Reply to Blair et al.: Dose-dependent effects and oxidative stress responses in antibiotic tolerance in Salmonella typhimurium

We appreciate the interest taken in our recent work (1) by Blair et al. (2), who are “perplexed” that we did not observe induction of ramA expression in Salmonella typhimurium treated with 50–125 μM indole. It is important to note, however, that induction of the Salmonella typhimurium ramA gene by indole is dose dependent (3) and that the indole concentrations used in our study are an order of magnitude lower than those that induce ramA. The study cited by Blair et al. as being contradictory to our results, in fact, shows that ramA is induced at indole concentrations greater than 1 mM, and negligible induction of ramA is observed when strains were grown in media containing 500 μM (3) (see figure 1B in ref. 3; lower concentrations were not presented). We previously observed, using full-genome transcriptional analysis, that nontoxic concentrations of indole do not significantly induce drug efflux pumps in Escherichia coli (4). This finding is consistent with those of an earlier study showing that high concentrations of indole (2 mM) induce efflux pumps but lower concentrations (0.5–1 mM) do not (5). As noted above, we did not observe induction of ramA, the main regulator of efflux pumps in S. typhimurium, at the relatively low physiological concentrations of indole we considered.

Our results instead point to a different mechanism for indole-induced antibiotic tolerance. We observed that physiological concentrations of indole induce oxidative stress responses in both E. coli and S. typhimurium (1, 4). Further, knocking out oxyR, a key regulator of the oxidative stress response, abolishes the majority of indole-induced tolerance in these species (1, 4). These findings suggest that indole-induced tolerance at the concentrations we considered is mediated primarily by the oxidative stress response.

Antibiotic tolerance mechanisms are highly complex and likely involve multiple physiological processes, which vary depending on the environmental conditions and physiological state of the bacteria. Further studies are needed to expand our understanding of the multifaceted mechanisms and strategies used by bacteria to overcome antibiotic assault.