Abstract
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 no ‘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.
Author Summary There is an urgent need for alternative therapeutic options against multi-drug resistant bacteria. Antivirulence approaches, which target bacterial virulence factors thereby reducing the ability to establish and maintain infections, are proposed as an evolutionary robust way to manage infections. Conversely to conventional antibiotics, there is great hope that “disarming” rather than killing pathogens will exert weaker selection for resistance. Here we compared the evolutionary robustness of two proposed antivirulence drugs, gallium and flucytosine, both targeting the pyoverdine mediated iron-uptake in the opportunistic human pathogen Pseudomonas aeruginosa. Pyoverdine is a strong iron chelator and an important virulence factor during acute infections, and has thus been identified as a promising target for antivirulence drugs. After exposing bacteria to treatments for 20 days in human blood serum, as an ex-vivo infection model, we found that resistant mutants against the two anti-virulence drugs readily arose. However, extensive selective spreading of resistant clones was only observed for flucytosine but not for gallium treatment. Our study demonstrates that anti-virulence treatments are not evolutionarily robust per se, but vary substantially in their propensity to select for resistance. We thus need standardized protocols to identify those treatments that are both efficient in controlling infections and withstand resistance evolution.