Spatial Organization Governs Metabolic Activity and Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

Dayton H, Kiss J, Wei M, Chauhan S, LaMarre E, Cornell WC, Morgan CJ, Janakiraman A, Min W, Tomer R, Price-Whelan A, Nirody JA, Dietrich LEP. Cellular arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms. PLoS Biol 2024;22(2):e3002205. doi: 10.1371/journal.pbio.3002205.

Background: Biofilms are structured bacterial communities where access to nutrients and antibiotics is limited. In Pseudomonas aeruginosa, spatial cell arrangement may influence metabolic activity and drug tolerance, but the mechanistic links between microanatomy and physiology remain poorly understood.

Hypothesis: This study tested the hypothesis that the physical organization of cells within P. aeruginosa biofilms governs resource distribution, metabolic activity and antibiotic susceptibility.

Methods: The authors used a ClearScope to visualize clonal cell arrangement across biofilm depth. A targeted screen of 48 mutants disrupted in biofilm-related genes was conducted to identify factors affecting spatial organization. To evaluate physiological consequences, stimulated Raman scattering (SRS) microscopy was separately employed to map metabolic activity via deuterium incorporation. Substrate penetration and antibiotic response were assessed using fluorescent tracers, inducible reporters and propidium iodide staining after tobramycin exposure.

Results: Wild-type biofilms formed vertically aligned striations of clonal cells that corresponded with peak metabolic activity. Mutants lacking functional pili or O-antigen structures displayed disordered arrangements, altered nutrient penetration and shifts in metabolic zones. These changes increased susceptibility to tobramycin near the biofilm surface.

Conclusions: Biofilm microanatomy, governed by genetic determinants such as pili and O-antigen structures, modulates internal resource distribution and drug susceptibility. These findings highlight spatial organization as a key determinant of biofilm physiology and suggest novel strategies for improving antimicrobial efficacy.