EPS inhibitor treatment of Salmonella impacts evolution without selecting for resistance to biofilm inhibition

Virulence factors of pathogens, such as toxin production and biofilm formation, often exhibit a public character, providing benefits to nearby non-producers. Consequently, anti-virulence drugs targeting these public traits may not select for resistance, as resistant mutants that resume production of the virulence factor share the benefits of their resistance with surrounding sensitive cells. In agreement with this, we show that even after long-term treatment with a 2-amino-imidazole (2-AI) biofilm inhibitor, Salmonella populations remained as susceptible to biofilm inhibition as the ancestral populations. Nonetheless, further genotypic and phenotypic analysis revealed that the Salmonella populations did adapt to the treatment and accumulated mutations in efflux pump regulators and alternative sigma factors. These mutations resulted in a reduced biofilm-forming capacity and increased efflux activity. Their selection was due to a growth delaying side effect of the biofilm inhibitor. Enhanced efflux activity helped overcome this growth delay, providing a fitness advantage over the ancestor. Finally, we demonstrate that chemical modification of the inhibitor enhances its specificity by partially alleviating the unintended growth delay while retaining the anti-biofilm activity, which in turn eliminated the selection pressure for increased efflux. Overall, our findings highlight that while unintended side effects can complicate anti-virulence strategies, adaptation to these effects does not necessarily restore the inhibited virulence trait. Moreover, chemical modification can mitigate these unintended side effects and enhance drug specificity. © 2025 Elsevier B.V., All rights reserved.