Transcriptional signatures associated with the survival of biofilm during treatment with plasma-activated water.

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 reactive oxygen and nitrogen species (RONS), particularly superoxide. However, the exact genetic mechanisms behind PAW's effectiveness against biofilms remain unclear. Here, we examined the stress responses of biofilms exposed to sub-lethal PAW treatment using bulk RNA sequencing and transcriptomics. We compared gene expression in PAW-treated biofilms with and without superoxide removal, achieved by adding the scavenger Tiron. Biofilms treated with PAW exhibited a 40 % variation in gene expression compared to those treated with PAW-Tiron and controls. Specifically, PAW treatment resulted in 478 upregulated genes (>1.5 logFC) and 186 downregulated genes (<-1.5 logFC) compared to the control. Pathway and biological process enrichment analysis revealed significant upregulation of genes involved in sulfur metabolism, ATP-binding transporter, amino acid metabolism, hypochlorite response systems and oxidative phosphorylation in PAW-treated biofilms compared to control. Biofilm viability and intracellular RONS accumulation were tested for mutants lacking key genes from these pathways. Knockout mutants of thioredoxin (), thiosulfate-binding proteins (), and NADH dehydrogenase subunit () showed significantly reduced biofilm viability after PAW treatment. Notably, biofilms had the highest intracellular ROS accumulation, as revealed by 2',7'-dichlorofluorescin diacetate staining after PAW treatment. This confirms the importance of these genes in managing oxidative stress caused by PAW and highlights the significance of superoxide in PAW's bactericidal effects. Overall, our findings shed light on the specific genes and pathways that help biofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.