Antibiotic efficacy can be antagonized by bioactive metabolites and other drugs present at infection sites. Pseudomonas aeruginosa, a common cause of biofilm-based infections, releases metabolites called phenazines that accept electrons to support cellular redox balancing. Here, we find that phenazines promote tolerance to clinically relevant antibiotics, such as ciprofloxacin, in P. aeruginosa biofilms and that this effect depends on the carbon source provided for growth. We couple stable isotope labeling with stimulated Raman scattering microscopy to visualize biofilm metabolic activity in situ. This approach shows that phenazines promote metabolism in microaerobic biofilm regions and influence metabolic responses to ciprofloxacin treatment. Consistent with roles of specific respiratory complexes in supporting phenazine utilization in biofilms, phenazine-dependent survival on ciprofloxacin is diminished in mutants lacking these enzymes. Our work introduces a technique for the chemical imaging of biosynthetic activity in biofilms and highlights complex interactions between bacterial products, their effects on biofilm metabolism, and the antibiotics we use to treat infections.

Bacteria typically rely on secreted metabolites, potentially shareable at the community level, to scavenge resources from the environment. The evolution of diffusible, shareable metabolites is, however, difficult to explain because molecules can get lost, or be exploited by cheating mutants. A key question is whether natural selection can act on molecule structure to control loss and shareability. We tested this possibility by collating information on diffusivity properties of 189 secreted iron-scavenging siderophores and the natural habitats occupied by the siderophore-producing species. In line with evolutionary theory, we found that highly diffusible siderophores have preferentially evolved in species living in structured habitats, such as soil and hosts, because structuring can keep producers and their shareable goods together. Poorly diffusible siderophores, meanwhile, have preferentially evolved in species living in unstructured habitats, such as seawater, indicating that these metabolites are less shareable and more likely provide direct benefits to the producers.

Many microorganisms secrete molecules that interact with resources outside of the cell. This includes, for example, enzymes that degrade polymers like chitin, and chelators that bind trace metals like iron. In contrast to direct uptake via the cell surface, such release strategies entail the risk of losing the secreted molecules to environmental sinks, including ‘cheating’ genotypes. Nevertheless, such secretion strategies are widespread, even in the well-mixed marine environment. Here, we investigate the benefits of a release strategy whose efficiency has frequently been questioned: iron uptake in the ocean by secretion of iron chelators called siderophores. We asked the question whether the release itself is essential for the function of siderophores, which could explain why this risky release strategy is widespread. We developed a reaction–diffusion model to determine the impact of siderophore release on iron uptake from the predominant iron sources in marine environments, colloidal or particulate iron, formed due to poor iron solubility. We found that release of siderophores is essential to accelerate iron uptake, as secreted siderophores transform slowly diffusing large iron particles to small, quickly diffusing iron–siderophore complexes. In addition, we found that cells can synergistically share their siderophores, depending on their distance and the size of the iron sources. Our study helps understand why release of siderophores is so widespread: even though a large fraction of siderophores is lost, the solubilization of iron through secreted siderophores can efficiently increase iron uptake, especially if siderophores are produced cooperatively by several cells. Overall, resource uptake mediated via release of molecules transforming their substrate could be essential to overcome diffusion limitation specifically in the cases of large, aggregated resources. In addition, we find that including the reaction of the released molecule with the substrate can impact the result of cooperative and competitive interactions, making our model also relevant for release-based uptake of other substrates.