Siderophores promote cooperative interspecies and intraspecies cross-protection against antibiotics in vitro.

The antibiotic cefiderocol hijacks iron transporters to facilitate its uptake and resists ß-lactamase degradation. While effective, resistance has been detected clinically with unknown mechanisms. Here, using experimental evolution, we identified cefiderocol resistance mutations in Pseudomonas aeruginosa. Resistance was multifactorial in host-mimicking growth media, led to multidrug resistance and paid fitness costs in cefiderocol-free environments. However, kin selection drove some resistant populations to cross-protect susceptible individuals from killing by increasing pyoverdine secretion via a two-component sensor mutation. While pyochelin sensitized P. aeruginosa to cefiderocol killing, pyoverdine and the enterobacteria siderophore enterobactin displaced iron from cefiderocol, preventing uptake by susceptible cells. Among 113 P. aeruginosa intensive care unit clinical isolates, pyoverdine production directly correlated with cefiderocol tolerance, and high pyoverdine producing isolates cross-protected susceptible P. aeruginosa and other Gram-negative bacteria. These in vitro data show that antibiotic cross-protection can occur via degradation-independent mechanisms and siderophores can serve unexpected protective cooperative roles in polymicrobial communities.

Evolved bacterial siderophore-mediated antibiotic cross-protection.

Antibiotic cross-protection enables resistant bacteria to protect other bacteria that would be otherwise susceptible to the drug. Cefiderocol is the first siderophore cephalosporin antibiotic approved for treating Gram-negative bacterial infections, including carbapenem-resistant Pseudomonas aeruginosa strains. While highly effective, CFDC resistance has been detected clinically, and mechanisms of resistance and cross-protection are not completely understood. In this study, we used experimental evolution and whole genome sequencing to identify cefiderocol resistance mechanisms and evaluated the trade-offs of evolving resistance. We found some cefiderocol-resistant populations evolved cross-protective social behavior, preventing cefiderocol killing of susceptible siblings. Notably, cross-protection was driven by increased secretion of bacterial iron-binding siderophores, which is unique from previously described antibiotic degradation mediated cross-protection. While concerning, we also showed that resistance can be selected against in drug-free environments. Deciphering the costs associated with antibiotic resistance might aid the development of evolution-informed therapeutic approaches to delay the evolution of antibiotic resistance.