Modulation of Staphylococcus aureus Biofilm Formation through Subinhibitory Concentrations of Biogenic Silver Nanoparticles and Simvastatin
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript addresses an important issue in the field of microbiology and pharmacology - the rising concern of antibiotic-resistant strains and the potential use of alternative treatments. Overall, the manuscript is well-written with proper descriptions relevant to each section. However, some minor corrections suggested below are necessary.
Introduction: The information flows logically from the prevalence of MRSA to the challenges posed by biofilm formation, leading to the exploration of potential therapeutic solutions involving silver nanoparticles (AgNP) and the drug simvastatin. However, it is suggested to mention about the most common side effect (rhabdomyolysis) associated with statins in the introduction section.
Methods: Methods are very well described with good amount of information demonstrating the transparency and reproducibility of the work done.
Results: The results are presented in a clear and concise manner, ensuring a clear understanding for the readers.
It is suggested to describe briefly or provide the values to support the non-interference of solvents (water and and DMSO) with antimicrobial activity.
Include the units for the value 9.31 to 597.2 in line 213.
Provide a description of the control in line 220.
In Figures 2,3 and 4 there is a labeling error on the X-axis where 'CIM' should be corrected to 'MIC.'
Furthermore, for improved clarity, consider labeling the headings of Figures 2, 3, and 4 differently, avoiding the use of bacterial strain names.
Discussion: In the discussion section, various aspects of this study have been comprehensively explored, incorporating both the pros and cons of the findings. It is recommended to dedicate a segment of the discussion to the usage of statins as antibacterial agents, emphasizing both the advantages and disadvantages, particularly focusing into the associated side effects.
Author Response
Dear reviewer, thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
Comments 1: Introduction: The information flows logically from the prevalence of MRSA to the challenges posed by biofilm formation, leading to the exploration of potential therapeutic solutions involving silver nanoparticles (AgNP) and the drug simvastatin. However, it is suggested to mention about the most common side effect (rhabdomyolysis) associated with statins in the introduction section.
Answer: Thank you for your comments and suggestions. We included a paragraph reporting adverse effects to statins, including rhabdomiolysis (see page 2, lines 76 – 78.
Comments 2: Results: The results are presented in a clear and concise manner, ensuring a clear understanding for the readers.
- It is suggested to describe briefly or provide the values to support the non-interference of solvents (water and and DMSO) with antimicrobial activity.
Answer: We have included the following text in the page 5 - 6, lines 212 – 215.
- Include the units for the value 9.31 to 597.2 in line 213.
Answer: Included in the page 6, line 222
- Provide a description of the control in line 220.
Answer: We have included the following description in the page 6, lines 228-231: “Results for isolated AgNP evince that the MRSA HC 3817719 strain showed a slight increase in bacterial growth in relation to control, with statistical differences in ¼ and â…› MIC concentrations”
- In Figures 2,3 and 4 there is a labeling error on the X-axis where 'CIM' should be corrected to 'MIC.'
Answer: Sorry for the mistake. We corrected the labelling of the X-axis.
Furthermore, for improved clarity, consider labeling the headings of Figures 2, 3, and 4 differently, avoiding the use of bacterial strain names.
Answer: We changed bacterial strain names by letters and the information about the strains will be placed in the caption of the figures.
Comments 3: Discussion: In the discussion section, various aspects of this study have been comprehensively explored, incorporating both the pros and cons of the findings. It is recommended to dedicate a segment of the discussion to the usage of statins as antibacterial agents, emphasizing both the advantages and disadvantages, particularly focusing into the associated side effects.
Answer: We have included the following information, in the page 10-11, lines 368 – 379: “Statins, especially in combination with AgNPs, present an interesting use for topical application or even in coating medical devices such as implants, probes, and catheters [45]. Some studies have proposed coating medical devices with AgNPs to reduce microbial adhesion, thus preventing nosocomial infections. As for topical use, application would be beneficial in treating topical skin infections, especially those caused by S. aureus. Local application of SIM and AgNPs could ensure concentrations above the MIC at the affected site while also reducing potential systemic adverse events, such as rhabdomyolysis[24,25]. Further studies are needed to verify the safety and toxicity of this association, as statins can generate adverse reactions such as cheilitis,[46] autoimmune inflammatory syndromes similar to dermatomyositis and lupus,[47,48] phototoxicity,[49] occupational contact dermatitis [50,51], among others [52–54]. However, the risk of adverse cutaneous reactions seems to be relatively low [55].
Reviewer 2 Report
Comments and Suggestions for Authors1. Clarity in Methodology Description: The methodology section could benefit from greater clarity and detail. Specifically, provide a step-by-step description of the experimental procedures and assay conditions, ensuring that another researcher could replicate the experiments without ambiguity.
2. In-Depth Discussion of Biofilm Inhibition Assays: The section describing the biofilm inhibition assays needs expansion and clarification. Provide a comprehensive explanation of the results and their implications, and discuss any potential limitations or sources of variability in the experimental setup.
3. Discussion of Clinical vs. Standard Strains: Address the significance of the differences in outcomes between clinical strains and the standard ATCC 29213 strain. Explore possible reasons behind these variations and discuss their clinical relevance.
4. Implications for Clinical Application: Elaborate on the potential clinical and practical implications of the study's findings. How could the antimicrobial effects of simvastatin and bio-AgNPs at subinhibitory concentrations be applied in the real-world context of nosocomial infections and antibiotic resistance?
5. Data Presentation and Clarity: Review the presentation of data and results, ensuring that the figures and tables are well-labeled and adequately explained. Provide statistical analysis where applicable to support the findings.
6. Discussion of Synergistic Effects: Further discuss the observed synergistic effects between simvastatin and bio-AgNPs, including the mechanisms involved and their relevance to combating biofilm formation.
7. General Clarity and Language Use: Enhance the overall clarity of the manuscript by carefully proofreading for grammar, punctuation, and sentence structure. Improve the flow of the narrative for readability.
Comments on the Quality of English Language
The quality of English language in the manuscript is generally clear and understandable. However, there are a few instances where the language could be improved for greater clarity and readability.
Author Response
Dear reviewer, thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
Comments 1: Methodology: Clarity in Methodology Description: The methodology section could benefit from greater clarity and detail. Specifically, provide a step-by-step description of the experimental procedures and assay conditions, ensuring that another researcher could replicate the experiments without ambiguity.
Answer: Thank you for your feedback and suggestions. We have thoroughly reviewed the entire methodology, incorporated additional details, providing a comprehensive step-by-step description. The modified text in methodology is highlighted in yellow (see pages 4 and 5).
Comments 2: Discussion: In-Depth Discussion of Biofilm Inhibition Assays: The section describing the biofilm inhibition assays needs expansion and clarification. Provide a comprehensive explanation of the results and their implications, and discuss any potential limitations or sources of variability in the experimental setup.
Answer: We reviewed the presentation of data and captions for figures and tables.
Comments 3: Implications for Clinical Application: Elaborate on the potential clinical and practical implications of the study's findings. How could the antimicrobial effects of simvastatin and bio-AgNPs at subinhibitory concentrations be applied in the real-world context of nosocomial infections and antibiotic resistance?
Answer: We elaborated a paragraph in the page 10, lines 331 – 341, describing and discussing how the antimicrobial effects of simvastatin and bio-AgNPs at subinhibitory concentrations can be applied in the real-world context of nosocomial infections and antibiotic resistance.
Comments 4: Discussion of synergistic effects: Discuss in more detail the synergistic effects observed between simvastatin and bio-AgNPs, including the mechanisms involved and their relevance in combating biofilm formation.
Answer: In fact, we did not find “synergy” between simvastatin and bio-AgNPs when combined regarding biofilm formation, as the combination generally behaved similarly to the isolated substances. However, we observed that at subinhibitory concentrations, both the isolated substances and their combination exhibit inhibitory effects on MRSA and MSSA biofilm formation. Theferore, we have made some modifications in the text to clarify these points, in page 10, lines 342 - 348.
Comments 5: In-depth discussion of biofilm inhibition assays: The section describing biofilm inhibition assays needs expansion and clarification. Provide a comprehensive explanation of the results and their implications, and discuss any possible limitations or sources of variability in the experimental setup.
Answer: We have modified the explanation of biofilm assays on page 10, lines 345 - 353.
Comments 6: Discussion between clinical strains and standard strains: Address the importance of differences in results between clinical strains and the ATCC 29213 standard strain. Explore the possible reasons behind these variations and discuss their clinical relevance.
Answer: We have added a paragraph discussing the results regarding clinical strains on page 9, lines 302 – 309.
Comments 7: Overall Clarity and Use of Language: Improve the overall clarity of your manuscript by carefully reviewing grammar, punctuation, and sentence structure. Improve the flow of the narrative for easier reading.
Answer: Thank you for the notes. The manuscript was once again entirely reviewed by translators and the authors for grammatical, punctuation improvements, and to enhance the text's fluency.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Editor,
This study aimed to assess the antimicrobial effects of simvastatin and biologically synthesized silver nanoparticles individually and in combination using minimum inhibitory concentration assays, an in vitro biofilm model, and their effects on clinical strains of S. aureus. Transmission electron microscopy confirmed nanoparticle formation and the presence of Ag0 and AgCl. S. aureus strains were sensitive to both bio-AgNPs and simvastatin. Biofilm formation was reduced at subinhibitory concentrations of simvastatin and bio-AgNPs, but there was no synergistic effect when used in combination.
My suggestions for authors: 1. Kindly format all species names in italic typeface. 2. To enhance the article's readability, the graph should be displayed in color.
Author Response
Dear reviewer, thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
Comments 1: My suggestions for authors: 1. Format all species names in italics. 2. To improve the readability of the article, the graphic should be presented in color.
Answer: Thank you for the suggestions. We have colored the graphs and revised terms in italics.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors responded all of my raised comments and I recommend Editor can accept the revised draft. Thank you
Comments on the Quality of English Language
Good
