3.2 E-PL damages biofilm structure
To further investigate the influence of E-PL, P. aeruginosa biofilm was pre-cultured with E-PL for 24 h (Fig. 3). The structure and biomass of biofilm were prospected by SEM. The untreated bacteria secreted more EPS, which tightly wrapped P. aeruginosa, and the aggregation density and thickness increased until a complete, dense, and multi-layered mature biofilm had formed (Fig. 3a). Bacteria in the biofilm were compact and the morphological surface was smooth, with no obvious depressions on the cell surface. In contrast, when P. aeruginosa was stimulated with E-PL, the biofilm surface was incompletely formed and EPS secretion was significantly reduced, limiting cell aggregation, and causing loosening and shedding of the biofilm surface. The number of bacteria was significantly lower, which led to biofilm rupture (Fig. 3b, 3c, and 3d). The morphology and structure of bacteria in the biofilm were deformed or broken, and the surface was depressed. In addition, under the stress of E-PL, the bacterial contents were released, and the cell surface became swollen or crumpled. These findings indicated that E-PL can effectively destroy the membrane structure of P. aeruginosa biofilms likely by disrupting its morphological structure and preventing bacteria from secreting EPS.
3.4 Metabolic activity of biofilm bacteria
The current findings confirmed that E-PL has an important destructive effect on biofilm, likely because biofilm is primarily composed of bacteria and EPS. To further explore the effects of E-PL on biofilm, the metabolic activity of biofilm bacteria was assessed. After 6 h of E-PL treatment, the OD values in the 18.75 and 37.5 µg/mL groups were significantly reduced from 0.32 to 0.205 (18.75 µg/mL) and 0.104 (37. 5µg/mL) compared with the control group (OD = 0.2) (Fig. 5). After 24 h of treatment, the control group had an OD value of 0.838, while the experimental groups had values of 0. 635 and 0.369 following 18.75 and 37.5 µg/mL treatment, respectively (Fig. 5A). Similar results have been shown in response to other bacteriostatic substances, including quercetin and chitosan, which also reduce the number of biofilm bacteria [17, 30]. These findings indicate that E-PL effectively inhibited the metabolic activity of bacteria during biofilm formation.
The effects of E-PL on the metabolic activity of bacteria in mature biofilms were also assessed. After treating the mature biofilm with 300 and 600, the metabolic activity of biofilm bacteria was significantly decreased. While the OD value of the control was 3.853, the OD values of the 300 and 600 µg/mL groups were 0.81 and 0.334, respectively (Fig. 5B). Thus, E-PL can effectively inhibit the metabolic activity of bacteria in biofilm and reduce bacterial cell adhesion to the matrix. Other bacteriostatic agents, nisin and the antimicrobial peptide, SAAP-148, have shown a similar capacity to destroy mature biofilms by killing the associated bacteria [31, 32].
3.5 Motility of biofilm bacteria
Biofilm formation is often related to the movement of bacteria. Under complex conditions, the motility of bacteria is necessary for their survival and pathogenicity [26, 27]. Flagellate-mediated motility and adhesion play an important role in microbial pathogenicity. Bacterial swimming and colony movement allow planktonic cells to attach to surfaces. As a result, the current study also assessed bacterial motility in E-PL-treated biofilms. Bacteria in the control group expanded from the inoculation point to the outer edge to form a large ring colony ring, indicating that P. aeruginosa was motile (Fig. 6). However, in the treatment groups, the size of the colony ring was dependent on E-PL concentration, and bacteria growth, reproduction, and motor diffusion were completely inhibited at 600 µg/mL. These findings indicated that E-PL successfully prevented bacterial adhesion and movement. Compared to the control group, 150 and 300 µg/mL E-PL also significantly inhibited P. aeruginosa locomotion and swarm movement while 600 µg/mL E-PL completely prevented swarm movement (Fig. 6). In addition, after staining, no obvious crystal violet attachment points were observed in the control group. Meanwhile, at 600 µg/mL E-PL, the plates were clean and smooth, and no trace of crystal violet was observed, indicating that E-PL effectively inhibited the puncture movement of bacteria in the medium. These findings indicate that E-PL can effectively inhibit the movement of bacteria in biofilm. Silver nitrate is reported to have a similar effect on biofilms [33].
3.6 Biofilm bacteria gene expression
To explore the effect of E-PL on biofilm bacterial gene expression, qPCR was used to detect plsA, rhlI, antB, katB, phzS, phzH, and phzM. E-PL treatment effectively downregulated plsA (0.05 fold), rhlI (0.62 fold), phzS (0.2 fold), phzH (0.43 fold), and phzM (0.5 fold) (Fig. 7). These results indicated that E-PL restricted the expression of genes involved in biofilm synthesis, thereby hindering biofilm formation. The relative expression of antB (1.26 fold) and katB (2.35 fold) was sharply up-regulated, indicating that E-PL has a regulatory effect on biofilm. Since extracellular polysaccharides are a major component of biofilms [34], inhibition of the pslA gene is likely to prevent biofilm synthesis. As shown previously, the current study also revealed that genes involved in transcriptional regulation and phenazine synthesis affect biofilm synthesis [33, 35].
3.7 Transcriptome analysis
Transcriptomics is commonly used to analyze gene expression at the transcriptional level. Biofilm formation is a co-regulatory process coordinated by complex genetic networks. The current study used transcriptomics (RNA-Seq sequencing technology) to assess how E-PL impacted the bacterial gene expression profiles of mature P. aeruginosa biofilm. The results showed that differentially expressed genes (DEGs) were determined (Fig. 8A). The impact of E-PL on the gene expression profile of mature P. aeruginosa biofilm was further evaluated by identifying the GO and KEGG pathways of the DEGs (Fig. 8B). The GO map displayed that most of DEGs were functionally enriched in Biological Process (BP), Cellular Components (CC) and Molecular Function (MF). The BPs were concentrated in “small molecule metabolic processes”, “transmembrane transport,” and “organic acid metabolic processes.” Meanwhile, DEGs involved in Cellular Components (CC) were mainly concentrated in “intracellular,” “intracellular parts,” and “protein-containing complexes”, and MF was primarily concentrated in “small molecule binding,” “anion binding,” and “nucleotide binding”. The down-regulated DEGs were most enriched in membrane-related components, including “membrane composition,” “intrinsic components of the membrane,” and “overall composition of the membrane.” There was also a high number of genes involved in the “REDOX process” and “transport activity.” While “flavin adenine dinucleotide” activity was only slightly enriched, with a small number of involved genes, E-PL was shown to impact its activity on the transcriptional level.
Analysis of the KEGG database was also used to identify genes that were enriched and differentially expressed in P. aeruginosa biofilm. DEGs were enriched in 20 KEGG pathways that were primarily involved in “microbial metabolism in different environments,” “ABC transport,” “amino acid biosynthesis,” and “ribosome and bacterial secretion” (Fig. 8C). Meanwhile, the downregulated genes were mainly involved in “ABC transport,” “degradation of valine, leucine, and isoleucine,” “quorum sensing,” “the bacterial secretion system for degrading aromatic compounds,” “the bacterial secretion system,” and “microbial metabolism in different environments” (Fig. 8D). “Phenazine synthesis" was also enriched in the KEGG pathway. These KEGG and GO displayed different functional gene changes, suggesting that E-PL may use several mechanisms to prevent biofilm formation.
The bacterial flagellum is a locomotive organelle that allows many bacterial species to swim or aggregate on liquid and solid surfaces [36, 37]. Bacteria typically rely on a complex sensory system to respond to environmental stimuli using the chemotactic signaling protein, to regulate the direction of flagellar movement [38]. Flagella are chemotactic, mediating the movement of bacteria toward favorable environments and away from unfavorable ones. Flagellate-mediated motility and adhesion correlate closely with the formation and drug resistance of bacterial biofilms. In the E-PL-treated biofilms in the present study, flagella-related genes, including FlgE, cupA5, cupB4, pscL, and related genes, were downregulated (Fig. 9A). Thus, downregulating flagella-associated genes and chemotactic transduction reduces fimbriae and prevents bacteria from binding to surfaces or tissues, potentially inhibiting biofilm formation. Fimbriae formation is shown to correlate with P. aeruginosa virulence and downregulation of the felt protein, pilW, which is involved in felt formation and movement. The current study also showed the downregulated expression of genes involved in chemotactic transduction, including wspE, PA1251, PA4915, and PA2788, following E-PL treatment. In summary, E-PL induced the downregulation of flagellin, fibrinogen, and chemotaxis-related proteins on biofilm to prevent bacterial movement and adhesion, processes required for the bacterial life stage shift from the planktonic (mobile) to the biofilm (fixed) phase.
Flagellate-mediated movement is necessary to initiate bacterial attachment to surfaces to form biofilms [39]. The transition from motility to biofilm formation may involve flagellar gene transcription is suppressed, and flagella are likely to be diluted to extinction by growth in the absence of neogenesis [40]. Cells are shown to adapt to the biofilm state by reducing metabolism, suggesting that metabolism-related genes may contribute to biofilm formation. Indeed, flagellar synthesis genes were inhibited in E-PL-treated biofilms, suggesting that these genes likely hinder flagellar growth and formation.
Transcriptomic analysis showed significant downregulation of genes involved in biofilm synthesis. Quorum sensing (QS), an intercellular communication system that effectively controls and regulates gene expression, directly impacts the release of virulence factors and biofilm synthesis during infection [41]. Three QS systems (las, rhl, and pqs) regulate P. aeruginosa virulence factor gene expression. The current study found that some key genes involved in the P. aeruginosa QS pathway, including rhlI/rhlR, PA1760, and PA0268 in the rhl system, were down-regulated in E-PL-treated biofilm (Fig. 9B). Genes involved in Psl polysaccharide biosynthesis and alginate biosynthesis, including pelC, pelE, pelK, pelF, pelD and pelM, were also downregulated. These findings indicated that E-PL affects the synthesis of polysaccharides in extracellular polymers, thus hindering P. aeruginosa biofilm formation.
Bacterial polysaccharides and eDNA coordinate to form the main structure of bacterial biofilm [37] or serve as signals to promote biofilm formation [43]. Polysaccharide is the main component of the extracellular polymer matrix in mature biofilm. Psl acts as a signal to promote the formation of P. aeruginosa biofilm, and enhances the secretion of the extracellular polymer matrix. Pyocyanin is one of the secondary metabolites of P. aeruginosa, the synthesis of which is achieved through a complex cascade involving multiple genes, including phzABCDEFG and phzHMS [44]. This blue-green pigment causes oxidative stress in the host and disrupts host catalase and mitochondrial electron transfer. After E-PL treatment in this study, most of the key genes involved in phenazine biosynthesis, including phzM, phzH, phzB1, and phzS, were downregulated [45]. The inhibition of these genes resulted in decreased levels of PQS and pyocyanin. Genes related to flavin electron transfer, including PA3492, PA3493, PA2097, fadH2, and morB, which are involved in extracellular electron transfer, were also downregulated. These results further revealed that E-PL affected both P. aeruginosa biofilm synthesis and transcriptional regulation.
In summary, this study identified that E-PL may inhibit biofilm formation at the molecular level by preventing flagellar gene transcription during biofilm formation and thereby destroying flagellate-mediated bacterial movement and surface attachment. The release of P. aeruginosa virulence factors during infection and polysaccharides synthesized in the extracellular polymer during biofilm formation may also interact with eDNA to form the main skeleton of bacterial biofilms or promote biofilm formation. In addition, pyocyanin, one of the secondary metabolites of P. aeruginosa, causes oxidative stress in the host, disrupting host catalase and mitochondrial electron transport. These findings suggest that E-PL affects biofilm formation by influencing bacterial activity and metabolic gene expression.