Transcriptional Signatures Associated with the Survival ofEscherichia coliBiofilm During Treatment with Plasma-Activated Water

Abstract

Biofilm formation on surfaces, tools and equipment can damage their quality and lead to high repair or replacement costs. Plasma-activated water (PAW), a new technology, has shown promise in killing biofilm and non-biofilm bacteria due to its mix of reactive oxygen and nitrogen species (RONS), and in particular superoxide. However, the specific genetic mechanisms behind PAWs effectiveness, especially against biofilms, are not yet fully understood. Here, we examined the stress responses ofEscherichia colibiofilms when exposed to sub-lethal PAW treatment with and without superoxide (by adding the scavenger Tiron to remove it). A 40 percent variation in gene expression was observed for PAW treated biofilms when compared to PAW-Tiron and controls. Specifically, PAW treatment resulted in 478 upregulated genes (> 1.5 log2FC) and 186 downregulated genes (< -1.5 log2FC) compared to the control. Pathway enrichment and biological process enrichment analysis revealed significant upregulation of sulfur metabolism, ATP-binding cassette transporter genes, amino acid metabolic/biosynthesis pathways, hypochlorite response systems and oxidative phosphorylation for biofilms treated with PAW compared to control. Knockout mutants of significantly upregulated genes associated with these pathwaystrxC(4.23–fold),cysP(1.58–fold) andnuoM(1.74–fold) were compared to the wild-type (WT) for their biofilm viability and intracellular RONS accumulation. Relative to PAW-treated WT, ΔtrxCand ΔnuoMknockout mutants displayed significantly reduced biofilm viability confirming their role in PAW-mediated response. Interestingly, ΔtrxCbiofilms had the highest intracellular ROS accumulation, as revealed by DCFDA staining after PAW treatment. This study gives a detailed insight into howE. colibiofilms respond to oxidative stress induced by PAW. It highlights the significance of superoxide in PAW's bactericidal effects. Overall, our findings shed light on the specific genes and pathways that helpE. colibiofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.