Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article "Virtual screening and meta-analysis approach identifies factors for inversion stimulation (Fis) and other genes responsible for biofilm production in Pseudomonas aeruginosa: A corneal pathogen" presents a rigorous and multidisciplinary approach to the investigation of P. aeruginosa biofilm formation, providing valuable information on potential targets for anti-biofilm treatments,  utilizing transcriptome data obtained from various investigations, in vitro analysis and meta-analysis approaches. This approach provides a more comprehensive insight into the complexity of biofilm formation.

Although meta-analysis improves statistical power, the number of studies included in the analysis could be expanded to increase the robustness of the results.

Dexamethasone, although showing anti-biofilm potential "in vitro", does not possess direct antimicrobial activity, suggesting that its mechanism of action is indirect and complex.

Overall, it is an interesting and well-written article that contributes to the knowledge in the domain, but requires further research to investigate the mechanisms of action in detail, to confirm and extend the results obtained. 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review comments

Overview: The study titled "Virtual Screening and Meta-Analysis Approach Identifies Factors for Inversion Stimulation (Fis) and Other Genes Responsible for Biofilm Production in Pseudomonas aeruginosa: A Corneal Pathogen" investigates the genes responsible for biofilm production in Pseudomonas aeruginosa, a bacterium that can cause severe infections like bacterial keratitis. The study’s commendable adoption of integrative approach combines meta-analysis of transcriptomic datasets, virtual screening and in vitro analysis to identify significant biofilm-related genes, particularly focusing on Fis (Factor for Inversion Stimulation). Thus, provides important insights for potential therapeutic targets to inhibit biofilm formation, which is crucial for treating P. aeruginosa-induced infections.

 

Strengths:

1.     Novel Approach Combining Meta-Analysis and Virtual Screening: The authors effectively combine meta-analysis of publicly available transcriptomic data and in silico virtual screening. This dual approach strengthens the robustness of their findings by integrating data from multiple studies to identify consistent genetic targets.

2.     Significant Findings in Biofilm-Related Genes: The study identified 83 differentially expressed genes (DEGs), including 45 upregulated and 38 downregulated genes in biofilm-forming P. aeruginosa. Of particular interest is the Fis gene, which was found to play a significant role in biofilm formation.

3.     Functional Enrichment Analysis: The use of Gene Ontology (GO) and KEGG pathway enrichment is well-implemented, allowing for deeper insights into the biological processes and pathways involved in biofilm production. The enriched pathways like ribosomal synthesis and quorum sensing are aligned with known biofilm mechanisms in P. aeruginosa.

4.     Virtual Screening of Drug Candidates: The study utilized virtual screening to identify Dexamethasone as a potential inhibitor of biofilm formation. The docking analysis and interaction studies with Fis were insightful, indicating that this drug might serve as a therapeutic agent for biofilm inhibition.

Weaknesses:

1.     Limited Validation: While the computational findings are compelling, the lack of comprehensive in vitro and in vivo experimental validation weakens the translational potential  of  the  study.  The  use  of minimal  inhibitory  concentration (MIC) and biofilm inhibition assays for Dexamethasone is a good start, but further validation in clinically relevant models may further strengthen the experimental aspect of the study.

2.     Antibacterial Activity Results: The study result suggests that Dexamethasone does not exhibit significant antibacterial activity against P. aeruginosa, but it does have potential as a biofilm inhibitor. However, the concentration used for the biofilm inhibition assays should be optimized further for clinical application, as the current dosages may not be practical in a clinical setting.

3.     Mechanistic Insights on Fis Limited: While Fis is highlighted as a major target, the detailed mechanism by which it regulates biofilm formation remains somewhat underexplored. Further discussion on how Fis interacts with other biofilm-related pathways would enhance the paper's scientific depth.

 

4.     Meta-Analysis Constraints: The use of random effects meta-analysis improves the power of the study, but the dataset used is somewhat limited (only four transcriptomic datasets). Expanding the meta-analysis to more diverse conditions or strains could provide a broader understanding of biofilm formation in various contexts.

 

Figures and Data Representation:

The figures are clear and well-labeled, especially the heatmaps and volcano plots used to illustrate the differentially expressed genes. The docking interaction figures are particularly useful for understanding the interaction between Fis and Dexamethasone.

Recommendations for Improvement if possible:

·       Further Validation: The study would greatly benefit from additional in vitro experiments that validate the computational predictions, particularly the role of Fis in biofilm formation and Dexamethasone’s efficacy as a therapeutic agent.

·       Mechanistic Insights: Expanding on the mechanistic role of Fis and its downstream effects on biofilm-related genes would provide more depth to the study.

·       Clinical Relevance: More discussion on the practical applications of Dexamethasone for biofilm inhibition, including potential dosage optimizations and safety considerations in clinical use, would enhance the study’s impact.

Conclusion:

This study presents a promising approach to identifying therapeutic targets for biofilm formation in Pseudomonas aeruginosa, combining meta-analysis and virtual screening. The identification of Fis as a key gene involved in biofilm production is significant, and the study’s methodology offers a strong foundation for future therapeutic development. However, further experimental validation is necessary to confirm these findings and translate them into clinically relevant treatments.

 

Minor issues

 

1.     The first letter of the second sentence in the last paragraph of the introduction should begin with an uppercase

 

2.     There exists only one sentence in the introduction on planktonic cells, and it may be important to expand on the choice as a control for biofilm to aid understanding of the background information.

 

3.     P. aeruginosa must be consistently written in italics

 

4.     Figure 7 quality can be improved

Author Response

Please see the attachment

Author Response File: Author Response.docx