Fig 4.

Transcriptional profiling of S. aureus reveals changes in biofilm formation after exposure to D. incerta. (A) Transwell coculture experimental design. Four wells per condition were extracted for RNA-seq analysis. (B) Bacterial density of D. incerta and S. aureus SA113 after 24 hours of incubation (OD₆₀₀ of bacterial cultures before RNA extraction). (C) Bacterial density of D. incerta and USA300 MRSA after 24 hours of incubation (OD₆₀₀ of bacteria before RNA extraction). (D) Volcano plot of genes in USA300 MRSA that were differentially expressed during D. incerta coculture compared to monoculture. Shaded areas highlight genes whose adjusted P < 0.05 and fold change >2.0 (blue, downregulated; red, upregulated). Bold points mark genes of interest, which were found within operons containing multiple differentially expressed genes. (E) Heatmap depicting differentially expressed genes in USA300 MRSA with adjusted P < 0.05 and fold change >2.0. For visualization purposes, genes were ordered by hierarchical clustering based on Pearson correlation. Values were scaled to the row mean. (F) USA300 MRSA cell density in response to acidified media (pH = 5.0, yellow) compared to neutral pH media (red) and coculture with D. incerta. Four wells per condition were used. (G) USA300 MRSA biofilm formation measured via crystal violet stain retention (OD₅₇₀) normalized to cell growth (OD₆₀₀). Two replicates of three wells per condition were used. t-test, ^*P < 0.05.