Ferulic Acid Inactivates Shigella flexneri through Cell Membrane Destruction, Biofilm Retardation, and Altered Gene Expression

Abstract

Antibiotic resistance and capacity for biofilm formation of Shigella flexneri render previous prevention and control strategies minimally effective. Ferulic acid (FA) has been demonstrated to be useful due to its application in foods as an alternative natural preservative. However, information regarding the S. flexneri phenotype and molecular responses to FA exposure is limited. The present study investigated the effects of FA on S. flexneri planktonic growth and biofilm formation. The results demonstrated that the cell membrane of S. flexneri in planktonic growth mode exhibited irreversible destruction after FA exposure, as characterized by decreased cell viability, leakage of cytoplasmic constituents, accelerated adenosine triphosphate (ATP) consumption, cell membrane depolarization, and cellular morphological changes. FA significantly inhibited S. flexneri adhesion and biofilm formation at a working concentration (1/8 MIC) that almost did not inhibit planktonic growth. Transcriptomics profiling showed that the exposure to a subinhibitory concentration of FA dramatically altered gene expression in the S. flexneri biofilm, as a total of 169 differentially expressed genes (DEGs) were upregulated and 533 DEGs were downregulated, compared to the intact biofilm. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in pathways of ribosomes, ABC transporters, and the citrate cycle. Furthermore, we show that FA altered the transcription of S. flexneri genes associated with adhesion, transcriptional regulation, and the synthesis and transport of extracellular polymeric substances that contribute to biofilm formation. These data provide novel insights into S. flexneri behavioral responses to FA exposure and suggest that FA could effectively constrain S. flexneri and its biofilm formation.

Juglone Inactivates Pseudomonas aeruginosa through Cell Membrane Damage, Biofilm Blockage, and Inhibition of Gene Expression

“…The bacterial viability test was similar to the previous study by Kang et al [ 16 ]. The bacterial suspension (approximately 10 7 CFU/mL) was centrifuged, the supernatant was discarded, and the precipitated cells were resuspended with sterile PBS.…”

“…Then, the bacterial suspension (approximately 10 7 CFU/mL) was inoculated into the test tubes of each experiment and incubated on a shaker at 37 • C. The test tube was taken out every 2 h for 24 h, and the OD (wavelength of 600 nm) value of bacteria was measured using a spectrophotometer (1530, Thermo Fisher Scientific Oy, FSP GROUP INC, Wuhan, China). The bacterial viability test was similar to the previous study by Kang et al [16]. The bacterial suspension (approximately 10 7 CFU/mL) was centrifuged, the supernatant was discarded, and the precipitated cells were resuspended with sterile PBS.…”

“…Another possible explanation is that juglone blocks protein synthesis in P . aeruginosa and even inhibits the synthesis of related enzymes and DNA expression [ 16 ]. Subsequently, the changes in intracellular nucleic acid content were further evaluated.…”

“…One possible explanation is that juglone may destroy the integrity and permeability of the cell membrane, leading to the leakage of a large number of contents of the cell. Another possible explanation is that juglone blocks protein synthesis in P. aeruginosa and even inhibits the synthesis of related enzymes and DNA expression [16]. Subsequently, the changes in intracellular nucleic acid content were further evaluated.…”

“…After incubation for 48 h, the planktonic cells were cleaned and resuspended with PBS, before being mechanically dispersed to destroy the biofilm. The plate counting method was used to quantitatively analyze the biofilm [ 16 ].…”