikacin.  What were the adverse events or effects? What were the laboratory

abnormalities?

We have moved this section to supplementary material as suggested.

I recommend to include information on the causes of biofilm-associated tolerance and resistance against different classes of antibiotics, in the context of an enhanced effect of the nano approach (one paragraph). Suggested references:

We have added most of these references on the introduction, thanks for the suggestions.

Ciofu O1, Tolker-Nielsen T1. Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics. Front Microbiol. 2019 May 3;10:913. doi: 10.3389/fmicb.2019.00913. eCollection 2019.

Ciofu O, Rojo-Molinero E, Macià MD, Oliver A. Antibiotic treatment of biofilm infections. APMIS. 2017 Apr;125(4):304-319. doi: 10.1111/apm.12673. Review.

Mihai MM, Holban AM, Giurcaneanu C, Popa LG, Oanea RM, Lazar V, Chifiriuc MC1, Popa M, Popa MI. Microbial biofilms: impact on the pathogenesis of periodontitis, cystic fibrosis, chronic wounds and medical device-related infections. Curr Top Med Chem. 2015;15(16):1552-76.

Henriksen K1, Rørbo N2, Rybtke ML1, Martinet MG1, Tolker-Nielsen T1, Høiby N1,3, Middelboe M2, Ciofu O1.P. aeruginosa flow-cell biofilms are enhanced by repeated phage treatments but can be eradicated by phage-ciprofloxacin combination. Pathog Dis. 2019 Mar 1;77(2). pii: ftz011. doi: 10.1093/femspd/ftz011.

We have added these references on the current version. We would like to thank the reviewer for these suggestions that have significantly improved the manuscrtip.

Corrections:

Incomplete phrase: While experiments examining the ability of phages to treat P. aeruginosa infection in lungs is lacking.

We added this information, and we agree that this was necessary. Thanks for the tip

Misleading term: This solidifies the idea that phages. Suggestion of change:  Reinforces the idea

We changed it for reinforce

My best regards

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript has been improved by the amendments provided.

However, there are still minor revisions which need to be addressed prior proceeding to publication:

line 26-28 “offers....offering”. Please, amend the redundancy; line 37 “airways”.Please, amend the redundancy; line 63, please amend “0.1 percent” with “0.1%”; line 193, “antibiotic after 15”. Minutes? Hours? Please, be specific; line 233 looks missing; line 297, “RG” looks missing prior to “756 NPs”

Overall, a punctuation check throughout the manuscript could be beneficial.

References no. 15, 22, 28 and 34: please provide for the correct journal abbreviation.

Reference 16 does not report on any journal/book ref.

It is also suggested to merge the SI with the main manuscript for a more easy and straightforward understanding by a broad range audience (e.g. ref. numbering can be confusing).

Author Response

The manuscript has been improved by the amendments provided. However, there are still minor revisions which need to be addressed prior proceeding to publication.

We would like to thank the reviewer for the kind words as well as the thoughtful comments on the minor things that need to be addressed.

line 26-28 “offers....offering”. Please, amend the redundancy; line 37 “airways”. Please, amend the redundancy; line 63, please amend “0.1 percent” with “0.1%”; line 193, “antibiotic after 15”.

We have revised the manuscript and we have changed the redundancies along the manuscript.

Minutes? Hours? Please, be specific; line 233 looks missing; line 297, “RG” looks missing prior to “756 NPs”

We have done these corrections

Overall, a punctuation check throughout the manuscript could be beneficial.

We have checked the punctuation

References no. 15, 22, 28 and 34: please provide for the correct journal abbreviation. Reference 16 does not report on any journal/book ref.

We have corrected these references

It is also suggested to merge the SI with the main manuscript for a more easy and straightforward understanding by a broad range audience (e.g. ref. numbering can be confusing).

We have moved the supplementary information to the main manuscript.

Reviewer 3 Report

Dear authors,

I see a problem at the References section (at the numbering).

Author Response

Thanks for pointing out the problem with the references. We have carefully checked all references