Synergistic antimicrobial effect of ascorbic acid and nicotinamide with rifampicin and vancomycin against SCCmec type IV methicillin-resistant Staphylococcus aureus (MRSA)
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Background. Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacteria involved in a wide spectrum of human diseases. Many virulence factors promote this widespread propagation. One important factor is acquiring antibiotic resistance genes, which leads to a reduction in the availability and efficacy of therapy options. Recently, research has suggested that the remarkable antimicrobial effect of antioxidants against superbugs such as MRSA shows synergistic effects when accompanied by antimicrobial therapy. This paper aims to examine the synergistic effects of ascorbic acid and nicotinamide with a panel of antibiotics used in antimicrobial therapy against MRSA.
Material and Methods. Two SCC mec type IV MRSA reference strains (EMRSA-15 and USA300) and 10 MRSA clinical isolates feature in this paper. SCC mec typing was conducted on the 10 clinical isolates via multiplex PCR after identification. Synergy experiments on antioxidants and antibiotics were evaluated via checkerboard assay. The minimum inhibitory concentration (MIC) of each agent was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) M100 guidelines through twofold microdilution assay.
Results and Discussion. Synergy (FIC <0.5) was demonstrated for ascorbic acid (1/2 to 1/4 MIC) with rifampicin (1/2 to 1/8 MIC), and also ascorbic acid (1/2 to 1/16 MIC) when associated with vancomycin (1/2 MIC). Similarly, nicotinamide (1/2 to 1/16 MIC) showed a synergistic effect when paired with low concentrations of rifampicin (1/2 to 1/16 MIC), and also (at 1/4 to 1/16 MIC) with vancomycin (1/2 MIC). All reduced MICs due to synergistic combinations demonstrated statistical significance ( P <0.05).
Conclusion. The synergistic activity demonstrated in associating antioxidants with antibiotics shows promise in managing superbugs. However, more research is required to better understand the mechanism of the synergy and for utilization in clinical care.
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. Dear Abdullah AlSaleh, Thank you for your responses, these are very much appreciated as well as your experimental efforts. Both reviewers and myself are satisfied with your edits and as a result formally accept the manuscript. Best wishes, John.
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This study would be a valuable contribution to the existing literature. The reviewers raise concerns regarding the scientific rigour and experimental design of the work. Dear Dr AlSaleh, Thank you for your considered response and efforts to this point. Many of the reviewers concerns have been addressed. However, we initially had concerns about the replication of the study, which in your rebuttal, you indicated each experiment was carried out in duplicate. In any biological setting we would expect experiments to be carried out, minimum, in triplicate as standard to assess reproducibility. As a result we would ask that the number of replicates be increased to at least three for the checkered board assays. Additionally, for figure legends, a general rule of thumb is that there should be enough information to interpret the figure …
This study would be a valuable contribution to the existing literature. The reviewers raise concerns regarding the scientific rigour and experimental design of the work. Dear Dr AlSaleh, Thank you for your considered response and efforts to this point. Many of the reviewers concerns have been addressed. However, we initially had concerns about the replication of the study, which in your rebuttal, you indicated each experiment was carried out in duplicate. In any biological setting we would expect experiments to be carried out, minimum, in triplicate as standard to assess reproducibility. As a result we would ask that the number of replicates be increased to at least three for the checkered board assays. Additionally, for figure legends, a general rule of thumb is that there should be enough information to interpret the figure without accessing the main body of text. While not reiterating the full methods section, for example, figure 1, I would expect information on the primers, their specific target, the expected product sizes (bp), DNA ladder information (i.e. supplier etc.) etc.. This is partly to make the information quickly and easily accessible to readers but also to improve the interpretation of the results. While i appreciate this information is in table 1, it does not replace the nature of a figure legend as a standard practice. You will also need to update your figures list section as multiple figure 1s now exist etc. Best wishes, John.
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Dear Dr AlSaleh, Following reviews, there are concerns about the scientific rigor of the work. This is a core foundation especially for work published in Access Microbiology. Please address the comments below. Best wishes,
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Materials and methods: Stock concentrations of antioxidants should be stated There is no mention of how many replicates of the Checkerboard assay were performed. There is no reference for the determination of fractional inhibitory concentration General comments on data in Tables: Can the authors comment on why there is a range for some MICs and for the majority of FICs in Tables 2 and 3? Following on from above, if the cut-off for additive or indifference for FIC is 0.5-4, and the FIC range provided includes numbers greater than 0.5, how can synergy be specifically stated? (e.g. supplementary material 1, bottom row of USA300- range is 0.156-1) Can the authors comment on why there is no MIC data for clinical isolates …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Materials and methods: Stock concentrations of antioxidants should be stated There is no mention of how many replicates of the Checkerboard assay were performed. There is no reference for the determination of fractional inhibitory concentration General comments on data in Tables: Can the authors comment on why there is a range for some MICs and for the majority of FICs in Tables 2 and 3? Following on from above, if the cut-off for additive or indifference for FIC is 0.5-4, and the FIC range provided includes numbers greater than 0.5, how can synergy be specifically stated? (e.g. supplementary material 1, bottom row of USA300- range is 0.156-1) Can the authors comment on why there is no MIC data for clinical isolates against several antibiotics in Table 2? If it is because they are sensitive it should be stated as lack of data could also infer they weren't tested. 2. Presentation of results Graph presentation: Greater explanation of the data is needed. There are no labels for axes and no explanation of how the data is presented which is very confusing for the reader. If these graphs represent the data in supplementary material, how do the numbers in the range translate across? There may be a better way to present the data rather than what looks like a bar graph. More information is needed Supplementary data: Presentation in excel sheets is inconsistent and not labelled clearly. Any highlighted cells in the spreadsheet should be clearly labelled with what the highlight means, in addition to an explanation of what the data is. In general, the order of antibiotics and data presentation should be consistent across all tables to allow ease of comparison for the reader. In supplementary materials 1 and 2, MIC and FIC tabs are reversed, and due to an error there are no antibiotics listed apart from ciprofloxacin in supplementary material 1. In supplementary material 2 under the vancomycin/ascorbic heading there are two columns, one containing " >1 " for all isolated- it is not clear what this means. 3. How the style and organization of the paper communicates and represents key findings Presentation of data has made it hard for the reader to interpret key findings. 4. Literature analysis or discussion Results and Discussion comments: The discussion around "The discrepancy in the percentage of clinical isolates affected by the synergistic interaction might be due to human error" in lines 25 and 27, and lines 27 and 28 "It could also be because clinical isolates might harbour various virulence factors which might have hindered the demonstration of synergy" doesn't fit with the rest of the discussion, which very much implies that synergy findings are demonstrated. The overall message of the discussion is that the findings show synergy, but then these sentences don't seem to fit- can the authors comment whether they are referencing the fact that FIC ranges used to demonstrate synergy have an upper figure greater than the cut-off for synergy? This is not clear and a bit confusing. The language used in Lines 27 and 28 is also a bit confusing. Do the authors mean that there may be virulence factors present which protect against the synergistic effects of combined antibiotic and micronutrient treatment? The authors mention using LB in their discussion, and the CLSI paper referenced in the methods describes using Muller-Hinton Broth. Can the authors comment why they used LB instead of MHB in this study? 5. Any other relevant comments It is important to identify alternative/supplemental treatments for AMR infections. From this standpoint the aims of this paper are relevant and timely.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
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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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data (P.5. L.5-L.10) What solvents were used to prepare antibiotic stock solutions? (P.5. L.12 - P.6. L.7) With regards to the checkerboard assay, the preparation of the assay is adequately explained, however could the authors please elaborate on how they established growth inhibition - did they count colony forming units or measure optical density. If they assayed inhibition using a plate reader, what plate reader? There is also no mention of if/how many replicates were performed for each assay, nor any attempt at statistical analysis of the results. 2. Presentation of results Despite carrying out multiplex PCR to type their 10 clinical isolates, the authors have provided no results of this PCR to confirm the isolates' typing.
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data (P.5. L.5-L.10) What solvents were used to prepare antibiotic stock solutions? (P.5. L.12 - P.6. L.7) With regards to the checkerboard assay, the preparation of the assay is adequately explained, however could the authors please elaborate on how they established growth inhibition - did they count colony forming units or measure optical density. If they assayed inhibition using a plate reader, what plate reader? There is also no mention of if/how many replicates were performed for each assay, nor any attempt at statistical analysis of the results. 2. Presentation of results Despite carrying out multiplex PCR to type their 10 clinical isolates, the authors have provided no results of this PCR to confirm the isolates' typing. (P.11) Tables 2 and 3 communicate the MIC and FIC values clearly, however I disagree with the authors' argument that vitamin C and nicotinamide are showing synergy with rifampicin and vancomycin (P.8). This is because, in Table 3, the authors report a range of FIC values both below and above the 0.5 cutoff for synergy. (P.12) Figures 2 and 3 clearly illustrate the FIC values the authors intend to present, however are presented without error bars. (Supplementary Tables 1 and 2) Referring back to my comment on the number of replicates performed on the checkerboard assay for each strain, the MIC and FIC values for the compounds tested by the authors are presented as ranges. Can the authors present these values as mean MIC/FIC plus standard error or deviation. If the authors measured the turbidity of the wells at the endpoint of their assay, could they provide this data (e.g. in the form of a heatmap)? This would make the data easier to interpret. 3. How the style and organization of the paper communicates and represents key findings The paper is logically ordered and structured, however the absence of evidence of the authors' typing of their clinical isolates is conspicuous. They clearly explain their logic and the experimental design is sound and clearly explained apart from the concerns I have already raised. 4. Literature analysis or discussion The introduction of the paper is succinct, covering the background of the study and what the authors aimed to achieve by screening antioxidant synergy. I would however like to see justification as to why they chose to examine vitamin C and nicotinamide in particular. The authors discuss nicotinamide's effects on fungal growth, however I am not sure that is a relevant comparison to the work they are presenting here. (P.8.L26-28) The authors postulate that some of their results may have been affected by human error - this is a serious concern and should be addressed to ensure there is no question as to the validity of their results. 5. Any other relevant comments Overall, I believe the work here requires substantial revisions, in particular to show statistical significance of any synergy observed. The authors also need to clarify how they quantified growth in their plates.
Please rate the manuscript for methodological rigour
Poor
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?
No: No human or animal work presented
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