Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits-Like Characteristics

Alginate beads represent a simple and highly reproduciblein vitromodel system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated withPseudomonas aeruginosaand followed for up to 72 h. Confocal microscopy revealed thatP. aeruginosaformed dense clusters similar in size toin vivoaggregates observedex vivoin cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3-as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulatedP. aeruginosaconfirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulatedP. aeruginosaexhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth ofP. aeruginosain alginate beads represent a simple and flexiblein vivo-like biofilm model system, wherein bacterial growth exhibits central features ofin vivobiofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3-as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosahas been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.