Abstract Background and objectives Treatments that inhibit the expression or functioning of bacterial virulence factors 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. Here we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa . Methodology We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex-vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance. Results We found that mutants resistant against anti-virulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp , a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance could be hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike. Conclusions and implications Our work highlights that anti-virulence treatments are not evolutionarily robust per se . Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

Probing the evolutionary robustness of two repurposed drugs targeting iron uptake inPseudomonas aeruginosa