Review 1: "Restriction of Arginine Induces Antibiotic Tolerance in Staphylococcus Aureus"

RR:C19 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: 

The types of nutrients available to bacterial pathogens can effect how easy they are to eradicate with antibiotic therapies. Arginine restriction induces antibiotic tolerance in Staphylococcus aureus.

Antibiotic tolerance, a phenomenon whereby bacteria can survive in the presence of otherwise active antibiotics, has been increasingly implicated in persistent clinical infections. In this study, focusing on biofilms in particular, Freiberg et al. link arginine restriction and disruptions in arginine biosynthesis to antibiotic tolerance in Staphylococcus aureus.

In the first part of the study, the authors performed screens using LFQ proteomics and TnSeq to assess the effects of antibiotic exposure on artificially grown biofilms. Cross-referencing the results of these orthogonal approaches identified coordinated responses in arginine metabolism suggesting that decreased arginine is associated with increased fitness under antibiotic stress. Next, through amino acid profiling of biofilms using HPLC, they identify that arginine is absent from biofilms and conclude its necessity as a growth-limiting factor. In the second part of the study, the authors demonstrate arginine deprivation results in multi-drug tolerance in both biofilms and planktonic culture and link the mechanism to a disruption in protein translation. Importantly, they further recapitulate the tolerant phenotype using several methods that disrupt protein translation. Overall, the manuscript provides convincing evidence that arginine plays an important role in protein translation, and that when protein translation is inhibited, either by lack of arginine or through other chemical means, a tolerant phenotype develops. 

However, we do have some comments:

1. It is not entirely clear as to the rationale of cross-referencing the results from the LFQ proteomics and TnSeq to identify “hits” of antibiotic tolerance. Mechanisms of tolerance may not always result in changes in protein expression levels and/or arise through loss-of-function mutations. For example, in the supplemental tables, the authors show that Tn disruption of sucA results in increased fitness under antibiotic stress consistent with previous publications (Zalis et al. mBio 2019) but the protein product 2-oxoglutarate dehydrogenase was not identified at all in the LFQ proteomics data. Furthermore, gain-of-function mutations in Rel, the central regulator of the bacterial stringent response, promotes antibiotic tolerance. Together this suggests that the author’s approach likely underestimates “hits” of tolerance.

2. The biofilms grown at a solid-air interface that the authors use as their model appear to be taken from the gram-negative literature. We wonder whether this model has been validated for gram-positive biofilms, specifically for S. aureus or other firmicutes. This is particularly relevant given that the strain they use, MRSA JE2, is known in the literature as a poor biofilm former.

3. In Figure S1B, to assess antibiotic tolerance of biofilms, antibiotic kill kinetics of the biofilms is compared to planktonic mid-exponential phase killing. This is a bit misleading given very different culture/nutrient conditions between the biofilm and control bacteria. Biofilm is not the only variable being studied in this experiment.  

In summary, the authors present convincing data that arginine restriction, or more generally, protein translation inhibition, conveys antibiotic tolerance. Although this is consistent with the currently accepted model and mechanism of multi-drug antibiotic tolerance, the specific link between arginine deprivation and tolerance presented here is new.