Probing the evolutionary robustness of anti-virulence treatments targeting iron uptake in Pseudomonas aeruginosa

Antivirulence treatments are therapeutic strategies that inhibit the expression or functioning of bacterial virulence factors. They hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that anti-virulence treatments disarm rather than kill pathogens, is controversial, and there is a lack of studies testing the potential for resistance evolution under conditions relevant for infections. Here, we approach this issue by probing the evolutionary robustness of two anti-virulence treatments, gallium and flucytosine, targeting the iron scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa. We first let bacteria evolve under different drug concentrations in replicated populations over 20 days, using human serum as an ex-vivo model. We then applied a combination of phenotypic and genotypic screens to show that resistance against flucytosine quickly arose and spread in all populations. Genetic analysis revealed that mutations in upp, a gene encoding an enzyme required for flucytosine activation, are responsible for resistance evolution. Conversely, resistance against gallium arose only sporadically and not in all populations. Resistance mechanisms were based on mutations in transcriptional regulators, which resulted in the upregulation of pyocyanin, a redox-active molecule promoting siderophore-independent iron acquisition. Our work highlights that mutants resistant against anti-virulence treatments can easily arise, however, selective spreading varies considerably between treatments. This demonstrates that there is yes or no answer to the question of whether anti-virulence treatments are evolutionarily robust. Instead, evolutionary robustness is a relative measure on a sliding scale, with specific treatments occupying different positions on this scale.