Review 1: "Anti-tubercular Potential and pH-driven Mode of Action of Salicylic Acid Derivatives"

RR\ID Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: In this paper the authors demonstrate that salicylic acid (SA) and two iodinated derivatives have activity against Mycobacterium tuberculosis, but not against other bacteria such as Pseudomonas or E. coli. This is a brief paper which demonstrates moderate MICs and an effect on pH homeostasis. Overall the work was conducted with appropriate controls and numbers of replicates, but SD should be provided for data where the mean was calculated.

The activity of SA was increased when the pH was reduced to 5.5 as opposed to under neutral conditions but was not affected by the addition of methionine at neutral pH. There was a small difference in sensitivity to SA when methionine was added to pH 5.5, but this was only four-fold which may not be meaningful.

Using a ratiometric GFP they demonstrated a change in the intrabacterial pH which was more pronounced when the extracellular pH was 5.5. However, in these studies they presented the ratio and not convert this into pH which should be done in order to estimate the physiological relevance of the change in internal pH.

The authors suggest that SA acidifies the cytoplasm but since there was relatively little change when the extracellular pH was neutral, this is not a very strong claim. There were other mechanisms by which intracellular pH could be affected.

The authors also demonstrated that para-aminosalicylic acid had a different mode of action in that it did not affect the intracellular pH.  

Conclusions from the data should be put into context of the other studies done on the effect of salicylate in M. tuberculosis.