4-Ethoxybenzoic acid interferes with the spatiotemporal dynamics of <i>Staphylococcus aureus</i> ATCC 6538 biofilm formation

Flow-cell grown biofilms were fed Luria Bertani (LB) broth (control) or LB broth with 0.8 mg/mL 4EB (treatment). Treatment inhibited the progress of multiplication phase and caused a 6-hour delay in the onset of exodus phase. Transcriptional analysis showed patterns of nuc, saeS, and saeR expression consistent with the delayed exodus phenotype. Imaging by confocal laser scanning microscopy followed by digital image analysis determined that 4EB interfered with biofilm structure formation, including reductions in height (57%/44%) and biovolume (73%/63%) during the multiplication and maturation phases, respectively, with statistically insignificant effects during exodus phase (1.9%/15%). These measurements indicated that the occurrence of exodus phase was not impacted by 4EB. Gene expression analysis using flow cell effluent found significant downregulation of genes including atl (-3.1 fold change) during multiplication phase and agrA and saeR (-5.8 and -5.2 fold change, respectively) during maturation. Principal component analysis with 24 measured parameters confirmed that 4EB treatment primarily affected multiplication and maturation phases.