The effect of antibacterial peptide ε-Polylysine against Pseudomonas aeruginosa biofilm in marine environment

The effect of antibacterial peptide ε-Polylysine against Pseudomonas aeruginosa biofilm in marine environment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The effect of antibacterial peptide ε-Polylysine against Pseudomonas aeruginosa biofilm in marine environment Quantong Jiang, Siwei Wu, Dongzhu Lu, Xiaofan Zhai, Jizhou Duan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4276320/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2024 Read the published version in npj Materials Degradation → Version 1 posted 9 You are reading this latest preprint version Abstract Natural agents with antimicrobial properties have a broad potential to resist biofilm adhesion in marine environments. ε-Polylysine (E-PL) is a natural cationic, homomeric polymer with 25–30 lysine residues that can resist microbial biofilm adhesion due to its stability, nontoxicity, and biodegradability. The current study investigated the action of E-PL against Pseudomonas aeruginosa biofilm isolated from a marine environment. Crystal violet staining was used to examine the effects of E-PL on the formation and destruction of mature biofilms. Scanning Electron and fluorescence microscopy revealed that E-PL treatment damaged the biofilm structure and affected the secretion of extracellular polymers. The CCK8 colorimetric assay showed that E-PL also decreased the metabolic activity and motility of biofilm bacteria. QPCR and transcriptome analysis revealed that E-PL affected biofilm formation and transcriptional regulation by downregulating genes involved in flagellar synthesis (flgE, PA4651, pilW), chemotaxis transduction (PA1251, PA4951, PA4788), biofilm biosynthesis (pelC, pelD, pslK, plsM), transcriptional regulation (PA3973, PA3508, PA0268), phenazine biosynthesis (phzM, phzH, phzS), and electron transfer (PA5401, PA5400, PA3492). This study used multiple methods to identify the mechanism of E-PL action against biofilm, informing the design of novel biofilm treatment methods. Pseudomonas aeruginosa ε-Polylysine antibacterial peptide biofilm Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Pseudomonas aeruginosa ( P. aeruginosa ), a gram-negative bacteria with high environmental adaptability and fast growth and reproduction [ 1 ]. P. aeruginosa can thrive in a variety of aquatic and terrestrial environments, including lakes, rivers, soils [ 2 ]. P. aeruginosa is also a conditional pathogen, causing wound infection by attaching to medical equipment [ 3 ]. In general, bacteria have two life stages, unicellular (planktonic) and multicellular (aggregation into colonies that facilitate attachment to surfaces). Under the right growth conditions, aggregated bacterial communities can form biofilms [ 4 ]. Biofilms are composed of microorganisms that attach to the surfaces solids and wrap themselves in the extracellular matrix to form a membrane [ 5 ]. In biofilm, these microorganisms secrete extracellular polymers (EPS) such as proteins, polysaccharides, and nucleic acids [ 6 – 8 ]. EPS keep microbes embedded in the extracellular matrix, providing important nutrients and resistance to external stimuli [ 9 ]. Thus, biofilms exist in a relatively stable and durable state that can be difficult to eradicate [ 3 ]. In extreme cases, biofilm bacteria can tolerate up to 1,000 times higher antibiotic concentrations than free-living bacteria. Biofilm bacteria can also resist adverse external conditions, such as humidity and temperature, and various structures within biofilms can provide additional resistance to antibiotics [ 2 , 10 ]. Strategies aimed at inhibiting biofilms are assessed based on their ability to prevent, destroy, weaken, or kill the microbial community within the biofilm. Several methods, including antibiotics, antimicrobial peptides, and catalytic antibacterial robots, have been developed [ 11 – 13 ]. In clinical settings, biofilms can be difficult to treat with antibiotics alone, promoting the development of chronic diseases and increasing morbidity and mortality rates [ 2 , 14 , 15 ]. In marine environments, the attachment of P. aeruginosa can greatly shorten the service life of large-scale marine facilities. Thus, there is an urgent need to identify novel natural agents with antibacterial and anti-biofilm activity. The use of traditional antibiotics, which remain the most common antimicrobial method, can promote the development of antibiotic resistance. In addition, as environmental protection and safety awareness have increased, peptides with natural antibacterial functions have attracted more research attention. New strategies that disrupt or eradicate biofilm structure are being developed [ 16 , 17 ]. ε-Polylysine (E-PL) is a natural cationic polypeptide consisting of 25–30 L-lysines connected by an isopeptide bond of ε-amino and α-carboxyl groups [ 18 , 19 ]. E-PL is a natural homologous polyamide with a molecular weight of 0.8–2.0 kDa that is safe, non-toxic, stable, and protective to the environment [ 19 , 20 ]. This polypeptide is a natural preservative of antimicrobial peptides, with broad-spectrum and high-intensity antibacterial properties [ 21 – 23 ]. Antimicrobial peptides can bind to the surface of bacterial membranes and alter their structure and permeability by inserting the hydrophobic end of the molecule into the lipid membrane [ 24 , 25 ]. To date, few studies have assessed the inhibitory effect of E-PL on P. aeruginosa biofilm. The current study used crystal violet staining to explore the inhibitory mechanism of E-PL on P. aeruginosa biofilm and used the CCK8 assay to assess the metabolic activity of bacteria in the biofilm. The biofilm structure was observed using scanning electron and fluorescence microscopy and the effect of E-PL on biofilm gene expression was investigated at the molecular level by fluorescence quantification and transcriptomics. The findings provide theoretical guidance for the future removal of P. aeruginosa biofilm in marine environments. 2. Materials and methods 2.1 Bacterial and regents Pseudomonas aeruginosa (MCCC1A10045) was obtained from the Marine Culture Collection of China (Xiamen, China). Crystal violet (1%) was purchased from Solarbio Life Services (Beijing, China). Glutaraldehyde Fixation Solution (2.5%) was purchased from Absin Biotechnology Co., Ltd (Shanghai, China). 2.2 Crystal violet tested biofilm formation Crystal violet staining was used to assess biofilm formation as described previously [ 17 ]. The P. aeruginosa concentration was 1 × 10 6 CFU/mL in 96-well plates with different concentrations of E-PL (0, 1.18, 2.35, 4.69, 9.38, 18.75, 37.5, 75, 150, 300, and 600 µg/mL) incubated at 30℃, 60 rpm for 24 h. After 24 h, the wells were washed twice with 0.2 mL PBS and fixed with 2.5% glutaraldehyde for 15 min. Crystal violet solution (0.1 mL) was added to each well to allow staining for 5–10 min, then washed with 0.2 mL PBS. The biofilm was dissolved with 0.1 mL 95% ethanol for 30 min and light absorption was measured at OD 595 nm. 2.3 Assessment of biofilm metabolic activity Different concentrations of E-PL were placed in the bacterial suspension 1 × 10 6 CFU/mL, the final E-PL concentration was 0, 75 µg/mL, and 18.75 µg/mL, respectively. These mixtures were added to the 96-well plate at 37℃ for 6 h and 24 h. The cells were cleaned with 1 × PBS buffer three times. The plate was incubated at 37℃ for 1 h using the CCK8 kit methodology (Beyond, Beijing), and the results was measured at OD450 nm. To determine the impact of E-PL on the metabolic activity of mature biofilm, P. aeruginosa was cultured in 96-well plates for 24 h to form mature biofilm. The followed process as above section described. Different from above, E-PL (0, 600 µg/mL, and 1200 µg/mL) was add to 96-well plates for 6 h. Then, the plate was incubated at 37℃ for 1 h. 2.4 Assessment of biofilm structure The bacterial suspension (200 µL; 1 × 10 8 CFU/mL) was added to a 24-well culture plate containing 1.8 mL LB medium at 37 ℃ and 60 rpm for 24 h. The plate was washed twice with PBS, then fresh medium containing E-PL (150, 300, and 600 µg/mL) was added to the 24-well plate, and the plate was incubated at 37℃ for 6 h. The plate was gently rinsed twice with 1 × PBS, and the cell crawling tablets were removed and fixed with 2.5% glutaraldehyde for 30 min. Then, the plates were dehydrated in turn with 30, 50, 60, 70, 80, 90, and 100% anhydrous ethanol for 10–15 min each at room temperature. After the dehydration was complete, the plates were naturally dried, treated with gold spray, and observed by SEM. As described in above section, the control group (0 µg/mL) and the experimental group receiving E-PL treatment (150, 300, and 600 µg/mL) were removed, respectively. The planktonic bacteria were washed with 0.5% NaCl and stained on the dark for 15 min using an MKBio SYTO 9 Live/Dead bacterial double stain kit (Maokang, Shanghai, China). The slides were covered with cover glass and observed under a confocal laser microscope. 2.5 Assessment of bacterial motility Three types of media were prepared: (1) 0.3% agar medium containing 1.0% tryptone, 0.5% NaCl, and 0.3% agar, (2) semi-solid agar medium with 1% tryptone, 0.5% sodium chloride, 0.8% nutrient agar, and 0.5% glucose, and (3) LB medium with a 1.0% agar layer. After the medium was removed, E-PL (0, 150 µg/mL, 300 µg/mL, and 600 µg/mL) was added and solidified at room temperature. The bacteria (1 µL; 1 × 10 6 CFU/mL) were then inoculated into the center of the agar medium and incubated at 30℃ for 24 h. The swimming ability and community dynamics of the bacteria were determined by measuring the circular expansion radius. The bacterial biofilm on the dish was stained with 1% (w/v) crystal violet (Beyond, Beijing). The dye was removed by washing with water and puncture movement was observed. 2.6 Real-time fluorescent quantitative PCR The prepared P. aeruginosa suspensions were pre-cultured with 1 × 10 6 CFU/mL for 24 h, cultured in medium with or without E-PL (300 µg/mL) for 6 h, and the biofilms were collected. Total RNA was extracted from the biofilms using the trizol method [ 26 – 28 ]. QuantStudioTM 1 Plus real-time PCR (Applied Biosystems, USA) was used to detect phzM transcription in E-PL-treated biofilms. Specific amplification primers for all tested genes are listed in Table 1 . The final reaction volume was 20 µL, including 10 µL ArtiCanCEO SYBR qPCR Mix, 10 µM primer F, 1 µL each for the forward and reverse primes, 7 µL ddH 2 O, and 1 µL of the triple diluted cDNA template. The PCR procedure was 95°C 5 min, 40 cycles (95°C 15 s, 60°C 20 s, 72°C 20 s), 65°C 1 min. Rpod was used as a reference gene to calculate the relative expression of each gene. Three wells were used for each sample. The relative expression of target genes was calculated using the 2 −△△Ct method [ 29 ]. Table 1 2.7 Transcriptome analysis To further explore the ability of E-PL to regulate gene expression in P. aeruginosa biofilms, transcriptomic analysis was performed on the control and E-PL (300 µg/mL) treated biofilms. The samples were pre-cultured in LB medium for 24 h, and floating bacteria on the surface of the samples were gently washed with PBS. Samples in the control group (0 µg/mL) and experimental group (300 µg/mL) were treated with E-PL for 6 h, and the total RNA was extracted. Transcriptome sequencing was performed with Novogene (Tianjin, China) using Gene Ontology (GO) and the Kyoto Encyclopedia Gene and Genome (KEGG) analysis of genetic variations ( https://www.bioinformatics.com.cn ). 2.8 Statistical analysis All experiments were performed at least thrice. Statistical analyses were performed using SPSS v.22.0. Single-factor analysis of variance and Tukey’s test were used for statistical analyses. The figures in this study were generated using Origin 2018. 3. Results and Discussion 3.1 E-PL impairs biofilm formation The current study assessed P. aeruginosa biofilm formation using crystal violet staining [ 17 ]. The number of bacteria increased gradually in the first 2 h and most were independent cells showing little evidence of adhesion and no obvious biofilm (Fig. S1 a). Mutual aggregation began after 4 h and biofilm biomass increased significantly at longer culture times, with a sharp rise at 10–12 h (Fig. S1 f and S1g). The biofilm also decreased with an increase in E-PL concentration. At 300 µg/mL, E-PL completely prevented the formation of P. aeruginosa biofilm (Fig. 1 ). These findings indicated that E-PL inhibits biofilm biomass formation in a concentration-dependent manner. E-PL was also found to destroy mature and fully-formed biofilm in a concentration-dependent manner (Fig. 2 ). This finding indicated that E-PL inhibited the multicellular stage of P. aeruginosa . 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. 3 a). 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. 3 b, 3 c, and 3 d). 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. 3.3 Additional effects of E-PL on biofilm structure To probe the effect of E-PL on biofilm bacteria, the microbial population was labeled with SYTO9 and PI bicolor dyes. SYTO9 labels both intact and damaged cell membranes with green fluorescence. In contrast, PI only passes through damaged cell membranes. Upon entering the cell, PI causes the SYTO9 green fluorescence to decrease. When both dyes are present in bacteria, cells with an intact membrane structure fluoresce green, while those with a damaged membrane structure fluoresce red. Bacteria treated with the 150, 300, and 600 µg/mL concentrations had independent and single red fluorescence, while the control group had green or yellow-green fluorescence (Fig. 4 ). In the control group, obvious biofilm and bacteria in the membrane emitted green fluorescence (Fig. 4 a). After treatment with E-PL, the bacteria no longer aggreged, no obvious biofilm was formed, and the number of cells was reduced. These findings indicate that E-PL can destroy biofilm bacteria and inhibit bacterial metabolic activity and proliferation by destroying the bacterial cell membrane. 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. 5 A). 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. 5 B). 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. 8 A). 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. 8 B). 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. 8 C). 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. 8 D). “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. 9 A). 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. 9 B). 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. 4. Conclusion This study explored the effect of E-PL on P. aeruginosa biofilm synthesis. E-PL was shown to inhibit and destroy the formation of biofilm in a concentration-dependent manner by 1) inhibiting the secretion of extracellular polymer to damage the biofilm structure, 2) reducing the metabolic activity of the biofilm bacteria, 3) limiting bacterial motility, and 4) regulating the expression of genes involved in synthesizing flagella, chemotactic transduction, extracellular polymeric polysaccharide synthesis, lifetime metabolism, and electron transfer. These findings suggest that the natural and environmentally friendly antimicrobial peptide, E-PL, has great potential for use in biofilm treatment in many industries. Declarations Author contributions Siwei Wu: conceptualization, methodology, writing-original draft. Quantong Jiang: funding acquisition, supervision. Dongzhu Lu: manuscript review & editing. Xiaofan Zhai: experimental design. Jizhou Duan: experimental design. Baorong Hou: supervision. All authors contributed to the article and approved the submitted version. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements The research was supported by Hainan Province Science and Technology Special Fund (ZDYF2021GXJS210), Wenhai Program of the S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (2021WHZZB2301), Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City (2021CXLH0005), Overseas Science and Education Centers of Bureau of International Cooperation Chinese Academy of Sciences (121311KYSB20210005) for providing support. Data Availability All data included in this study are available upon request by contact with the corresponding author. References J.B. Lyczak, C.L. Cannon, G.B. Pier, Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist, Microbes and Infect. 2(9) (2000) 1051-1060. P.K. Taylor, A.T.Y. Yeung, R.E.W. 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Alnabelseya, M.J. Pestrak, M.J. Lee, L.K. Jennings, J. Tam, R.A. Melnyk, M.R. Parsek, D.C. Sheppard, D.J. Wozniak, P.L. Howell, Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms, Sci. Adv. 2(5) (2016). Y. Lekbach, Y. Dong, Z. Li, D. Xu, S. El Abed, Y. Yi, L. Li, S.I. Koraichi, T. Su, F. Wang, Catechin hydrate as an eco-friendly biocorrosion inhibitor for 304L stainless steel with dual-action antibacterial properties against Pseudomonas aeruginosa biofilm, Corros Sci. 157 (2019) 98-108. F.F.V. Chevance, K.T. Hughes, Coordinating assembly of a bacterial macromolecular machine, Nat. Rev. Microbiol. 6(6) (2008) 455-465. Y. Chang, B.L. Carroll, J. Liu, Structural basis of bacterial flagellar motor rotation and switching, Trends Microbiol. 29(11) (2021) 1024-1033. J.S. Parkinson, G.L. Hazelbauer, J.J. Falke, Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update, Trends Microbiol. 23(5) (2015) 257-266. X. Lou, Y. Wu, Z. Huang, W. Zhang, X. Xiao, J. Wu, J. Li, Z. Fang, Biofilm formation and associated gene expression changes in Cronobacter from cereal related samples in China, Food Microbiol. 118 (2024). S.B. Guttenplan, D.B. Kearns, Regulation of flagellar motility during biofilm formation, Fems Microbiol Rev. 37(6) (2013) 849-871. K. Tang, X.-H. Zhang, Quorum quenching agents: resources for antivirulence therapy, Mar Drugs. 12(6) (2014) 3245-3282. W.C. Fuqua, S.C. Winans, E.P. Greenberg, Quorum sensing in bacteria- the luxr-luxi family of cell density-responsive transcriptional regulators, J Bacteriol. 176(2) (1994) 269-275. W. Hu, L. Li, S. Sharma, J. Wang, I. McHardy, R. Lux, Z. Yang, X. He, J.K. Gimzewski, Y. Li, W. Shi, DNA builds and strengthens the extracellular matrix in Myxococcus xanthus biofilms by interacting with exopolysaccharides, Plos One. 7(12) (2012). D.V. Mavrodi, R.F. Bonsall, S.M. Delaney, M.J. Soule, G. Phillips, L.S. Thomashow, Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1, J Bacteriol. 183(21) (2001) 6454-6465. G.W. Lau, D.J. Hassett, H.M. Ran, F.S. Kong, The role of pyocyanin in Pseudomonas aeruginosa infection, Trends Mol Med. 10(12) (2004) 599-606. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations (Not answered) Supplementary Files Suplementmaterials.docx Table.docx Cite Share Download PDF Status: Published Journal Publication published 29 Nov, 2024 Read the published version in npj Materials Degradation → Version 1 posted Editorial decision: revise 19 Jun, 2024 Review # 2 received at journal 17 Jun, 2024 Reviewer # 2 agreed at journal 20 May, 2024 Review # 1 received at journal 29 Apr, 2024 Reviewer # 1 agreed at journal 25 Apr, 2024 Reviewers invited by journal 22 Apr, 2024 Editor assigned by journal 19 Apr, 2024 Submission checks completed at journal 19 Apr, 2024 First submitted to journal 16 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4276320","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":293905708,"identity":"14be46aa-ec3d-424b-b7e4-84aa3c7ea362","order_by":0,"name":"Quantong Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACxmYUbgUDYxvxWthAxBkitCAASAtQPWMDIYXM7czPHn4pOyxvLt987OHXedayfewNjB9+MNjl4XYYm7mxzLnDhjvb2NKNZbelG7fxHGCW7GFILsbjFzNpybbDjBuO8QAZ2w4ntkkkMEgzMBxIxOVCxmb2byAt9hAtc8BamH/j18JjJvmx7XAiSIvkxwawFjYCtvCUSTOcS0/ecCwtTZrhGMgvB9ssewyScWox7D++TfJHmbXthsOHj0n+qLGWnd/efPjGjwo73FqAEsw8bBAOMw8DM8hmoJgBDvVAIA9S8gOqhfEHWMsoGAWjYBSMAlQAABmCVQvfjO+IAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5513-1049","institution":"Institute of Oceanology, Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Quantong","middleName":"","lastName":"Jiang","suffix":""},{"id":293905709,"identity":"8c87b210-9df1-46c2-be17-17096baa03d4","order_by":1,"name":"Siwei Wu","email":"","orcid":"","institution":"Laoshan Laboratory, Qingdao, China","correspondingAuthor":false,"prefix":"","firstName":"Siwei","middleName":"","lastName":"Wu","suffix":""},{"id":293905710,"identity":"8f305638-d26c-4abf-9d08-788ba9d5adee","order_by":2,"name":"Dongzhu Lu","email":"","orcid":"https://orcid.org/0000-0003-0194-2107","institution":"CAS Key Laboratory of Marine Environmental Corrosion and Bio-fouling, Institute of Oceanology, Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Dongzhu","middleName":"","lastName":"Lu","suffix":""},{"id":293905711,"identity":"67afb637-8757-4faf-99bb-a75149bfbc23","order_by":3,"name":"Xiaofan Zhai","email":"","orcid":"https://orcid.org/0000-0002-4786-1946","institution":"Institute of Oceanology, Chinese Acadamy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaofan","middleName":"","lastName":"Zhai","suffix":""},{"id":293905712,"identity":"ac8aa739-24e8-4348-9d47-af73e99e5592","order_by":4,"name":"Jizhou Duan","email":"","orcid":"https://orcid.org/0000-0002-7458-4729","institution":"Institute of Oceanology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jizhou","middleName":"","lastName":"Duan","suffix":""},{"id":293905713,"identity":"130de641-ec6c-4d16-89cf-4c77a5fa9025","order_by":5,"name":"Baorong Hou","email":"","orcid":"","institution":"Institute of Oceanology, Chinese Acadamy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Baorong","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2024-04-16 13:26:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4276320/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4276320/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41529-024-00539-6","type":"published","date":"2024-11-29T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55258124,"identity":"a393acb6-f9ad-44d4-b0b4-a39c4334f0c4","added_by":"auto","created_at":"2024-04-24 21:02:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4072645,"visible":true,"origin":"","legend":"\u003cp\u003eE-PL prevented \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm formation. A. The biofilm biomass was tested by crystal violet assay when the biofilms were treated with E-PL. (a) 0 μg/mL, (b) 150 μg/mL, (c) 300 μg/mL, (d) 600 μg/mL. B. The biofilm biomass was tested by crystal violet assay at OD595nm.\u003cem\u003e P. aeruginosa\u003c/em\u003e was cultured for 24 h in different E-PL concentrations of 0, 1.18, 2.35, 4.69, 9.38, 18.75, 37.5, 75, 150, 300, and 600 μg/mL, respectively.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/cb76781eaa0abd6357778851.png"},{"id":55258133,"identity":"29e3588b-7b5a-4168-bc9f-8a9239954aab","added_by":"auto","created_at":"2024-04-24 21:02:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4868261,"visible":true,"origin":"","legend":"\u003cp\u003eE-PL dispersed \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm. A. The biofilm biomass was tested by crystal violet assay when the biofilms were treated with E-PL. (a) 0 μg/mL, (b) 150 μg/mL, (c) 300 μg/mL, (d) 600 μg/mL. B. The biofilm biomass was tested by crystal violet assay at OD595 nm.\u003cem\u003e P. aeruginosa\u003c/em\u003e was cultured in different E-PL concentrations of 0, 1.18, 2.35, 4.69, 9.38, 18.75, 37.5, 75, 150, 300, and 600 μg/mL.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/601409b50899a8fbd6a8142f.png"},{"id":55258132,"identity":"4fcf1a2f-4be9-43af-a3ad-4e4c2f3c7f8a","added_by":"auto","created_at":"2024-04-24 21:02:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2653045,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms. The biofilms with magnifications of 10.00k, (a-d) The biofilms were treated with E-PL 0, 150, 300, 600 μg/mL, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/e8bf2059e67d80b59439eb83.png"},{"id":55258127,"identity":"75babcdc-4ed1-428b-aa5d-59201b08051a","added_by":"auto","created_at":"2024-04-24 21:02:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2515267,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscope images of biofilms when exposed to different E-PL concentrations of 0, 150, 300, 600 μg/mL.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/31d0d505378d0840a7333393.png"},{"id":55258129,"identity":"bf01955f-6e1a-4e1f-b7e3-1c591e085fff","added_by":"auto","created_at":"2024-04-24 21:02:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108240,"visible":true,"origin":"","legend":"\u003cp\u003eThe metabolic activity of bacteriocin E-PL on \u003cem\u003eP. aeruginosa\u003c/em\u003e in biofilm formation (A) and mature biofilm (B).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/5ba9eb72ec429fc9a774a3e3.png"},{"id":55258417,"identity":"f6d8aee8-32b8-4df2-9d68-95382c6a629a","added_by":"auto","created_at":"2024-04-24 21:10:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2049739,"visible":true,"origin":"","legend":"\u003cp\u003eThe motilities of swimming (a), swarming (b), and twitching (c) of \u003cem\u003eP. aeruginosa\u003c/em\u003e in the presence or absence of E-PL. The motilities of swimming (B), swarming (C), and twitching were determined by measuring the radius of bacterial colony. (P \u0026lt; 0.05, a, b, c.)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/ad813666a6cae9f5e31fb41f.png"},{"id":55258134,"identity":"320f0d48-c225-431f-b2cf-f60bfcc8d047","added_by":"auto","created_at":"2024-04-24 21:02:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87711,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of pslA, antB, katB, rhIl, and phzH, phzM, phzS, in \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms were determined by qPCR.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/1df1696b2f49e29bf6c2f90c.png"},{"id":55258131,"identity":"8bd8d71c-4b69-4688-b3d6-76aa409ed49f","added_by":"auto","created_at":"2024-04-24 21:02:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1129255,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and function analysis of the proteins identified by Transcriptome analysis in E-PL treated \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm. (A) Volcano map of differentially expressed genes. (B) GO enrichment cluster analysis of the differential proteins. (C) KEGG pathway of the differential genes. (D) KEGG pathway of the down-regulated differential genes.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/bd84c0e11d8df72b69259b02.png"},{"id":55258130,"identity":"b4f824e8-4a7b-4ad6-8c4b-9ab79b187000","added_by":"auto","created_at":"2024-04-24 21:02:54","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":65444,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of E-PL biofilm of \u003cem\u003eP. aeruginosa\u003c/em\u003e with treated E-PL\u003cem\u003e.\u003c/em\u003e (A) Changes of genes associated with the motility of \u003cem\u003eP. aeruginosa\u003c/em\u003e receiving E-PL treatment. (B) Changes of genes associated with the adhesion of \u003cem\u003eP. aeruginosa\u003c/em\u003e receiving E-PL treatment. (C) Changes of genes associated with the phenazine and electron transfer of \u003cem\u003eP. aeruginosa\u003c/em\u003e receiving E-PL treatment. Red boxes represent increased proteins; gray boxes represent unchanged proteins.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/69ecf71e4d7304cecaac93bf.png"},{"id":70241088,"identity":"7a906a94-ba56-4e4e-951b-613253a25c91","added_by":"auto","created_at":"2024-11-30 08:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27837110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/928ea65e-2ef0-40c4-b2a5-4c0513c0d5c8.pdf"},{"id":55258135,"identity":"cb8ed098-4357-4e4a-af81-0047cf6208f4","added_by":"auto","created_at":"2024-04-24 21:02:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":95816683,"visible":true,"origin":"","legend":"","description":"","filename":"Suplementmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/a373b43feb0eb578fad4e43c.docx"},{"id":55258126,"identity":"94386b8a-2339-4cfe-8d57-33d4b1fe643c","added_by":"auto","created_at":"2024-04-24 21:02:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":41065,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-4276320/v1/546eab3bce3d70d22059cdb3.docx"}],"financialInterests":"(Not answered)","formattedTitle":"The effect of antibacterial peptide ε-Polylysine against Pseudomonas aeruginosa biofilm in marine environment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e), a gram-negative bacteria with high environmental adaptability and fast growth and reproduction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eP. aeruginosa\u003c/em\u003e can thrive in a variety of aquatic and terrestrial environments, including lakes, rivers, soils [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eP. aeruginosa\u003c/em\u003e is also a conditional pathogen, causing wound infection by attaching to medical equipment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In general, bacteria have two life stages, unicellular (planktonic) and multicellular (aggregation into colonies that facilitate attachment to surfaces). Under the right growth conditions, aggregated bacterial communities can form biofilms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilms are composed of microorganisms that attach to the surfaces solids and wrap themselves in the extracellular matrix to form a membrane [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In biofilm, these microorganisms secrete extracellular polymers (EPS) such as proteins, polysaccharides, and nucleic acids [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. EPS keep microbes embedded in the extracellular matrix, providing important nutrients and resistance to external stimuli [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, biofilms exist in a relatively stable and durable state that can be difficult to eradicate [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In extreme cases, biofilm bacteria can tolerate up to 1,000 times higher antibiotic concentrations than free-living bacteria. Biofilm bacteria can also resist adverse external conditions, such as humidity and temperature, and various structures within biofilms can provide additional resistance to antibiotics [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Strategies aimed at inhibiting biofilms are assessed based on their ability to prevent, destroy, weaken, or kill the microbial community within the biofilm. Several methods, including antibiotics, antimicrobial peptides, and catalytic antibacterial robots, have been developed [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In clinical settings, biofilms can be difficult to treat with antibiotics alone, promoting the development of chronic diseases and increasing morbidity and mortality rates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In marine environments, the attachment of \u003cem\u003eP. aeruginosa\u003c/em\u003e can greatly shorten the service life of large-scale marine facilities. Thus, there is an urgent need to identify novel natural agents with antibacterial and anti-biofilm activity. The use of traditional antibiotics, which remain the most common antimicrobial method, can promote the development of antibiotic resistance. In addition, as environmental protection and safety awareness have increased, peptides with natural antibacterial functions have attracted more research attention. New strategies that disrupt or eradicate biofilm structure are being developed [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eε-Polylysine (E-PL) is a natural cationic polypeptide consisting of 25\u0026ndash;30 L-lysines connected by an isopeptide bond of ε-amino and α-carboxyl groups [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. E-PL is a natural homologous polyamide with a molecular weight of 0.8\u0026ndash;2.0 kDa that is safe, non-toxic, stable, and protective to the environment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This polypeptide is a natural preservative of antimicrobial peptides, with broad-spectrum and high-intensity antibacterial properties [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Antimicrobial peptides can bind to the surface of bacterial membranes and alter their structure and permeability by inserting the hydrophobic end of the molecule into the lipid membrane [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To date, few studies have assessed the inhibitory effect of E-PL on P. aeruginosa biofilm.\u003c/p\u003e \u003cp\u003eThe current study used crystal violet staining to explore the inhibitory mechanism of E-PL on \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm and used the CCK8 assay to assess the metabolic activity of bacteria in the biofilm. The biofilm structure was observed using scanning electron and fluorescence microscopy and the effect of E-PL on biofilm gene expression was investigated at the molecular level by fluorescence quantification and transcriptomics. The findings provide theoretical guidance for the future removal of P. aeruginosa biofilm in marine environments.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial and regents\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (MCCC1A10045) was obtained from the Marine Culture Collection of China (Xiamen, China). Crystal violet (1%) was purchased from Solarbio Life Services (Beijing, China). Glutaraldehyde Fixation Solution (2.5%) was purchased from Absin Biotechnology Co., Ltd (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Crystal violet tested biofilm formation\u003c/h2\u003e \u003cp\u003eCrystal violet staining was used to assess biofilm formation as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The \u003cem\u003eP. aeruginosa\u003c/em\u003e concentration was 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in 96-well plates with different concentrations of E-PL (0, 1.18, 2.35, 4.69, 9.38, 18.75, 37.5, 75, 150, 300, and 600 \u0026micro;g/mL) incubated at 30℃, 60 rpm for 24 h. After 24 h, the wells were washed twice with 0.2 mL PBS and fixed with 2.5% glutaraldehyde for 15 min. Crystal violet solution (0.1 mL) was added to each well to allow staining for 5\u0026ndash;10 min, then washed with 0.2 mL PBS. The biofilm was dissolved with 0.1 mL 95% ethanol for 30 min and light absorption was measured at OD 595 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Assessment of biofilm metabolic activity\u003c/h2\u003e \u003cp\u003eDifferent concentrations of E-PL were placed in the bacterial suspension 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, the final E-PL concentration was 0, 75 \u0026micro;g/mL, and 18.75 \u0026micro;g/mL, respectively. These mixtures were added to the 96-well plate at 37℃ for 6 h and 24 h. The cells were cleaned with 1 \u0026times; PBS buffer three times. The plate was incubated at 37℃ for 1 h using the CCK8 kit methodology (Beyond, Beijing), and the results was measured at OD450 nm.\u003c/p\u003e \u003cp\u003eTo determine the impact of E-PL on the metabolic activity of mature biofilm, \u003cem\u003eP. aeruginosa\u003c/em\u003e was cultured in 96-well plates for 24 h to form mature biofilm. The followed process as above section described. Different from above, E-PL (0, 600 \u0026micro;g/mL, and 1200 \u0026micro;g/mL) was add to 96-well plates for 6 h. Then, the plate was incubated at 37℃ for 1 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Assessment of biofilm structure\u003c/h2\u003e \u003cp\u003eThe bacterial suspension (200 \u0026micro;L; 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was added to a 24-well culture plate containing 1.8 mL LB medium at 37 ℃ and 60 rpm for 24 h. The plate was washed twice with PBS, then fresh medium containing E-PL (150, 300, and 600 \u0026micro;g/mL) was added to the 24-well plate, and the plate was incubated at 37℃ for 6 h. The plate was gently rinsed twice with 1 \u0026times; PBS, and the cell crawling tablets were removed and fixed with 2.5% glutaraldehyde for 30 min. Then, the plates were dehydrated in turn with 30, 50, 60, 70, 80, 90, and 100% anhydrous ethanol for 10\u0026ndash;15 min each at room temperature. After the dehydration was complete, the plates were naturally dried, treated with gold spray, and observed by SEM.\u003c/p\u003e \u003cp\u003eAs described in above section, the control group (0 \u0026micro;g/mL) and the experimental group receiving E-PL treatment (150, 300, and 600 \u0026micro;g/mL) were removed, respectively. The planktonic bacteria were washed with 0.5% NaCl and stained on the dark for 15 min using an MKBio SYTO 9 Live/Dead bacterial double stain kit (Maokang, Shanghai, China). The slides were covered with cover glass and observed under a confocal laser microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Assessment of bacterial motility\u003c/h2\u003e \u003cp\u003eThree types of media were prepared: (1) 0.3% agar medium containing 1.0% tryptone, 0.5% NaCl, and 0.3% agar, (2) semi-solid agar medium with 1% tryptone, 0.5% sodium chloride, 0.8% nutrient agar, and 0.5% glucose, and (3) LB medium with a 1.0% agar layer. After the medium was removed, E-PL (0, 150 \u0026micro;g/mL, 300 \u0026micro;g/mL, and 600 \u0026micro;g/mL) was added and solidified at room temperature. The bacteria (1 \u0026micro;L; 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) were then inoculated into the center of the agar medium and incubated at 30℃ for 24 h. The swimming ability and community dynamics of the bacteria were determined by measuring the circular expansion radius. The bacterial biofilm on the dish was stained with 1% (w/v) crystal violet (Beyond, Beijing). The dye was removed by washing with water and puncture movement was observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Real-time fluorescent quantitative PCR\u003c/h2\u003e \u003cp\u003eThe prepared \u003cem\u003eP. aeruginosa\u003c/em\u003e suspensions were pre-cultured with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL for 24 h, cultured in medium with or without E-PL (300 \u0026micro;g/mL) for 6 h, and the biofilms were collected. Total RNA was extracted from the biofilms using the trizol method [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eQuantStudioTM 1 Plus real-time PCR (Applied Biosystems, USA) was used to detect phzM transcription in E-PL-treated biofilms. Specific amplification primers for all tested genes are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The final reaction volume was 20 \u0026micro;L, including 10 \u0026micro;L ArtiCanCEO SYBR qPCR Mix, 10 \u0026micro;M primer F, 1 \u0026micro;L each for the forward and reverse primes, 7 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO, and 1 \u0026micro;L of the triple diluted cDNA template. The PCR procedure was 95\u0026deg;C 5 min, 40 cycles (95\u0026deg;C 15 s, 60\u0026deg;C 20 s, 72\u0026deg;C 20 s), 65\u0026deg;C 1 min. Rpod was used as a reference gene to calculate the relative expression of each gene. Three wells were used for each sample. The relative expression of target genes was calculated using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transcriptome analysis\u003c/h2\u003e \u003cp\u003eTo further explore the ability of E-PL to regulate gene expression in \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms, transcriptomic analysis was performed on the control and E-PL (300 \u0026micro;g/mL) treated biofilms. The samples were pre-cultured in LB medium for 24 h, and floating bacteria on the surface of the samples were gently washed with PBS. Samples in the control group (0 \u0026micro;g/mL) and experimental group (300 \u0026micro;g/mL) were treated with E-PL for 6 h, and the total RNA was extracted. Transcriptome sequencing was performed with Novogene (Tianjin, China) using Gene Ontology (GO) and the Kyoto Encyclopedia Gene and Genome (KEGG) analysis of genetic variations ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least thrice. Statistical analyses were performed using SPSS v.22.0. Single-factor analysis of variance and Tukey\u0026rsquo;s test were used for statistical analyses. The figures in this study were generated using Origin 2018.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 E-PL impairs biofilm formation\u003c/h2\u003e \u003cp\u003eThe current study assessed \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm formation using crystal violet staining [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The number of bacteria increased gradually in the first 2 h and most were independent cells showing little evidence of adhesion and no obvious biofilm (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Mutual aggregation began after 4 h and biofilm biomass increased significantly at longer culture times, with a sharp rise at 10\u0026ndash;12 h (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef and S1g). The biofilm also decreased with an increase in E-PL concentration. At 300 \u0026micro;g/mL, E-PL completely prevented the formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings indicated that E-PL inhibits biofilm biomass formation in a concentration-dependent manner. E-PL was also found to destroy mature and fully-formed biofilm in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding indicated that E-PL inhibited the multicellular stage of \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 E-PL damages biofilm structure\u003c/h2\u003e \u003cp\u003eTo further investigate the influence of E-PL, \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm was pre-cultured with E-PL for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The structure and biomass of biofilm were prospected by SEM. The untreated bacteria secreted more EPS, which tightly wrapped \u003cem\u003eP. aeruginosa\u003c/em\u003e, and the aggregation density and thickness increased until a complete, dense, and multi-layered mature biofilm had formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Bacteria in the biofilm were compact and the morphological surface was smooth, with no obvious depressions on the cell surface. In contrast, when \u003cem\u003eP. aeruginosa\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms likely by disrupting its morphological structure and preventing bacteria from secreting EPS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Additional effects of E-PL on biofilm structure\u003c/h2\u003e \u003cp\u003eTo probe the effect of E-PL on biofilm bacteria, the microbial population was labeled with SYTO9 and PI bicolor dyes. SYTO9 labels both intact and damaged cell membranes with green fluorescence. In contrast, PI only passes through damaged cell membranes. Upon entering the cell, PI causes the SYTO9 green fluorescence to decrease. When both dyes are present in bacteria, cells with an intact membrane structure fluoresce green, while those with a damaged membrane structure fluoresce red. Bacteria treated with the 150, 300, and 600 \u0026micro;g/mL concentrations had independent and single red fluorescence, while the control group had green or yellow-green fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the control group, obvious biofilm and bacteria in the membrane emitted green fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After treatment with E-PL, the bacteria no longer aggreged, no obvious biofilm was formed, and the number of cells was reduced. These findings indicate that E-PL can destroy biofilm bacteria and inhibit bacterial metabolic activity and proliferation by destroying the bacterial cell membrane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Metabolic activity of biofilm bacteria\u003c/h2\u003e \u003cp\u003eThe 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 \u0026micro;g/mL groups were significantly reduced from 0.32 to 0.205 (18.75 \u0026micro;g/mL) and 0.104 (37. 5\u0026micro;g/mL) compared with the control group (OD\u0026thinsp;=\u0026thinsp;0.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). 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 \u0026micro;g/mL treatment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar results have been shown in response to other bacteriostatic substances, including quercetin and chitosan, which also reduce the number of biofilm bacteria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings indicate that E-PL effectively inhibited the metabolic activity of bacteria during biofilm formation.\u003c/p\u003e \u003cp\u003eThe 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 \u0026micro;g/mL groups were 0.81 and 0.334, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). 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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Motility of biofilm bacteria\u003c/h2\u003e \u003cp\u003eBiofilm formation is often related to the movement of bacteria. Under complex conditions, the motility of bacteria is necessary for their survival and pathogenicity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e was motile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e). 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 \u0026micro;g/mL. These findings indicated that E-PL successfully prevented bacterial adhesion and movement. Compared to the control group, 150 and 300 \u0026micro;g/mL E-PL also significantly inhibited \u003cem\u003eP. aeruginosa\u003c/em\u003e locomotion and swarm movement while 600 \u0026micro;g/mL E-PL completely prevented swarm movement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In addition, after staining, no obvious crystal violet attachment points were observed in the control group. Meanwhile, at 600 \u0026micro;g/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 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Biofilm bacteria gene expression\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). 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 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], 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 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Transcriptome analysis\u003c/h2\u003e \u003cp\u003eTranscriptomics 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm. The results showed that differentially expressed genes (DEGs) were determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The impact of E-PL on the gene expression profile of mature \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm was further evaluated by identifying the GO and KEGG pathways of the DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). 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 \u0026ldquo;small molecule metabolic processes\u0026rdquo;, \u0026ldquo;transmembrane transport,\u0026rdquo; and \u0026ldquo;organic acid metabolic processes.\u0026rdquo; Meanwhile, DEGs involved in Cellular Components (CC) were mainly concentrated in \u0026ldquo;intracellular,\u0026rdquo; \u0026ldquo;intracellular parts,\u0026rdquo; and \u0026ldquo;protein-containing complexes\u0026rdquo;, and MF was primarily concentrated in \u0026ldquo;small molecule binding,\u0026rdquo; \u0026ldquo;anion binding,\u0026rdquo; and \u0026ldquo;nucleotide binding\u0026rdquo;. The down-regulated DEGs were most enriched in membrane-related components, including \u0026ldquo;membrane composition,\u0026rdquo; \u0026ldquo;intrinsic components of the membrane,\u0026rdquo; and \u0026ldquo;overall composition of the membrane.\u0026rdquo; There was also a high number of genes involved in the \u0026ldquo;REDOX process\u0026rdquo; and \u0026ldquo;transport activity.\u0026rdquo; While \u0026ldquo;flavin adenine dinucleotide\u0026rdquo; activity was only slightly enriched, with a small number of involved genes, E-PL was shown to impact its activity on the transcriptional level.\u003c/p\u003e \u003cp\u003eAnalysis of the KEGG database was also used to identify genes that were enriched and differentially expressed in \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm. DEGs were enriched in 20 KEGG pathways that were primarily involved in \u0026ldquo;microbial metabolism in different environments,\u0026rdquo; \u0026ldquo;ABC transport,\u0026rdquo; \u0026ldquo;amino acid biosynthesis,\u0026rdquo; and \u0026ldquo;ribosome and bacterial secretion\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Meanwhile, the downregulated genes were mainly involved in \u0026ldquo;ABC transport,\u0026rdquo; \u0026ldquo;degradation of valine, leucine, and isoleucine,\u0026rdquo; \u0026ldquo;quorum sensing,\u0026rdquo; \u0026ldquo;the bacterial secretion system for degrading aromatic compounds,\u0026rdquo; \u0026ldquo;the bacterial secretion system,\u0026rdquo; and \u0026ldquo;microbial metabolism in different environments\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). \u0026ldquo;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.\u003c/p\u003e \u003cp\u003eThe bacterial flagellum is a locomotive organelle that allows many bacterial species to swim or aggregate on liquid and solid surfaces [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eFlagellate-mediated movement is necessary to initiate bacterial attachment to surfaces to form biofilms [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eTranscriptomic 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 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Three QS systems (las, rhl, and pqs) regulate \u003cem\u003eP. aeruginosa\u003c/em\u003e virulence factor gene expression. The current study found that some key genes involved in the \u003cem\u003eP. aeruginosa\u003c/em\u003e QS pathway, including rhlI/rhlR, PA1760, and PA0268 in the rhl system, were down-regulated in E-PL-treated biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm formation.\u003c/p\u003e \u003cp\u003eBacterial polysaccharides and eDNA coordinate to form the main structure of bacterial biofilm [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] or serve as signals to promote biofilm formation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Polysaccharide is the main component of the extracellular polymer matrix in mature biofilm. Psl acts as a signal to promote the formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm, and enhances the secretion of the extracellular polymer matrix. Pyocyanin is one of the secondary metabolites of \u003cem\u003eP. aeruginosa\u003c/em\u003e, the synthesis of which is achieved through a complex cascade involving multiple genes, including phzABCDEFG and phzHMS [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm synthesis and transcriptional regulation.\u003c/p\u003e \u003cp\u003eIn 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e, 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study explored the effect of E-PL on \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm synthesis. E-PL was shown to inhibit and destroy the formation of biofilm in a concentration-dependent manner by 1) inhibiting the secretion of extracellular polymer to damage the biofilm structure, 2) reducing the metabolic activity of the biofilm bacteria, 3) limiting bacterial motility, and 4) regulating the expression of genes involved in synthesizing flagella, chemotactic transduction, extracellular polymeric polysaccharide synthesis, lifetime metabolism, and electron transfer. These findings suggest that the natural and environmentally friendly antimicrobial peptide, E-PL, has great potential for use in biofilm treatment in many industries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiwei Wu: conceptualization, methodology, writing-original draft. Quantong Jiang: funding acquisition, supervision. Dongzhu Lu: manuscript review\u0026nbsp;\u0026amp;\u0026nbsp;editing. Xiaofan Zhai: experimental design. Jizhou Duan: experimental design. Baorong Hou: supervision. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by Hainan Province Science and Technology Special Fund (ZDYF2021GXJS210), Wenhai Program of the S\u0026amp;T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (2021WHZZB2301), Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City (2021CXLH0005), Overseas Science and Education Centers of Bureau of International Cooperation Chinese Academy of Sciences (121311KYSB20210005) for providing support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available upon request by contact with the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ.B. 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Kong, The role of pyocyanin in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e infection, Trends Mol Med. 10(12) (2004) 599-606.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1 ","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-materials-degradation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmatdeg","sideBox":"Learn more about [npj Materials Degradation](http://www.nature.com/npjmatdeg/)","snPcode":"41529","submissionUrl":"https://submission.springernature.com/new-submission/41529/3","title":"npj Materials Degradation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pseudomonas aeruginosa, ε-Polylysine, antibacterial peptide, biofilm","lastPublishedDoi":"10.21203/rs.3.rs-4276320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4276320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNatural agents with antimicrobial properties have a broad potential to resist biofilm adhesion in marine environments. ε-Polylysine (E-PL) is a natural cationic, homomeric polymer with 25\u0026ndash;30 lysine residues that can resist microbial biofilm adhesion due to its stability, nontoxicity, and biodegradability. The current study investigated the action of E-PL against Pseudomonas aeruginosa biofilm isolated from a marine environment. Crystal violet staining was used to examine the effects of E-PL on the formation and destruction of mature biofilms. Scanning Electron and fluorescence microscopy revealed that E-PL treatment damaged the biofilm structure and affected the secretion of extracellular polymers. The CCK8 colorimetric assay showed that E-PL also decreased the metabolic activity and motility of biofilm bacteria. QPCR and transcriptome analysis revealed that E-PL affected biofilm formation and transcriptional regulation by downregulating genes involved in flagellar synthesis (flgE, PA4651, pilW), chemotaxis transduction (PA1251, PA4951, PA4788), biofilm biosynthesis (pelC, pelD, pslK, plsM), transcriptional regulation (PA3973, PA3508, PA0268), phenazine biosynthesis (phzM, phzH, phzS), and electron transfer (PA5401, PA5400, PA3492). This study used multiple methods to identify the mechanism of E-PL action against biofilm, informing the design of novel biofilm treatment methods.\u003c/p\u003e","manuscriptTitle":"The effect of antibacterial peptide ε-Polylysine against Pseudomonas aeruginosa biofilm in marine environment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-24 21:02:49","doi":"10.21203/rs.3.rs-4276320/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-06-19T13:44:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-17T15:27:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-20T14:28:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-04-30T03:02:25+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-25T07:46:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-04-22T06:52:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-20T02:03:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-19T13:31:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Materials Degradation","date":"2024-04-16T13:22:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-materials-degradation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmatdeg","sideBox":"Learn more about [npj Materials Degradation](http://www.nature.com/npjmatdeg/)","snPcode":"41529","submissionUrl":"https://submission.springernature.com/new-submission/41529/3","title":"npj Materials Degradation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62f2d170-fc2e-4fee-ae5c-4cb84362573f","owner":[],"postedDate":"April 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-30T08:07:07+00:00","versionOfRecord":{"articleIdentity":"rs-4276320","link":"https://doi.org/10.1038/s41529-024-00539-6","journal":{"identity":"npj-materials-degradation","isVorOnly":false,"title":"npj Materials Degradation"},"publishedOn":"2024-11-29 05:00:00","publishedOnDateReadable":"November 29th, 2024"},"versionCreatedAt":"2024-04-24 21:02:49","video":"","vorDoi":"10.1038/s41529-024-00539-6","vorDoiUrl":"https://doi.org/10.1038/s41529-024-00539-6","workflowStages":[]},"version":"v1","identity":"rs-4276320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4276320","identity":"rs-4276320","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}