The current antibiotic crisis highlights the importance of finding novel strategies that can be both effective and less prone to lead to the emergence of antimicrobial resistance spreading in bacterial pathogen populations. Especially, β-lactamase-producing Gram-negative bacteria and their extended-spectrum variants pose a serious threat for patients, as limited therapies are available or in development. Nutrient limitation in combination therapy has been suggested as an approach to reduce the emergence of antibiotic resistance. Pyoverdines, iron chelating metabolites secreted by fluorescent Pseudomonas species, can inhibit the growth of competing pathogens by sequestering this essential micronutrient and creating severe iron-limited environments. In this study, we combined pyoverdines with
two clinically relevant β-lactam antibiotics against different E. coli strains and tested for resistance evolution. We considered one sensitive and two isogenic ampicillin-resistant strains carrying a chromosomal or plasmid encoded β-lactamase gene.
In a first step, we defined a top pyoverdine candidate and examined its antibacterial effect as single treatment and in combination with ampicillin or ceftazidime against the three E. coli strains. Then, we investigated the cost of plasmid-encoded antibiotic resistance under treatment using competition assays. Finally, we performed experimental evolution and compared the growth of the evolved populations with that of the ancestors under the treatment conditions in which the populations evolved. This approach allowed us to examine how pyoverdine, the combination therapy and the E. coli strain background affected the evolution of resistance. We found that pyoverdine exerted a dose-dependent antibacterial effect. Plasmid carriage seemed to exert a cost in the untreated media and prevented the evolution of resistance to pyoverdine. Pyoverdine and the combination treatment appeared to be more evolutionary robust than ceftazidime. However, the combination therapy still selected for drug resistance and pyoverdine exposure did not reduce the level of antibiotic resistance.These results show that pyoverdine might be an effective and evolutionary more robust treatment than conventional antibiotics, especially against bacteria carrying multicopy plasmids. Moreover, our findings suggest that pyoverdine acts independently in most cases and has little effects on antibiotic efficacy and resistance. Our study leads to a better understanding of the effect of pyoverdine-induced iron limitation and combination therapy on the evolution of drug resistance
