Review Reports - 1,12-<i>Bis</i>-Triphenyl Phosphonium Dodecane Bromide Nanovesicles as Potential Inhibitors of MDR Staphylococcal Biofilms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Dear Authors,

The work is interesting and relevant, as overcoming antibiotic resistance is one of the key challenges of modern medicine. The topic is important and the presented results are of potential interest. However, I have a few comments that should be addressed.

In the Introduction, the authors discuss multidrug resistance and biofilms, as is common in many papers on this topic. However, 1,12-Bis-Triphenylphosphonium Dodecane Bromide Nanovesicles and similar systems are not mentioned. I recommend revising the Introduction and briefly discussing these approaches to better explain the novelty and relevance of the present work.
I also recommend adding the structures of the studied compounds or systems. This would make the manuscript easier to understand for a broader range of readers, particularly specialists in medicinal chemistry.

Author Response

Report Form Compilation

“Yes” for all items

Quality of English Language

The English is fine and does not require any improvement.

Thank you for appreciating our English.

General Comments

Dear Authors,

In the Introduction, the authors discuss multidrug resistance and biofilms, as is common in many papers on this topic. However, 1,12-Bis-Triphenylphosphonium Dodecane Bromide Nanovesicles and similar systems are not mentioned. I recommend revising the Introduction and briefly discussing these approaches to better explain the novelty and relevance of the present work.

Dear Reviewer, we appreciate your comment and interest in the physicochemical characteristics of our compound BPPB and triphenyl phosphonium salts in general. Anyway, we make kindly note that such information has been extensively reported and discussed in our previous work already published and cited in the present paper (Ref. 35. Alfei, S.; Zuccari, G.; Bacchetti, F.; Torazza, C.; Milanese, M.; Siciliano, C.; Athanassopoulos, C.M.; Piatti, G.; Schito, A.M. Synthesized Bis-Triphenyl Phosphonium-Based Nano Vesicles Have Potent and Selective Antibacterial Effects on Several Clinically Relevant Superbugs. Nanomaterials 2024, 14, 1351, doi:10.3390/nano14161351). In this regard, it appeared us redundant, to redescribe and discuss BPPB and similar derivatives. Anyway, some details on its characterization have been added in the abstract, a synthetic scheme for the synthesis of BPPB has been added in Materials and Methods (Section 2.1., Scheme 1) and some details on its structure are present in lines 193-199 (revised).

I also recommend adding the structures of the studied compounds or systems. This would make the manuscript easier to understand for a broader range of readers, particularly specialists in medicinal chemistry.

Dear Reviewer, the answer to your second question could be the same given to the first point. Anyway, as reported in the first point the Scheme for the synthesis of BPPB showing its structure, that of other reagents and reaction conditions, as well as the reaction yield, has been added to Section 2.1 (Scheme 1).

Reviewer 2 Report

Comments and Suggestions for Authors

My comments to the authors are attached in the Word file

Comments for author File: Comments.pdf

Comments on the Quality of English Language

My comments to the authors are attached, and scientific soundness should be improved.

Author Response

Report Form Compilation

“Yes” to the first four items, and “can be improved” to the last two ones.

Quality of English Language

The English could be improved to more clearly express the research.

Thank you for this suggestion. We confirm that all the manuscript has been double-checked and revised by our English expert, English mother tongue, Professor Deirdre Kantz, to reduce grammar errors and typos. Note that Deirdre teaches Scientific English in the University of Genoa and Pavia. Revisions are not evidences because substantial and along all manuscript.

General Comments

In this study, the authors report the antibiofilm evaluation of BPPB nanovesicles, previously characterized nanomaterials with confirmed nanoscale dimensions (50–60 nm) maintained even in tryptic soy broth (TSB), against strong biofilm-producing, multidrug-resistant (MDR) Staphylococcus aureus and S. epidermidis clinical isolates. Isolates were selected based on biofilm production (crystal violet assay), species identification (MALDI-TOF MS), and antimicrobial susceptibility profiling (VITEK 2), revealing resistance to 3–14 antibiotics, including one strain resistant to both vancomycin and teicoplanin. Despite this extensive resistance, all isolates showed high susceptibility to BPPB, with low MIC values (0.125–0.250 μg/mL) and strong, dose-dependent biofilm inhibition (83% to >99%) at sub-MIC, MIC, and supra-MIC concentrations. Comparative testing against conventional vancomycin showed that BPPB achieved lower MICs, retained full activity against a vancomycin-resistant isolate, and demonstrated markedly superior biofilm inhibition. This manuscript can be accepted after minor revision as suggested below.

Comments to Authors:

Authors mentioned that 1,12-Bis-Triphenyl Phosphonium Dodecane Bromide reported in the previous work, however, it would be good present a chemical structure so readers will understand easily while reading. Provide structure in 2.1. Synthesis of BPPB

We thank a lot the Reviewer for this suggestion which is sheared also with another Reviewer. So, despite an image of BPPB chemical structure is already present in Supplementary Materials associated to this paper, as asked, a Scheme for the synthesis of BPPB showing its structure, that of other reagents and reaction conditions, as well as the reaction yield, has been added to Section 2.1 (Scheme 1).

Figure 1 need to be clearer and more aligned right with the test in the centre.

Figure 1 have been improved according to Reviewer requests and centred.

Found many technical and grammatical errors for example Line 942: "Subsequent evaluation of its activity against BF forming by all isolates tested” change it to "against BF formation by all tested isolates."

Done.

The Conclusions section largely re-reports quantitative results already presented in the Results/Discussion (MIC ranges, % BF inhibition, SI values, resistance profiles). Per standard journal convention, the conclusion should synthesize the significance of findings rather than restate data in detail. Recommend condensing by ~30–40%, retaining only the key take-home figures (e.g., MIC range, BF inhibition range, comparison with vancomycin).

Conclusions have been revised accordingly to Reviewer comments, while some Figures have been moved in the Supporting Information file, retaining in the main text the most relevant.

I feel over cited which are not required and find self-citation almost 14 papers. Please add which are most important.

Self-citations correspond to ≈ 16% of total citations thus remaining within the requirements of the most relevant journals, including MDPI ones.

Reviewer 3 Report

Comments and Suggestions for Authors

I recommend the major revision.

1. The claimed antibiofilm effect is not sufficiently separated from antibacterial activity. Crystal violet measures total attached biomass, but it does not distinguish inhibition of biofilm formation from reduced bacterial growth. The “live-dead-like” experiment was performed only for one isolate, at 4× MIC and in Mueller–Hinton broth; therefore, it cannot validate the biofilm results obtained for all strains at ½–2× MIC in TSB. Viable-cell counts within the biofilm, preferably together with metabolic or microscopic analysis, are required. Moreover, the MIC of S. aureus 24 is inconsistently reported as 0.250 and 0.500 µg/mL.

2. The statistical analysis requires substantial correction. The manuscript defines significance thresholds incorrectly, including p < 0.1 as significant and stating that no symbol was used for p > 0.5 instead of p > 0.05. It is also unclear whether technical triplicates or independent experiments were used as the statistical unit, which risks pseudoreplication. The authors should reanalyse the raw data using biological replicates, clearly define the two ANOVA factors, verify assumptions, and report exact adjusted p-values and effect sizes.

3. Nanovesicle characterization and mechanistic interpretation are not sufficiently supported. DLS in TSB was analysed using the refractive index and viscosity of water, despite the different composition of the medium, and filtration before measurement may selectively remove aggregates. The relatively high PDI in water, large standard deviations, and remaining micrometre-sized aggregates also indicate a heterogeneous system. Therefore, the manuscript should avoid firm statements about stable 45–60 nm nanovesicles, passive bacterial-cell entry, biofilm penetration, or protein corona formation unless orthogonal methods such as TEM/cryo-TEM or nanoparticle tracking analysis confirm these points.

Author Response

Report Form Compilation

“Must be improved” to the first three items, missing judgment to the fourth item and “can be improved” to the last two.

Quality of English Language

The English is fine and does not require any improvement.

Thank you for this comment.

General Comments

I recommend the major revision.

The claimed antibiofilm effect is not sufficiently separated from antibacterial activity. Crystal violet measures total attached biomass, but it does not distinguish inhibition of biofilm formation from reduced bacterial growth. The “live-dead-like” experiment was performed only for one isolate, at 4× MIC and in Mueller–Hinton broth; therefore, it cannot validate the biofilm results obtained for all strains at ½–2× MIC in TSB. Viable-cell counts within the biofilm, preferably together with metabolic or microscopic analysis, are required. Moreover, the MIC of S. aureus 24 is inconsistently reported as 0.250 and 0.500 µg/mL.

The comments by this Reviewer are almost all correct but open to a constructive discussion, and in one case not entirely correct. For example, accepting as real the limitations of cryla violet method exposed by the Reviewer, CV assay remains a simple, reliable, and widely used method for quantifying biofilm formation. It is worldwide recognized for testing the ability of a new compound to inhibit biofilm. The CV assay is a straightforward method for the quantification of biofilm. CV staining has become ubiquitous in biofilm research because of its low cost and ease of use. (https://doi.org/10.1038/s41596-021-00515-3; https://doi.org/10.3390/biom8020029; https://pdf.benchchem.com/1676/Quantifying_Biofilm_Formation_Using_the_Crystal_Violet_Assay_Application_Notes_and_Protocols.pdf; https://doi.org/10.1016/j.bioflm.2024.100187). Unfortunately, crystal violet (CV) method is indirect, rather unrefined and imprecise (it does not distinguish inhibition of biofilm formation from reduced bacterial growth). Anyway, to address this issue and for incontestably quantify inhibition of biofilm formation in this study, we have separated bacterial growth from inhibition of biofilm formation carrying out time-kill assays at 24 hours, called live-dead-like experiments in the paper. Such experiments were carried out in Mueller–Hinton broth, since it is suggested by the commonly used procedure. They were performed at 4 × MIC, just to be sure that when we observed biofilm inhibition, all strains were unmistakably alive and capable of reproducing, since it doubled the maxima concentrations which inhibited biofilm formation. Concerning the MIC of S. aureus 24, we have double-checked the manuscript, noting that we have reported the incorrect value of 0.500 µg/mL, more than one time in the paper for confusion with vancomycin. Sorry for this refuse and very thanks for having evidenced such errors. The data and other correlated ones found by us have been corrected. Apologising in advance to this Reviewer, we make kindly note her/him the only incorrect comment. As reported in the text, we did not perform live-dead-like experiment only on S. aureus 24 but on “the different strains of S. aureus and S. epidermidis” and that the results obtained for S. aureus 24 shown in Figure 4 (revised manuscript) is only representative for results obtained on the other strains which were similar. More details on this have been added in the text for better clarifying the question.

The statistical analysis requires substantial correction. The manuscript defines significance thresholds incorrectly, including p < 0.1 as significant and stating that no symbol was used for p > 0.5 instead of p > 0.05. It is also unclear whether technical triplicates or independent experiments were used as the statistical unit, which risks pseudoreplication. The authors should reanalyse the raw data using biological replicates, clearly define the two ANOVA factors, verify assumptions, and report exact adjusted p-values and effect sizes.

We thank this Reviewer for her/his comments which stimulate us to re-check statistical analysis and correctly define the two ANOVA factors, verify assumptions, and report exact adjusted p-values. Please, check revised Section 2.4 and Figures captions in both mein text and Supplementary Materials.

Nanovesicle characterization and mechanistic interpretation are not sufficiently supported. DLS in TSB was analysed using the refractive index and viscosity of water, despite the different composition of the medium, and filtration before measurement may selectively remove aggregates. The relatively high PDI in water, large standard deviations, and remaining micrometre-sized aggregates also indicate a heterogeneous system. Therefore, the manuscript should avoid firm statements about stable 45–60 nm nanovesicles, passive bacterial-cell entry, biofilm penetration, or protein corona formation unless orthogonal methods such as TEM/cryo-TEM or nanoparticle tracking analysis confirm these points.

We appreciate these comments by Reviewer which enabled us to refine nanovesicles characterization by new DLS in TSB medium conferring results more scientific robustness. The setting of instrument on water parameters of viscosity and refractive index values was now considered and the errors introduced were estimated, which were used to correct observed dimensions accordingly. Please, check revisions made in several parts of the manuscript to conclusions, concerning this question. Specifically, check Sections 2.2, 3.1, 4.1, Table 1, lines 896-897. It is true that filtrations could remove aggregates, but a reported protocol was applied (Ref. 36). Following the suggestions of Reviewer, firm statements about stable 45–60 nm nanovesicles were removed. Other statements were left because supported by literature data. Passive bacterial cells entry refers to NPs size in water and is supported by literature reports.

Reviewer 4 Report

Comments and Suggestions for Authors

Reviewed work brings the evaluation of BPPB NPs, in scope of its antimicrobial and antibiofilm proliferation activity of two clinically important Staphylococci species. Beside this, DLS analysis of BPPB nanoparticles was realised in complex culture medium – TBS. Results show, that medium composition has impact on NP size, DPI, and ζ-p, but antimicrobial activity remains at very good level. MIC of BPPB for both Staphylococci species are at concentrations comparable with standard antibiotics and antibiofilm activity is very strong, even at subinhibitory concentration (1/2MIC). These results give are very promising.

The same antibiofilm activity is presented by three different figures. It is redundant. I advise choosing between Percentage of Biofilm inhibition or Percentage of residual Biofilm biomass and the rest should be in the supplementary.

In case of S. aureus 187, results of OD are in contradiction with results presented in percentage, when decrease of OD is the lowest at 2x MIC BPPB, but in S1figure, the percentage of inhibition is the highest at 2x MIC. When percentage is calculated based on measured OD, it doesn’t look OK.

The XTT assay or other metabolic activity assay would be more appropriate than time killing assay.

I advise shortening the discussion, it is too long and contains a lot of theoretical information, like describing emergence of ESKAPE and MDR bacteria at the beginning of sub chapter 4.2.1. Discussion contains a lot of comparisons with much other research works with same goal, but there are too many details and so the discussion is too long and tiring not sure if is necessary to include so many different compounds and also to many details about all of them, so discussion is so long and tiring. (lines 666 to 674 about antimicrobial peptides, the hole part about mechanistic bases for the limited BF formation activity of Vancomycin / it is too long and doesn’t fit so much here). Information are repeating time to time in the text. It is not necessary to extensively introduce the research of the compounds compared to BPPB. These parts can be shorten, and comaprison too.

I don’t understand why a table (Table 4) with results in the discussion part is. It should be in the results.

Check the references, many references don’t match with the text. E.g 42, 45, 48, 49, 52, 53, 56, 61, 62

References 63-70 except 67 are not significant

Highlights of work are results antibiofilm activity of BPPB NPs and very comprehensive discussion.

I consider the work is too long, losing in details and quantity of information. Results from antibiofilm activities are overrepresented. I advise considerable shortening, and re/check the references, it looks like they are moved in order, and some are completely inappropriate and do not contain cited information.

Comments on the Quality of English Language

I have only few comments to quality of English. There are several sentences too long and, so it is difficult to understand the meaning. Use of shorter sentences can improve the clarity of text. (for example, lines 348-349, 519-522).

Author Response

Report Form Compilation

“Yes” to the first two items, and to the fourth and fifth ones. “Can be improved” was given to the fourth and las items.

Quality of English Language

The English could be improved to more clearly express the research. I have only few comments to quality of English. There are several sentences too long and, so it is difficult to understand the meaning. Use of shorter sentences can improve the clarity of text. (for example, lines 348-349, 519-522).

Thank you for this suggestion. We confirm that all the manuscript has been double-checked and Revised by our English expert, English mother tongue, Professor Deirdre Kantz, to reduce grammar errors and typos. Note that Deirdre teaches Scientific English in the University of Genoa and Pavia. Revisions are not evidences because substantial and along all manuscript. Sentences pointed out by the Reviewer have been reformulated. Please, check lines 354-358 and 537-541 (revised).

General Comments

Thank for this suggestion which have been addressed.

We thank a lot this Reviewer for having signalled such contradiction between OD results reported in the main text and percentage results reported (now) in the Supplementary Materials (both inhibition percentages and residual biofilm percentages). These two percentages results are correct, and percentages data agree to each other. OD data for strain 187 were not correct because of an incorrect formula used to calculate them by Microsoft Excell software. Formula has been corrected in compliance with those used for other strains and new results were newly elaborated using PRISM to provide the new image reported as Figure 2, in the revised manuscript. Sorry for this error.

The XTT assay or other metabolic activity assay would be more appropriate than time killing assay.

We accept the Reviewer suggestion and agree with this reviewer. Anyway, as reported (https://doi.org/10.1016/j.ijantimicag.2024.107441), “In vitro time-kill curve (TKC) experiments are an important part of the pharmacokinetic-pharmacodynamic (PKPD) characterisation of antibiotics” and to evaluate bacteria colonies count over time (24 hours) evidencing their decrease or increase to assess if a new drug can be defined bacteriostatic or bactericidal killing irreversibly bacteria. As XTT assay, traditional TKCs use Mueller-Hinton broth (MHB), which lacks specific plasma components that could potentially influence the bacterial growth and killing dynamics, and affect translation to in vivo. Note that, while XTT on biofilms measures metabolic activity and the functional state of the biofilm, i.e. the presence of metabolically active cells, although not all cultivable, time-kill at 24 hours on biofilms measures both viable cells and cultivable ones, providing also outputs regarding bactericidal/bacteriostatic effect on the biofilm. In this case, a 24-hour time-kill on biofilms appears to be very robust and often more convincing to reviewers than a colorimetric assay alone.

As suggested by the Reviewer (many thanks for this) signalled “boring” details have been removed and several parts of Sections 4.2.1 and 4.2.2 have been eliminated for a total of almost 100 lines. All discussion regarding mechanistic bases for the limited BF formation activity of Vancomycin has been removed (please, note its disappearance) from main text and was moved to Supplementary Materials file, after Figure S7.

I don’t understand why a table (Table 4) with results in the discussion part is. It should be in the results.

Table 4 has been moved to Results Section as suggested.

Check the references, many references don’t match with the text. E.g 42, 45, 48, 49, 52, 53, 56, 61, 62. References 63-70 except 67 are not significant.

Many thanks to Reviewer for these reports. Accordingly, references have been checked: refs. 42, 45, 48, 49 and 52 have been replaced by more proper ones, while refs. 53, 56, 61 and 62 were maintained because appropriate. Perhaps Reviewer intended as not significant all self-citations. Why the Reviewer considerer our works not significant for what reported in the text? We ask kindly the Reviewer to accept the maintenance of these references despite self-citations because all self-citations represent only the 16.4% of all citations, thus falling within the thresholds wanted by the most part of relevant journals, MDPI included.

Highlights of work are results antibiofilm activity of BPPB NPs and very comprehensive discussion. I consider the work is too long, losing in details and quantity of information. Results from antibiofilm activities are overrepresented. I advise considerable shortening, and re/check the references, it looks like they are moved in order, and some are completely inappropriate and do not contain cited information.

All these requests have been addressed as more in detail specified in previous points.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have generally addressed and satisfactorily explained my comments. Therefore, I recommend accepting the manuscript.

Author Response

We thank the Reviewer for havingaccepted our manuscriptn for publication.

Reviewer 4 Report

Comments and Suggestions for Authors

In Figure 3 (live-dead-like experiment), the caption indicates that CFU/mL are on a logarithmic scale, but the y-axis shows actual CFU/mL counts instead of their log10 values. Please align the caption and the graph. If you prefer to keep the CFU/mL counts, please express them as powers of 10 for better readability.

The sentences in line 766 and 768 are almost the same. Please, keep only one of them in the text.

766 " Anyway, considering only the highest BF inhibition (%) observed for S. aureus (28.3-71.7%) by BC-NPs, it was inferior to that exerted by BPPB at the lowest concentrations tested (0.125 μg/mL) by 1.2-3.5-times."

768 "Collectively, considering the highest BF inhibition values reported against ATCC S. aureus (28.3–71.7%), the BF inhibition activity of BC-NPs remained lower than that by BPPB at the lowest concentration tested (0.125 μg/mL), with BPPB exhibiting approximately 1.2- to 3.5-fold
greater BF inhibition."

Comments on the Quality of English Language

Quality of English is improved after revision.

Author Response

Dear Reviewer, In Figure 4 not 3, the Y-axis is almost certainly on a logarithmic scale.

This is evident from several clues in the graph: The Y-axis labels are approximately:

2,000

4,000,000

8,000,000,000

Each major tick mark increases by a factor of approximately 2,000 (1 → 2,000 → 4×10⁶ → 8×10⁹), rather than by a constant difference. The numerous intermediate tick marks, evenly spaced between the major ones, are characteristic of a logarithmic scale. On a linear scale, it would be virtually impossible to display values close to 1 and values in the billions simultaneously while maintaining readable spacing. However, the scale does not appear to be a standard log10 scale, as the major labels do not correspond to powers of 10 (1, 10, 100, 1,000, etc.).

The sentences in line 766 and 768 are almost the same. Please, keep only one of them in the text.

Lines 768/772 have been removed from main text.