Transcriptional signatures associated with the survival of Escherichia coli biofilm 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 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 Escherichia coli biofilms exposed to sub-lethal PAW treatment using bulk RNA sequencing and transcriptomics. We compared gene expression in PAW-treated E. coli 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 log₂FC) and 186 downregulated genes (<-1.5 log₂FC) 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 E. coli mutants lacking key genes from these pathways. Knockout mutants of thioredoxin (trxC), thiosulfate-binding proteins (cysP), and NADH dehydrogenase subunit (nuoM) showed significantly reduced biofilm viability after PAW treatment. Notably, ΔtrxC 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 E. coli biofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.

Keywords: Biofilm; Escherichia coli; Plasma-activated water; Steel surfaces; Transcriptomics.

Fig. 1

Schematic representation of the reactor utilised to generate PAW and Bubble water used to treat 48 h E. coli biofilms grown on stainless-steel coupons. (A) A BSD reactor was used to generate PAW with oxygen as the gas source at 1 slm. PAW was generated with and without 20 mM Tiron scavenger to assess the impact of superoxide anions on biofilm cell stress responses. (B) Bubble water controls with and without 20 mM Tiron scavenger was generated by simply passing oxygen through the water at 1 slm without plasma discharge. In both instances, biofilms grown on stainless-steel coupons were treated in situ for 2 min.

Fig. 2

Two min in situ PAW treatment reduces E. coli biofilm viability by ~ 10-fold when compared to PAW-Tiron and controls (Bubble and Bubble-Tiron). Data represents mean ± standard error of the mean, ∗∗∗∗ (P ≤ 0.0001); non-significant (P > 0.05); n = 3 biological replicates, with 2 technical replicates each.

Fig. 3

Differential gene expression analysis of E. coli biofilms during 2 min in situ treatment with PAW, PAW-Tiron, Bubble and Bubble-Tiron. (A) MDS plot depicting distances between transcript expression profiles of E. coli biofilms treated with PAW (Cyan) compared to PAW-Tiron (Violet) and controls Bubble (Red) and Bubble-Tiron (Green). (B–F) The volcano plot represents the differentially expressed transcripts between PAW vs Bubble (B), PAW vs Bubble-Tiron (C), PAW-Tiron vs Bubble (D), PAW-Tiron vs Bubble-Tiron (E) and PAW vs PAW-Tiron (F) groups as depicted in the title. The log-fold change (base 2) is plotted on the x-axis and the negative log of P-value (base 10) is plotted on the y-axis. Data shows individual Log FC changes in expression between pooled E. coli biofilms (4 biological replicates, each comprising 4 pooled technical replicates). Up- and down-regulated genes are represented by violet and pink circles, respectively, while non-significant genes are represented by grey circles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Pathway and GO enrichment analysis of the upregulated differentially expressed genes. (A) KEGG pathway analysis of the upregulated DEGs in PAW treatment compared to the control conditions. The size of each dot is proportional to the number of upregulated DEGs for the given pathway in the reference list. Only pathways with rich factor more than 1 are shown for simplicity. (B) The top GO terms annotated in the biological process category for the upregulated genes are selected based on their fold-enrichment values. GO terms with fold-enrichment value more than 1 are shown only for simplicity. Fold-enrichment values were calculated from the number of genes observed in the upregulated DEGs list divided by the expected number in the reference list for a particular GO term. The size of each dot is proportional to the number of upregulated DEGs for the given GO term in the reference list. Colour bar representing false discovery rate. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Two min PAW treatment significantly impacts E. coli biofilm viability and causes intracellular ROS accumulation. (A) Effect on biofilm viability of PAW (black), PAW-Tiron (light grey), Bubble (white), and Bubble-Tiron (dark grey) treatments on E. coli biofilm Keio WT and gene knock out mutants ΔtrxC, ΔnuoM and ΔcysP was investigated. (B) DCFDA staining was used to detect and measure intracellular biofilm ROS accumulation whilst RNS were stained with (C) DAF-FM. Data represents mean ± SEM, ∗ (P ≤ 0.05); ∗∗∗ (P ≤ 0.001); ns (P > 0.05); n = 3 biological replicates, with 2 technical replicates each.