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. 2023 Feb 3;5(2):000475.v4. doi: 10.1099/acmi.0.000475.v4

Comment

Response

Please upload figures as separate, high resolution, editable files. Acceptable file types are PDF, GIF, TIFF, EPS, JPEG, PNG, SVG, and PPT. Please ensure the legends are in the main manuscript.

Figures were uploaded separately, and a figure list was added in the manuscript (P9L7-10)

What solvents were used to prepare antibiotic stock solutions?

We included a table (Table-1) that include all the solvents used (P5-6)

Could the authors please elaborate on how they established growth inhibition

Lack of visual representation of turbidity was considered to be negative; no subsequent culturing was necessary because we are investigating inhibitory effect not bactericidal effect (P4L19-23)

How many replicates were performed for each assay?

All experiment were done in duplicates

(P4L21), (P5-L15)

Any attempt at statistical analysis of the results.

Descriptive statistics as well as unpaired t test were performed (P5L13-17), (Supplementary material 1)

The authors have provided no results of this PCR to confirm the isolates' typing.

We do apologize for overlooking the importance of including the molecular analysis data, hence, now supplementary material 2 is dedicated to rectify this issue

The authors report a range of FIC values both below and above the 0.5 cutoff for synergy.

These ranges were meant to represent the minimum and maximum values of FIC to examine synergy and antagonism simultaneously. Since it was confusing (rightfully so), we opted to only represent the mean minimum value (FICmin) which basically is the most important value of synergy via the checkerboard assay (Table4), (Supplementary material 1)

Figures 2 and 3 clearly illustrate the FIC values the authors intend to present, however are presented without error bars.

Standard error bars and the mean were added to the figures, with their corresponding numerical values

Can the authors present these values as mean MIC/FIC plus standard error or deviation.

All the values were changed from ranges to means ± standard 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)?

An extended synergy tables was added to supplementary material 1 for all strains included in the study. These table illustrate all the concentrations of all the combinations that yielded FICs under 0.5

I would however like to see justification as to why they chose to examine vitamin C and nicotinamide in particular.

We included the reasoning in the introduction.

Basically, they are cheap, abundant and most importantly water-soluble, so they are versatile to work with in microbiological assays (e.g., dissolve in broths and conventional bacterial media). (P3L16-21)

Stock concentrations of antioxidants should be stated

We added the stock concentrations of antioxidants (P4L11-13)

There is no reference for the determination of fractional inhibitory concentration

There was at the beginning of the checkerboard assay paragraph.

“Garcia LS, Isenberg HD. Clinical Microbiology Procedures Handbook. Third Edition ed. Washington DC: ASM Press; 2010” (P4L16)

Can the authors comment on why there is no MIC data for clinical isolates against several antibiotics in Table 2?

Because the 10 clinical isolates were not tested against antibiotics other than Rif and Van, however, the clinical AST of these isolates was available, and we incorporated the results into supplementary material 1

There are no labels for axes and no explanation of how the data is presented which is very confusing for the reader

We added labels to the axis and all the necessary elements of the figures (Figure2&3)

In general, the order of antibiotics and data presentation should be consistent across all tables to allow ease of comparison for the reader.

This inconsistency is fixed, now all the tables and figures follow the same order of agents

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?

These ranges were meant to represent two different values FICmin and FICmax (synergy vs antagonism), we believe presenting the data in this form have made it very confusing, so, we opted to only present FICmin (synergy indicator) and rework all the tables and figures (table4)

Can the authors comment why they used LB instead of MHB in this study?

We actually didn’t use LB broth we only used MHB in this study, it was all a mistake in writing, because we simultaneously were working on inducing staphylococcal toxins via LB broth incubation for another project, so the names were interchanged in the writing process. Very sorry about that (P4L19)