Biofilm destruction activity of alpha-tocopherol against Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa

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1. 

   Access Microbiology

   Feb 20, 2024

   Sadly, the addition of the proper DMSO negative control in Figure 1, shows that the effect of DMSO on your biofilms is large and accounts for the majority of the antibiofilm phenotype that was reported in the original submission. You have asterisk added to these graphs to denote statistical significance, but it is not clear which comparisons are being made- for example in Figure 1b you have *** between 0mg/ml and the NC, but both of these values are 0%. Additionally, given the data added for the 0mg/ml DMSO solvent controls in figure 1, it is evident that the microscope work in Figure 3 will need to be redone to compare the effect of alpha-tocopherol compared to DMSO alone, and not BHI 1% glucose. Therefore, because the correct DMSO solvent controls have not been used in the microscopy work, and because the 0mg/ml data added to figure …

   Sadly, the addition of the proper DMSO negative control in Figure 1, shows that the effect of DMSO on your biofilms is large and accounts for the majority of the antibiofilm phenotype that was reported in the original submission. You have asterisk added to these graphs to denote statistical significance, but it is not clear which comparisons are being made- for example in Figure 1b you have *** between 0mg/ml and the NC, but both of these values are 0%. Additionally, given the data added for the 0mg/ml DMSO solvent controls in figure 1, it is evident that the microscope work in Figure 3 will need to be redone to compare the effect of alpha-tocopherol compared to DMSO alone, and not BHI 1% glucose. Therefore, because the correct DMSO solvent controls have not been used in the microscopy work, and because the 0mg/ml data added to figure 1 shows a large antibiofilm effect of DMSO alone, the conclusions of this manuscript that alpha-tocopherol exhibits an antibiofilm effect are not supported by the data and the manuscript in its current form is not suitable for publication.

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2. 

   Version published to 10.1099/acmi.0.000728.v2 on Access Microbiology

   Feb 1, 2024
3. 

   Access Microbiology

   Dec 14, 2023

   The two reviewers reports both give some very nice constructive suggestions on how to improve this manuscript with further lab work. However, the remit of Access Microbiology is for sound science without regard for work being high impact. Therefore it is at the discretion of the authors if they choose to add in additional times points for their biofilm assays, if they want to test a broader range of alpha-tocopherol concentrations, if they want to perform biofilm assays on additional materials, or if they want to perform polymicrobial biofilms. I agree that all of these ideas would enhance the manuscript and improve its impact, but choosing not to do these things will not block publication. Additional data that does need to be added are the data to show the effect of DMSO (that the alpha-tocopherol is dissolved in) on the cells, and I …

   The two reviewers reports both give some very nice constructive suggestions on how to improve this manuscript with further lab work. However, the remit of Access Microbiology is for sound science without regard for work being high impact. Therefore it is at the discretion of the authors if they choose to add in additional times points for their biofilm assays, if they want to test a broader range of alpha-tocopherol concentrations, if they want to perform biofilm assays on additional materials, or if they want to perform polymicrobial biofilms. I agree that all of these ideas would enhance the manuscript and improve its impact, but choosing not to do these things will not block publication. Additional data that does need to be added are the data to show the effect of DMSO (that the alpha-tocopherol is dissolved in) on the cells, and I have selected major revisions to allow time for you to do these experiments if the data are not already available. Please respond to all reviewers comments point by point. And please revise your graphs to show all data points rather than the averages, in line with reviewer 2 suggestions.

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4. 

   Access Microbiology

   Dec 13, 2023

   Comments to Author

   This manuscript explores the effects of α-tocopherol in biofilms of three key species of biofilm-related infections. The study expands on previous research that has shown antibiofilm activity of α-tocopherol against common pathogens (ref 16 and 28 in this manuscript). The protocol is well explained but the authors should have repeated the experiments more times to draw stronger conclusions. I would like authors to address a couple of points: 1) Why have you used reference strains and not clinical strains? For instance, in the case of P. aeruginosa, it is well known that lab/clinical isolates produce very different biofilms (see for instance Grace et al 2022 Front. Microbiol. 13:1023523). As α-tocopherol has been investigated due to its anti-biofilm potential in biofilm-associated infections, in my …

   Comments to Author

   This manuscript explores the effects of α-tocopherol in biofilms of three key species of biofilm-related infections. The study expands on previous research that has shown antibiofilm activity of α-tocopherol against common pathogens (ref 16 and 28 in this manuscript). The protocol is well explained but the authors should have repeated the experiments more times to draw stronger conclusions. I would like authors to address a couple of points: 1) Why have you used reference strains and not clinical strains? For instance, in the case of P. aeruginosa, it is well known that lab/clinical isolates produce very different biofilms (see for instance Grace et al 2022 Front. Microbiol. 13:1023523). As α-tocopherol has been investigated due to its anti-biofilm potential in biofilm-associated infections, in my opinion it would be more relevant to test it against clinical isolates. 2) Why have you chosen polystyrene and not other materials? Bacterial adhesion to plastic is marketedely different than for instance glass, and usually these two are compared. I would have tested materials where biofilm-associated infections develop (like, for instance, catheters). 3) Even though the authors only did duplicates of the SEM images, in my opinion quantifying them would be valuable (check for instance DOI: 10.1038/srep32694 on how it can be easily done using free software like ImageJ) 4) Since biofilm-associated infections usually involve polymicrobial biofilms, it would be interesting to see if the biofilm destruction activity of α-tocopherol described by the authors for each monoculture would still happen if all three bacteria were combined in a polymicrobial biofilm. Moreover, authors should restructure some sentences to make the text more fluid and understandable. Specifically lines 74 to 76, 80 to 82, lines 168-169, lines 184-186. Relating to the crystal violet assay in line 159 authors described "the staining procedure (…) as described above" but it hasn't been described, so please include it. I would also like to see the "few elongated cells can be observed in the SEM image" (lines 279/280) being highlighted in the figure by an arrow. In line 283/284 the authors mention "reduction of bacterial cell number (log CFU)" but this hasn't been calculated - could you please clarify? Lastly I would like to give a general comment to figures 1 and 2. Recently it has been more common to display all data points in the plots (ie, include the biological triplicates as points in plots instead of summarising it using bar plots). I would redo these figures so that panels A, B and C are displayed together in an unique plot, and the significance is displayed by "*" instead of an "a".

   Please rate the manuscript for methodological rigour

   Good

   Please rate the quality of the presentation and structure of the manuscript

   Satisfactory

   To what extent are the conclusions supported by the data?

   Partially support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

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5. 

   Access Microbiology

   Dec 7, 2023

   Comments to Author

   Dear Authors, I have carefully reviewed your manuscript titled " Biofilm destruction activity of alpha-tocopherol against Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa" and would like to provide constructive feedback to help enhance the clarity, rigor, and overall impact of your work. First and foremost, let me commend you on the strengths of your study, particularly the clear objective, the claimed novelty in reporting on the biofilm destruction activity of alpha-tocopherol against specific bacterial strains, and the detailed explanation of the methods employed for testing biofilm destruction, cell viability, and SEM imaging. However, to further strengthen your manuscript before publication, I would like to highlight several areas for improvement: Detailed comments and …

   Comments to Author

   Dear Authors, I have carefully reviewed your manuscript titled " Biofilm destruction activity of alpha-tocopherol against Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa" and would like to provide constructive feedback to help enhance the clarity, rigor, and overall impact of your work. First and foremost, let me commend you on the strengths of your study, particularly the clear objective, the claimed novelty in reporting on the biofilm destruction activity of alpha-tocopherol against specific bacterial strains, and the detailed explanation of the methods employed for testing biofilm destruction, cell viability, and SEM imaging. However, to further strengthen your manuscript before publication, I would like to highlight several areas for improvement: Detailed comments and suggestion for improvement Limited Biofilm Maturation Time: The study solely focuses on 24-hour biofilm formation and its treatment with alpha-tocopherol, possibly leading to premature biofilm conditions. Consider extending your experimental design to include both 24, 48, and 72-hour biofilms. This adjustment could provide a more comprehensive understanding of alpha-tocopherol's effects on mature biofilms. Lack of Positive Control: It would strengthen the experimental design to include a positive control using a known biofilm-forming agent for all experiments. This inclusion would provide a valuable benchmark for comparison and strengthen the validity of your results. Expanded Concentration Range: The concentration range tested for alpha-tocopherol (2 - 0.01 mg mL-1) is narrow. A broader range, especially at lower concentrations, would offer deeper insights into the dose-dependent relationship. Test a broader range of alpha-tocopherol concentrations, to establish a more comprehensive dose-response relationship. Negative Control Data: The study lacks information on the effect of the negative control (0.5% DMSO) on bacterial growth/viability. Providing these data as supplementary files would enhance transparency. Mechanistic Insights (optional): While the study hints at a potential mechanism for biofilm disruption (solubilisation of polysaccharides), a more in-depth exploration is needed. Additionally, considering the variation in extracellular surface characteristics of different bacteria used could provide context for diverse outcomes. Statistical Analysis: The results are presented descriptively without statistical analysis. Conducting appropriate statistical tests would add rigor and ascertain the significance of observed differences. SEM Image Clarity: The study could benefit from a more detailed interpretation of SEM images, explicitly linking visual observations to biofilm destruction. It is also crucial to specify the concentration of alpha-tocopherol used before SEM imaging. Concluding Statements: Revise your conclusion to be more concise, focusing on summarising key findings and their implications. Explicitly state the study's limitations and propose avenues for future research. I believe addressing these points will significantly enhance the quality and impact of your manuscript. Your dedication to furthering scientific knowledge is evident, and I look forward to seeing your work progress. Best wishes

   Please rate the manuscript for methodological rigour

   Good

   Please rate the quality of the presentation and structure of the manuscript

   Satisfactory

   To what extent are the conclusions supported by the data?

   Strongly support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

   Read the original source
6. 

   Version published to 10.1099/acmi.0.000728.v1 on Access Microbiology

   Oct 23, 2023