Fluoroquinolone Metalloantibiotics: Fighting Staphylococcus aureus Biofilms

Round 1

Reviewer 1 Report

Dear Authors:

I finished reviewing your interesting article.  I would say this article is appropriate for the journal.  However, would you please consider the revision a bit.  My concerns are related only to biofilm assay.  

#1: I felt that the difference and demarcation antimicrobial effect and anti-biofilm one were vague.  Could you make the difference more clearly?  You could describe it in the introduction part or discussion part.  

#2: Related to the above question, you mentioned your previous study as reference 19.  I checked it, but the paper didn't deal with biofilms.  Plus, I felt that you should have explained the assay to evaluate antibiofilms effect in this manuscript.  Please describe the method in this manuscript.  

Author Response

We thank the reviewer for the comments.

 

Point 1: “I felt that the difference and demarcation antimicrobial effect and anti-biofilm one were vague. Could you make the difference more clearly? You could describe it in the introduction part or discussion part.”

 

Authors’ response: The antimicrobial effect of a compound is expressed by the MIC and is related to the effect against planktonic bacterial cells. The antibiofilm effect exhibits the activity of a compound against the biofilm structure and is usually evaluated in terms of the cell viability (in this study determined by the MTT assay) and of the quantification of biomass of the structure (in this work evaluated by the crystal violet assay). These two definitions are inherent to microbiological assays. As the journal “micro” is focused on microbiological studies we thought that these two definitions were implicit for the common readers of the journal. However, the definitions of antimicrobial activity and antibiofilm effect were added to the section 3.3.

 

 

Point 2: “Related to the above question, you mentioned your previous study as reference 19.  I checked it, but the paper didn't deal with biofilms.  Plus, I felt that you should have explained the assay to evaluate antibiofilms effect in this manuscript.  Please describe the method in this manuscript.”

 

Authors’ response: The reference 19 was cited to evidence that “The antimicrobial activity (MIC values) of metalloantibiotics has been widely explored against susceptible and resistant strains of Gram‑negative and Gram‑positive bacteria. These compounds revealed an improved antimicrobial activity against MRSA clinical isolates, revealing MIC values 4 to 28-fold lower than those of free FQs.”, as stated in the introduction and in section 3.3. of the manuscript. To evaluate the antibiofilm activity of the metalloantibiotics based on the MTT and the crystal violet assays, it is necessary to know the MIC of each compound (as the assays evaluate the effect of compounds in a range of concentration between the MIC and 32x MIC). As the MICs of the metalloantibiotics were previously determined in a previous work of our research group (reference 19), the reference 19 was cited. The methods used to determine the antibiofilm effect are described in the section 2.6.2. of the manuscript.

 

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for the comments and have performed the recommended changes, as following described. The changes were highlighted in the revised document.

 

Point 1: “Some descriptions in the manuscript are misleading, thus modifications are suggested before further consideration for publication. “Metalloantibiotics” is a general term for a large family of antibiotics that show diverse structure and functions (even review articles about them were published). The authors distorted the meaning of metalloantibiotics by apparently restricting it to only the metal complexes of the fluoroquinolone family. This ignorance is analogous to calling one specific metal-dependent protein family as metalloproteins, overlooking the rest large number of other metalloproteins. The complexes are one example of metalloantibiotics and their functions (and structures) described in the introduction do not represent those of the vast number of families of other metalloantibiotics. For instance, what they described in the introductory section “To the best of our knowledge, the study of the interaction of metalloantibiotics with Gram-positive bacterial membranes is still lacking.” is misleading since there were many extensive studies about interactions of several metalloantibiotics (other than the one the authors considered) with bacterial membranes. There are also additional antibiotics which have been proposed to act on membrane in a metal-dependent (e.g. Ca2+ ) manner. This narrow view about the term “metalloantibiotics” throughout the manuscript must be corrected.

Authors’ response: We totally agree with reviewer, knowing that exists several metalloantibiotics with different compositions and that CuFQphen complexes are one example of metalloantibiotics. We did not want to restrict metal complexes to the fluoroquinolone family. Therefore, the manuscript was carefully revised. To clarify that CuFQphen metalloantibiotics were the one considered for the present work, the word “metalloantibiotics” was replaced by “CuFQphen metalloantibiotics “ in some parts of the manuscript (especially in the abstract, in the third paragraph of the introduction and in the conclusions).

 

Point 2: “The anti-biofilm activity is marginal, which nevertheless may be a starting point for further exploration. Since the title of the manuscript indicates “fighting Staphylococcus aureus biofilms”, the authors should perform other standard biofilm assays, such as fluorescence assays, to quantify and corroborate other assays despite the weak anti-biofilm activities. Biofilms are composed of complicated biochemical and biomaterials, lipids being one group. However, the research focused more on interactions with lipids, which would connect more to “interaction with cell membranes."

Authors’ response: We agree that the antibiofilm activity of the CuFQphen metalloantibiotics is slight (around 20%), concerning the cell viability of the biofilm (MTT assay). However, the crystal violet assay exhibited a significant reduction of the total biomass (for concentrations > 4x MIC). As stated in section 3.3., these results suggest a destabilization of the biofilm structure.

These results are a starting point for further exploration. As future work, the study of the antibiofilm activity of these metalloantibiotics should include biofilms of MRSA (as previous work revealed improved antimicrobial activity – MIC - for metalloantibiotics against MRSA clinical isolates, compared to susceptible bacterial strains) and the evaluation of the combination of the metalloantibiotics with other antimicrobial compounds (to evaluate possible synergistic or additive effects, previously determined for metalloantibiotics against MRSA planktonic bacterial cells), as suggested in section 3.3 of the manuscript and in the section of conclusions. The live/dead assay should also be further considered in future work, as suggested by the reviewer.

The proposed experiments should be explored in future work, to complement/corroborate the results obtained in the present work (that we agree that is more focused on the interaction of the CuFQphen metalloantibiotics with the lipidic components of the bacteria).

 

Point 3: “Of the three figures showing spectra, only Fig. 7 shows/labels the traces clearly. The authors can easily describe the traces in the captions by following the changes in intensity at specific wavelengths (e.g. from top, or bottom, at 400 nm or 450 nm etc.) since the colors of the traces are not clearly distinguishable in the plots.”

Authors’ response: The authors thank the reviewer for the comment. The Figures 1 and 3 were changed.

 

Reviewer 3 Report

Congratulations, 

It was well documented study. Enough details have been given. My only suggestion is also to add some gene expression profile in your future studies to see what is changing inside the cell under metalloantibiotic treatment. 

Thanks

 

Author Response

We thank the reviewer for the comments.

 

Point 1: “It was well documented study. Enough details have been given. My only suggestion is also to add some gene expression profile in your future studies to see what is changing inside the cell under metalloantibiotic treatment.”

 

Authors’ response: The suggestion to perform future studies based on the gene expression profile will also be considered in future work.

 

 

Round 2

Reviewer 2 Report

The current revised version is appropriate.