Biofilm metabolic heterogeneity drives macrophage stress and immune evasion

Spatially confined biofilms generate distinct extracellular microenvironments that may shape host immune responses, yet their effects on macrophage function remain poorly defined. Here, we established an agar block model to generate two metabolically distinct Escherichia coli (E. coli) biofilm states as: Type 1 biofilms, in which the permeation of glucose does not exceed oxygen permeation and metabolism remains predominantly aerobic; and Type 2 biofilms, in which glucose penetrates deeper than oxygen and an anaerobic core forms. Supernatant from Type 1 biofilm triggered higher mitochondrial dysfunction, oxidative stress, and cell death, whereas supernatant from Type 2 biofilm induced a metabolically suppressed yet viable state that reduced oxidative phosphorylation and impaired bacterial degradation. Although Type 2 treatment limited acute cytotoxicity, it promoted intracellular persistence of bacteria, indicating that survival of host cells does not necessarily translate into effective clearance. These results reveal that biofilm metabolic state determines whether macrophages undergo terminal injury or functional suppression and suggest that successful therapy for chronic infection should consider the metabolic mode of the infecting biofilm.