The dynamic response of Cupriavidus metallidurans CH34 cells and biofilms to silver nanoparticles and the importance of biofilm maturation stage

The dynamic response of Cupriavidus metallidurans CH34 cells and biofilms to silver nanoparticles and the importance of biofilm maturation stage | 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 Research Article The dynamic response of Cupriavidus metallidurans CH34 cells and biofilms to silver nanoparticles and the importance of biofilm maturation stage Nissem Abdeljelil, Timothej Patocka, Luna Hendrickx, Najla Ben Miloud Yahia, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8079940/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Biofilms are in many sectors a persistent technical challenge that can lead to reduced efficiency in industrial processes, increased maintenance costs, and potential product contamination. Furthermore, biofilms can contribute to the development of antimicrobial resistance, posing health risks in medical and food production environments, as well as reducing the effectiveness of cleaning and sterilization efforts. Therefore, we investigated the impact of antimicrobial silver nanoparticles (AgNPs) on macroscopically visible biofilms of Cupriavidus metallidurans CH34, a metal-resistant and oligotrophic bacterium. We accelerated the time-consuming process of naturally forming macroscopic biofilms via a laboratory setup using porous synthetic surfaces. This setup simulates growth dynamics on air-exposed and nutrient-leaking substrates such as rocks, wood, polymeric coatings, joints and dripping pipes. Remarkably, 24-hour matured biofilms were unaffected by AgNPs at concentrations as high as 750 mg/L. Biofilm resilience to AgNPs was age-dependent and although densely populated, young biofilms (6–9 hours) were inactivated by AgNPs while older ones (14–24 hours) exhibited resistance in the same conditions. Additional experiments suggested that the resistance in older biofilms was not attributed to cell density, but potentially to other factors like extracellular polymeric substances (EPS) or their metabolic state. AgNPs induced oxidative stress in exposed biofilms but did not result in microscopically observable morphological changes in the biofilm-resident cells, indicating that inhibition mechanisms did not involve outer membrane disruption. The proteomic analysis of macroscopic biofilms exposed to AgNPs revealed an age-independent protein set that was consistently upregulated/down regulated across all analyzed biofilm stages as well as age-specific pathways. These findings suggest complex adaptive molecular mechanisms in C. metallidurans biofilms that confer resistance to AgNPs, involving oxidative stress management, iron metabolism, and electron transport chain adjustments. Biofilm nanoparticles proteomics bacteria metal resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Environments where a source of humidity is present are prone to microbial proliferation in the form of biofilms. Biofilms are microbial communities encased in or glued together by a self-made matrix composed mainly of water and extracellular polymeric substances (EPS). These substances include polysaccharides, proteins and nucleic acids (Flemming and Wingender, 2010). Biofilm formation is key for persistence during unfavorable conditions; it retains moisture and shields resident cells from physical and chemical stress. It is acknowledged that the resistance of biofilm cells to stressful conditions is orders of magnitude higher than free cells. At early stages, biofilms are rarely visible without staining techniques but given enough time and nutrients, these organized microbial communities can develop into macroscopically visible structures. The thicker the biofilm, the more resilient it is as thickness acts as a protective physical barrier. Macroscopic biofilms occur naturally in humid environments and are important for the balance of ecosystems. They can be encountered in rivers and streams (Merbt et al., 2017; Souza-Egipsy et al., 2008; Stone et al., 2011) or as oceanic microbial mats, which are considered the largest biofilm structures on the planet (Staley et al., 1963). However, macroscopic biofilms can also develop on marine equipment (de Carvalho, 2018), buildings and monuments (Grewe and Pulz, 2012), water distribution infrastructures (Liu et al., 2017), industrial equipment (Matoulková et al., 2012) and more critically on teeth (Jakubovics et al., 2021), wounds (Metcalf and Bowler, 2013) and medical devices (Carlo Luca Romano and Delia Romano, 2017; Donelli, 2015). In the built environment, uncontrolled biofilm formation is not only a health hazard, but also a technical challenge as well. The proliferation of biofilms can cause clogging, leakage and corrosion of expensive equipment in addition to the spoilage of goods with considerable economic consequences. The global impact of biofilms on the economy was estimated to 3,967 billion dollars (Cámara et al., 2022). Although biofilm formation is attracting a growing interest in the scientific community, many fundamental aspects of this microbial lifestyle such as communication, stress adaptation or genetic and phenotypic variability remain enigmatic. In addition, these biological structures grow differently depending on their environment (i.e., nutrient flow, shear forces, humidity level) and dynamically adapt on many levels throughout time: types and numbers of resident cells, chemical gradients (Stewart and Franklin, 2008), matrix architecture and composition (Bisht, 2023; Koh et al., 2007; Ziege et al., 2021). Moreover, less is known about these dynamics within macroscopic biofilms due to their complexity. This is very challenging in an industrial context where microbial contamination must be strictly controlled and the use of antibiotics or chemical sanitation is not an option (solvents, quaternary ammonium compounds, oxidizers). Therefore, a knowledge gap in understanding fundamental aspects of macroscopic biofilms behavior exists. In this work, we investigate the response of macroscopic biofilms of Cupriavidus metallidurans to silver nanoparticles (AgNPs), which are a widely employed sanitation substance (Sim et al., 2018). More specifically Agpure W10 silver nanoparticles will be used, as they are well-characterized and listed by the OECD as reference material (NM-300K) for nanotoxicology and environmental safety assessments (Schneider, 2017). AgNPs elicit a multifactor stress, being at the same time an antimicrobial, a nanomaterial and a metal. Several mechanisms behind the direct antibacterial properties of AgNPs have been hypothesized but have not been proven to full extent. For instance, the antimicrobial properties of AgNPs have been attributed mainly to a chemical origin through the release of free silver ions and the proximal delivery of chemisorbed Ag + a phenomenon also described as “nanoparticles-enhanced silver ions stress” (Kurvet et al., 2013). Variations in the AgNP properties, such as shape, size, coating and administered concentration, affect these described mechanisms and formulating a general definition about AgNPs mechanism of action is therefore challenging. The differences may be further exaggerated by cell density because of higher cell numbers, less dissolved oxygen, higher EPS content, higher content in antioxidant metabolites and cell debris capable of trapping AgNPs and Ag + ions or neutralizing oxidative stress. C. metallidurans is a metal-resistant species adapted to oligotrophic environments such as water distribution systems and has some strains caused medical concerns in healthcare environments (Ayhan et al., 2024; D’Inzeo et al., 2015; Langevin et al., 2011). Furthermore, closely related species have been reported to produce extracellular AgNPs (Ameen et al., 2020). We explored the interaction between silver nanoparticles and C. metallidurans type strain CH34 liquid cultures and macroscopic biofilms at different maturation stages via assessing growth and survival, the cellular response, morphology and its whole proteome to elucidate molecular mechanisms behind interactions with silver nanomaterials. Materials and Methods Viability and adhesion of planktonic cells exposed to a single dose of AgNPs in a liquid media C. metallidurans CH34 was grown in Tris-buffered MM284 (pH 7) supplemented with 0.2% gluconate for 48 h at 30°C. Next, cell density was adjusted with fresh medium to OD 600 ≈ 0.1 (exponential phase cells) or OD 600 ≈ 1 (stationary phase cells) and exposed at 30°C (180 rpm shaking) to increasing concentrations of AgNPs (AgPure W10, mean particle size = 15 nm, RAS materials + technologies, Germany). For the exponential phase cells, optical density (600 nm) was measured at 2 h, 3 h, 5 h and 24 h. For the stationary phase cells, viable counts were performed after 24 h of exposure. Adhesion on polypropylene tubes was also assessed for the stationary phase cultures. For this, the culture was discarded and the test tubes were gently rinsed 3 times with deionized water and left to dry at room temperature. Adhered biomass was stained by 0.1 % crystal violet (CV) (0.1 g of CV in 2 mL 95% ethanol and 98 mL of deionized water) for 15 minutes. Afterwards, the CV solution was removed and the tubes were rinsed 3 times with deionized water. The amount of CV was quantified by solubilizing in 4 mL of 95% ethanol and measuring absorbance at 600 nm. Viability and adhesion of planktonic cells exposed to AgNP-loaded solid media Solid agar media loaded with AgNPs was prepared by adding AgNP suspensions, according to the concentrations to be tested, to MM284 agar just before pouring the plates To test viability, a viable count was performed by spotting a tenfold serial dilution of a C. metallidurans CH34 stationary phase culture (OD 600 = 1) on AgNP-loaded MM284 agar and MM284 agar (control), incubating at 30°C for 48 h in the dark and counting colonies. To test the effect of AgNPs on biofilm growth, sterile Supor membranes (Ø ≈ 0.2 µm) were placed on the surface of AgNP-loaded MM284 agar and MM284 agar (control). Membranes were then inoculated with 30 µL from an OD 600 = 1 culture and plates were incubated in the dark at 30°C for 24 h. Next, biofilms were collected by removing the membranes from the agar, placing them in sterile Eppendorf tubes with 1 mL of filter-sterilized phosphate-buffered saline (PBS) and vortexing at maximum speed for at least 30 seconds until all visible biomass was homogenously resuspended. Cell enumeration by total viable count was performed by spotting 10 µL of a serial 10-fold dilution in sterile PBS on MM284 agar and counting colonies after 24 h at 30°C. Cell viability in macroscopic biofilm exposed to a single dose of AgNPs and AgNO 3 on solid media Biofilms were grown on LB agar for 24h then transferred to petri dishes with only agar (to prevent potential cell recovery). They were gently covered by 30 µL of increasing concentrations of AgNPs suspension or AgNO 3 solution (250, 500, 750 mg/L). Control biofilms received 30 µL of sterile H 2 O. After additional 24 h in the dark at 30°C, biofilms were collected and resuspended as previously described and cell survival was determined via viable count. Effect of biofilm development stage on susceptibility to silver nanoparticles Biofilms were grown on Supor membranes placed on LB agar plates for 6, 9, 14, 18 and 24 h as previously described, then transferred to AgNP-loaded MM284 agar plates or to MM284 agar plates for controls. In addition, to ensure total exposure, biofilms on AgNP-loaded agar plates were also covered with 30 µL of AgNP suspension (with corresponding concentration) (Fig. 1). Controls were covered with 30 µL of sterile deionized water. After 24 h of incubation in the dark at 30°C, membranes were placed in 1 mL PBS, biofilms were resuspended by vortex for at least 30 seconds and cells enumerated by viable count. To assess the role of the biofilm matrix on the susceptibility to AgNPs, 1 mL of an OD 600 ≈ 1 culture of C. metallidurans CH34 was centrifuged, and the resulting pellet was resuspended in 30 µL PBS to obtain a cell number equivalent to a 24 h-old biofilm population. This suspension was transferred to Supor membranes placed on AgNP-loaded MM284 agar plates or on MM284 agar plates for controls. After drying for 5 minutes, 30 µL of AgNP suspension (with the corresponding concentration) was added. Controls received 30 µL of sterile deionized water. All samples were incubated in the dark at 30°C. After 24 h, biomass on the membranes was collected and enumerated by viable count. Figure 1: Experimental setup for the exposure of C. metallidurans CH34 macroscopic biofilms to AgNPs on AgNPs-loaded MM284 agar plates. 1) Exposure of upper layers cells via AgNPs droplets. 2) Pre-formed macroscopic biofilm. 3) Porous Supor membrane. 4) AgNPs loaded MM284 agar. 5) Exposure of bottom layers cells through upwards diffusion of silver material. Assessment of total hydrolytic activity and oxidative stress in liquid cultures Cells were grown in MM284 until reaching an OD 600 of ≈ 1 and subsequently exposed to 12.5 mg/L of AgNPs for 24 h. For total hydrolytic activity, one mL of cell suspension was washed and re-suspended in 900 µL PBS, to which 100µL of fluorescein diacetate (FDA; 200 µM in dimethyl sulfoxide, Sigma) was added. For assessing oxidative stress, one mL of cell suspension was washed and re-suspended in 900 µL PBS, to which 100 µL of 200 µM of dichlorodihydrofluorescein diacetate (H 2 DCFDA called also DCFH-DA) in dimethyl sulfoxide were added. These mixtures were incubated in the dark at 30°C with agitation (180 rpm) for 1 h and analyzed through flow cytometry using the FITC filter (530 nm filter, 488 nm laser; Novocyte flow cytometer, Agilent) and a flow rate 66 µL/min. Unwanted signal was filtered by setting thresholds for FSC-H at 750 and SSC-H at 1000. The percentage of fluorescent cells in the population was counted (threshold was based on unstained control). Assessment of reductase activity, electron chain function and oxidative stress in macroscopic biofilms exposed to AgNP-loaded agar Biofilms on Supor membranes were grown for 14 h and 18 h and then exposed to MM284 agar plates loaded with different concentrations of AgNPs as previously described. After 24 h in the dark at 30°C, biofilms were resuspended and a serial dilution in PBS was prepared. Reductase activity and electron chain function are both revealed by the fluorescence of the Baclight RedoxSensor™ Green reagent (Thermo Fisher Scientific, Geel, Belgium). One µL of stock solution was diluted in 99 µL PBS. Two µL was used for 200µL of sample and incubated for 30 min. Next, samples were analyzed with a flow cytometer (Novocyte, Agilent) at a flow rate 66 µL/min and fluorescence was detected with an FITC filter (530 nm filter, 488 nm laser). Unwanted signal was filtered by setting thresholds for FSC-H at 750 and SSC-H at 1000. For the assessment of oxidative stress levels through 2′,7′-dichlorofluorescein diacetate, the resuspended biofilms were processed as described for planktonic cells. Scanning electron microscopy Cell morphology and biofilm architecture were analyzed by scanning electron microscopy (SEM) using a Field Emission Scanning Microscope (Jeol JSM-7200F). Samples were prepared by directly fixing the biofilms grown on Supor membranes in a twice-refreshed fixation buffer (3.125 % (w/v) glutaraldehyde in 0.131 M sodium cacodylate, Sigma-Aldrich). After fixation, the specimens were washed three times with 0.15 M sodium cacodylate solution. Next, they were dehydrated in an ascending ethanol series (30, 50, 70, 90, 95, and 2 times 100% (w/v), Sigma-Aldrich) for 10 minutes in each solution. The dehydrated samples were dried in pure hexamethyldisilane (Merck Millipore) for 30 minutes then air-dried and coated with gold in an ion sputter coater (Jeol, JFC-1100E). Samples were observed at least at 10 different positions and images were collected. Proteomic analysis Protein extraction and quantification For the proteomic analysis, the effect of AgNPs on biofilms of C. metallidurans CH34 was investigated at the concentration of 500 mg/L versus controls. Membrane-grown biofilms were placed in 1 mL filter-sterile PBS and the biomass was resuspended by vortex for at least 30 seconds. Biofilm suspensions were then centrifuged at 9000 rpm for 15 minutes at 4°C. Supernatants were discarded and pellets were washed with cold and filter sterilized PBS then centrifuged again. The resulting pellets were later dissolved in 250 µL lysis buffer (2% SDS in 50mM Ammonium Bicarbonate). The mixtures were vortexed for 3 x 10 seconds until pellets were dissolved. These solutions were transferred to 1.5 mL tubes and incubated for 5 minutes at 95°C. After incubation, tubes were cooled on ice for 5 minutes, followed by a brief centrifugation. Samples were lysed using ultrasonication (4 x 10 s, amplitude 40%, 1 cycle, in between 1 min on ice) (Imlab, Boutersem, Belgium). After sonication, samples were centrifuged for 20 minutes at 20,817g (14,000 rpm) and 4°C. Finally, supernatants were transferred to fresh tubes. The protein quantification was carried out using the BCA assay (Merck Life Science BV, Hoeilaart, Belgium) following the manufacturer’s instructions. Mass spectrometry analysis of protein samples Extracted proteins were further processed for LC-MS/MS analysis as previously described (Ellena et al., 2024). Briefly, LC-MS/MS analysis was performed using a nanoElute UHPLC (Bruker Daltonics, Bremen, Germany) connected to a QTOF-MS instrument (Impact II, Bruker Daltonics, Germany) via a CaptiveSpray nanoflow electrospray source (Bruker Daltonics, Bremen, Germany). In total, 2 µg of tryptic digest (in solvent A) was injected onto a trapping column setup (300 µm x 5 mm, C18 PepMap 300, 5 µm, 100 Å; Bruker Daltonics, Bremen, Germany). Subsequently, peptides were separated using a C18 Reprosil AQ, 1.9 µm, 120 Å, 0.075 x 150 mm column operated at 40°C (Bruker Daltonics, Bremen, Germany) at a flow rate of 0.2 µL/min. Gradient conditions were: 2–35% solvent B for 100 min; 35–95% solvent B for 10 min; and 95% solvent B held for 10 min (solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile). In all the full-scan measurements, a lock-mass (m/z 1221.9906, Hexakis (1H, 1H, 4H-hexafluorobutyloxy)phosphazine) (Bruker Daltonics, Bremen, Germany) was used as internal calibrator. The mass spectrometry proteomics data was submitted to the ProteomeXchange Consortium78 via the PRIDE79 partner repository (Deutsch et al., 2023; Perez-Riverol et al., 2022) with the dataset identifier PXD060314 and 10.6019/PXD060314. Raw data processing All raw mass spectrometry spectra files were processed using MaxQuant software version 2.0.1.0 (Max-Plank Institute of Biochemistry, Department of Proteomics and Signal Transduction, Munich, Germany) and the proteins were identified with the built-in Andromeda search engine (Cox and Mann, 2008; Tyanova et al., 2016a). The database searches were performed against a database containing all Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34) ( Ralstonia metallidurans ) (Taxonomy ID: 266264). UniProt protein sequences (downloaded from ftp.uniprot.org on 2024-04-23). Default MaxQuant parameter settings were used: cysteine carbamidomethylation as fixed modification, and methionine oxidation and Protein N-terminal acetylation, as variable modification; False-discovery rate (FDR) cutoffs set to 1% on peptide, protein and site decoy level; trypsin as a digestion enzyme; 7 amino acids as minimum peptide length. Normalization and Differential Expression Analysis The resulting data from MaxQuant with minimum 2 unique peptides was retained and was processed to remove reverse hits and contaminants from the data, then transformed intensities to log2 for further analysis. To remove the technical variation among replicates, median normalization on log2-transformed intensities was performed using an in-house python script (Kammers et al., 2015; Tyanova et al., 2016b). Missing values in the data were replaced by value from a normal distribution with settings width =0.3 and downshift =1.8. Protein differential expression was calculated using LIMMA R package (Kammers et al., 2015), based on the empirical Bayes moderated test-statistics. The expression was considered significant at a p-value 0.5849 and < -0.5849) were considered for interpretation. Results and Discussion Effect of AgNP exposure on the viability and metabolism of planktonic cells To have a better understanding of the impact of AgNPs on C. metallidurans CH34, we started by studying the effect of a single dose of this nanomaterial on planktonic cells in the exponential (assessment of growth) (Fig. 2a) and stationary phase (viability assay) (Fig. 2b). Exponential phase cultures (OD ≈ 0.1) were extremely sensitive to AgNPs and growth in liquid media was rapidly inhibited by 0.25 mg/L AgNPs. Although a comparison is not straightforward, this is much less than the minimal inhibitory concentration (MIC) of 50-nm AgNPs for P. aeruginosa that was 2 mg/L (Kora and Arunachalam, 2011) and the MIC of biogenic AgNPs (with a size between 24 and 43 nm) for Ralstonia solanacearum YY06 that was 10 mg/L (Cheng et al., 2020). For Cupriavidus necator H16, the MIC of Ag pure W10 silver nanoparticles (size < 15 nm) was 60 mg/L (Schacht et al., 2013). However, as indicated, results should be compared with caution as all these studies were conducted in complex organic medium. For stationary phase cultures, a 3-log reduction in CFU/mL was observed from 4 mg/L to 12.5 mg/L and full inhibition at 25 mg/L AgNPs. Not many comparable results are described in literature. Nevertheless, 2 mg/L of nano-Ag (average size 3 nm) killed 99 % of E. coli ATCC 25922 in 6 h (10 6 CFU/mL cultivated in LB) (Lee et al., 2014). For reference, Agpure W10 concentrations for antimicrobial testing recommended by the manufacturer ranged between 50 and 1000 mg/kg. Figure 2: Effect of AgNP exposure on the growth of C. metallidurans CH34 in MM284 (a) and on the viable count in stationary phase liquid cultures after 24 h (b). The average values of at least three independent experiments with standard deviations are shown. Next, C. metallidurans cultures exposed to 12.5 mg/L were used to assess the time-dependent effect of AgNPs on hydrolytic activity and oxidative stress through FDA and DCFH-DA assays, respectively. Hydrolytic activity reflects the ability of cells to break down complex molecules using enzymes of the hydrolases family (de Duve, 1959), serving as a key indicator of cell metabolic activity. Assessment of the hydrolytic activity through the intracellular transformation of the pre-fluorophore FDA into fluorescein essentially by esterases revealed that cultures exposed to 12.5 mg/L AgNPs had an increased metabolic activity at 4 h of exposure (53.48 ± 4.18 %) compared to 1 h (10.01 ± 0.49 %) and 0 h (8.61 ± 2.47 %). At 24 h, the hydrolytic activity was high and 87.51 ± 0.56 % of the bacterial populations were fluorescent (Fig 3a). Studies assessing FDA hydrolysis in bacterial monocultures exposed to silver nanoparticles are scarce, but relevant insights can be drawn from research on the effects of silver and silver nanomaterials on hydrolytic activity in soil, lake and sediment samples. For example, increased FDA hydrolysis was observed in sediments exposed to AgNPs at a concentration of 1000 mg/kg dry weight, whereas bulk silver at the same conditions showed no significant effect (Bao et al., 2016). Another study reported increased esterase activity in soil microbiota exposed to silver ions at 10 mg/kg of soil (Przemieniecki et al., 2022). Conversely, FDA hydrolysis decreased in lake water samples spiked with at least 1 mg/L of AgNPs-PVP, while AgNPs-citrate had no significant effect (Burkowska-But et al., 2014). A similar decrease in FDA hydrolysis was observed in soil treated with nanosilver at 1000 mg/kg (Shin et al., 2012) and in the maize rhizosphere exposed to 100 mg/kg of AgNPs (Sillen et al., 2015). These findings highlight the variable impact of AgNPs on hydrolytic activity depending on the environmental context and nanoparticle type. Our results showed that an increase of hydrolytic activity was only observed starting from 4 h after exposure, therefore sampling time may also influence the outcomes and depending on the species, the metabolic reaction time to AgNPs could be different. Figure 3: Effect of 12.5 mg/L AgNP exposure on (a) hydrolytic activity scored by FDA assay and (b) oxidative stress levels in stationary phase liquid cultures of C. metallidurans CH34 scored by DCFH-DA assay (expressed as percentage of fluorescent cells). The average values of at least three independent experiments with standard deviations are shown. To investigate oxidative stress levels after exposure to AgNPs, we performed a time-dependent measurement of the intracellular transformation of the pre-fluorophore DCFH-DA in stationary phase cultures of C. metallidurans CH34 exposed to 12.5 mg/L AgNPs versus non-exposed controls. Despite high metabolic activity in all NP-exposed replicates, oxidative stress was low at 1 h and 4 h (Fig. 3b). However, after 24 h of exposure oxidative stress increased significantly in AgNP-exposed cells. In addition, we noticed a large divergence in the 24-h replicates, where 3 replicates exhibited high fluorescence levels and 3 replicates had lower fluorescence, which resulted in a high standard deviation. It was demonstrated that AgNPs increased oxidative stress levels (DCFH-DA measurements) in Escherichia coli , Salmonella enterica , Pseudomonas aeruginosa (Liao et al., 2019; Seong and Lee, 2017; Xiao et al., 2021) and Staphylococcus epidermidis (luminol-based measurements) (Mazur et al., 2020). AgNPs can catalyze the generation of reactive oxygen species (ROS) after adsorption of molecular oxygen (O 2 ). In fact, adsorbed oxygen can interact with the conduction band electrons of the AgNPs, leading to a transfer of electrons from the nanoparticles to the oxygen molecules. Thus, the AgNP surface is not only highly attractive to oxygen but also facilitates electron transfer reactions. It amplifies the reduction of O₂ and consequently the production of the superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH), this phenomenon is called surface catalysis (Guo et al., 2021). AgNPs can also disturb the electron transport chain, leading to an amplification of the naturally occurring electron leakage, which in turn can cause an increase in the reduction of oxygen to superoxide anions (Valdiglesias, 2022). Moreover, AgNPs bind to glutathione, a major antioxidant, and deplete the cellular reserve, thereby reducing the cell’s capacity to neutralize ROS (Molina-Hernandez et al., 2021). AgNPs can activate NADPH oxidase enzymes on cell membranes, which catalyze the production of superoxide anions from oxygen (Garcés et al., 2021). In addition, silver ions released from AgNPs can promote Fenton-like reactions in the presence of H₂O₂, generating highly reactive hydroxyl radicals. When ROS levels are elevated they can cause DNA damage, which in turn increases bacterial mutation rates. It was demonstrated that the exposure of E. coli to high concentrations of AgNPs (480 mg/L) resulted in the formation of mutant colonies that acquired resistance through single-nucleotide polymorphism (SNP) affecting genes involved in silver transport and in osmoregulation (Wu et al., 2022). Experiments where E. coli MG1655 was exposed to H 2 O 2 also resulted in SNP mutations characterizing oxidative stress-surviving populations (Rodríguez-Rojas et al., 2020). Added to this, it was observed that mutation rates in biofilms are higher compared to planktonic cells, sometimes by up to 60-fold. They were also linked to endogenous oxidative stress (Ryder et al., 2012). Therefore, exposure to AgNPs may have increased the mutagenic pressure in C. metallidurans biofilms facilitating the emergence of stress-resistant variants. This could explain why half of the replicates showed low levels of oxidative stress after 24 h compared to the initially identical replicates receiving the same treatment. Figure 4: Crystal violet quantification of C. metallidurans CH34 biofilm formation on polypropylene after 24 h of exposure to 4 mg/L of AgNPs. The average values of at least three independent experiments with standard deviations are shown. Our results showed that exposure to 4 mg/L AgNPs did not stop the growth but did inhibit biofilm formation of C. metallidurans (Fig. 4). Silver nanoparticles can prevent biofilm formation through a combination of physical, chemical and biological mechanisms. They disrupt initial adhesion by possibly acting both on the cells (surface charge, membrane damage, interference with quorum sensing) and the substrate (surface charge, adsorption). In other bacteria, the effect of AgNPs on biofilm formation is better studied in clinically relevant species than in environmental ones. For the latter, in 12h-old P. putida KT2440 biofilms, cell survival was totally inhibited by 25.86 mg/L of AgNPs (40-60 nm) and further biomass development was repressed in older ones (Thuptimdang et al., 2015). Exposure for 24 h to concentrations of AgNPs (8.3 ± 1.9 nm) higher than 16 mg/L completely inhibited biofilm formation by Serratia proteamaculans 94 (Radzig et al., 2013). A study by Inbakandan et al. (2013) showed that exposure to 50 mg/L of AgNPs (15-34 nm) for 4 days inhibited biofilm formation in sixteen marine bacterial species isolated from the hull of a ship (Inbakandan et al., 2013). Overall, low-density cultures of C. metallidurans were extremely sensitive to AgNPs when exposed in mineral media, while high density cultures could tolerate higher concentrations of the antimicrobial. AgNPs at 12.5 mg/L rapidly induced a significant metabolic activity in C. metallidurans cells while oxidative stress levels remained low for hours. Although these cultures were not critically affected by 4 mg/L AgNPs, at this concentration their biofilm formation capacity was compromised. Effect of AgNP exposure on pre-formed macroscopic biofilms First, biofilm formation on Supor membranes was tested (Fig 1 and 2 Supplementary material). The diffusion of nutrients from the LB agar plates through the Supor membrane allowed C. metallidurans to produce visible biofilms onto the porous synthetic surface in hours. At 48 h of maturation, these biofilms were at least 3-7 µm thick (after dehydration) and densely packed with cells (Fig. 5). Based on the previous results where stationary phase cultures were inhibited by 25 mg/L AgNPs, we estimated that a higher AgNP concentration was needed to affect these macroscopic biofilms. Therefore, 250, 500 and 750 mg/L AgNPs were used. In addition, the effect of AgNPs was compared to AgNO 3 . Figure 5: SEM micrograph of a macroscopic C. metallidurans CH34 biofilm grown for 48 h on a Supor membrane. Magnification = 8000 X. Figure 6: Viability of 24 h-old C. metallidurans CH34 biofilms exposed for 24 h to AgNPs or AgNO 3 . The average values of at least three independent experiments with standard deviations are shown. Asterisks above the error bars indicate significant differences (p < 0.05) based on one way ANOVA. Surprisingly, exposure for 24 h to AgNP concentrations as high as 750 mg/L had no effect on the viable population in 24 h-old macroscopic biofilms. AgNO 3 at 750 mg/L caused a significant decrease in CFU/mL (Fig 6). The setup based on AgNP-loaded MM284 agar was used to investigate the effect of this nanomaterial on pre-formed macroscopic biofilms at different growth stages (6, 9, 14, 18 and 24 h) (Fig. 7). In 6 h- and 9 h-old biofilms, a complete inactivation was observed, while cells in 14 h-old biofilms resisted 250 mg/L AgNPs and cells in 18 h- and 24 h-old biofilms survived the highest concentration. Further analysis showed that young biofilms had significantly less protein content than older ones (Fig 4. Supplementary material) and less resident cells (Fig. 8). We also noticed that the viable population in these biofilms reached a maximum at 14 h and stabilized onwards. It is possible to assume that the resistance to high concentrations of AgNPs may be due to the high cell number (more cells means more binding and inactivation of AgNPs) or to higher EPS content, or to cells metabolic state (stationary/dormant). Figure 7: Cell survival in biofilms of C. metallidurans exposed to AgNPs at different maturation stages. The average values of at least three independent experiments with standard deviations are shown. Figure 8: Age-dependent evolution of cells population in biofilms of C. metallidurans . The average values of at least three independent experiments with standard deviations are shown. To test if the cell density was responsible for the resistance to 250 mg/L AgNPs observed in 14 h-old biofilms, equivalent biomass of planktonic cells (≈ 10 9 CFU/mL) was deposited on Supor membranes placed on AgNP-loaded MM284 agar. After 24 h of exposure, the viable count showed no survival in the biomass exposed to AgNPs (Supplementary Figure 3). Therefore, the resistance phenotype observed in 14 h-, 18 h- and 24 h-old biofilms could not be explained by cell density. Oxidative stress and cellular respiration Results demonstrated that 24 h exposure to AgNPs induced a significant increase in oxidative stress levels in the cell population within macroscopic biofilms of C. metallidurans CH34 (Fig. 9). Stress levels increased when the AgNP concentration was increased from 250 mg/L to 750 mg/L. Interestingly, 18 h-old biofilms exposed to 750 mg/L AgNPs showed higher oxidative stress levels (45.01 ± 6.74 % fluorescent cells) than 14 h-old biofilms (30.72 ± 2.60 % fluorescent cells) although they showed a better survival (Fig. 7). From the redox sensor-based analysis (Fig. 10), it appears that the 24 h exposure to 250 mg/L AgNPs did not affect the redox balance in both 14 h- and 18 h-old biofilms as their fluorescence response overlays with that of the untreated biofilms. However, tripling the AgNP concentration did induce a change in the redox profile and in the fluorescence corresponding to the reduction of the sensor. Thus, these biofilms demonstrated a more reducing environment, especially in the 14 h samples. A reducing environment helps in scavenging ROS and other free radicals, minimizing oxidative damage to cellular components. Moreover, exposure to silver materials has been linked to uncoupling of the electron transport chain (ETC) and accumulation of reducing power (NADH) (Holt and Bard, 2005). Taken together, these results indicate that at 250 mg/L AgNPs 14 h- and 18 h-old biofilms experienced comparable oxidative stress levels and comparable reductase activity. In both biofilms, oxidative stress levels and reductase activity increased when the AgNPs concentration also increased, with the highest oxidative stress levels scored for the 18 h-old biofilms and the highest reductase activity observed in the 14 h samples, respectively. Figure 9: Oxidative stress measurement scored by DCFH-DA fluorescence assay in biofilms of C. metallidurans CH34 after 24 h exposure to AgNPs. The average values of at least three independent experiments with standard deviations are shown. Figure 10: Flow cytometry analysis of Baclight RedoxSensor™ Green fluorescence in C. metallidurans biofilms and planktonic cells exposed to AgNPs (mg/L). Cellular morphology in macroscopic biofilms C. metallidurans cells exposed to AgNPs appeared similar to untreated cells and no external damages or deformations could be observed even when clusters of nanoparticles were adherent to bacteria (Fig. 11). The biological effects of AgNPs strongly depend on their physicochemical properties such as size, shape capping agents and dissolution rate. It was suggested that variations in these parameters could induce different cellular outcomes. For example, AgNPs slowly releasing bactericidal Ag + should induce oxidative stress and morphological anomalies, while for rapidly dissolving AgNPs such phenomena may not be observed, putatively due to the fast cell killing (Bondarenko et al., 2018). In Gram-negative bacteria, the cell envelope is the first component that enters in contact with external stressors. This envelope is divided into three main compartments: the outer membrane (OM) composed of a hydrophilic external layer of lipopolysaccharides (LPS) plus a hydrophobic inner layer of phosphatidylethanolamines (PE), a hydrophilic periplasmic space containing proteins and peptidoglycans and a hydrophobic inner membrane (IM) composed in majority of PE. In literature, AgNPs have been associated with cell membrane damage and the distortion of cell morphology in gram-negative bacteria. Studies on lipid vesicles exposed to AgNPs demonstrated changes in their fluidity (Chen et al., 2012). Rupture of lipid vesicles and nanodroplets was reported as well (Martin et al., 2024; Zaitsev et al., 2021). Hence, the toxic effect of these nanomaterials has been linked to mechanisms affecting the cellular membrane. Our results showed that although AgNPs significantly reduced cell survival and increased oxidative stress in exposed biofilms, external cell morphology of C. metallidurans CH34 was not affected. Figure 11: Representative scanning electron microscope images of biofilms of C. metallidurans CH34. Untreated 14 h-old biofilm (A) and untreated 24 h-old biofilm (C) versus 14 h-old biofilm exposed to 750 mg/L AgNPs (B) and 24 h-old biofilm exposed to 750 mg/L AgNPs (D). Magnification = 30000 X. Thus, we conclude that direct disruption of the outer membrane is not the only mechanism by which AgNPs can inhibit bacterial growth. Proteomic response to AgNPs in macroscopic biofilms of C. metallidurans CH34 As demonstrated, C. metallidurans biofilms showed a different survival rate and metabolic response to AgNPs depending on their maturation stage. To gain a better understanding of the molecular pathways behind the observed phenomena, we analyzed the proteome of biofilms of different ages after 24 h of exposure to 500 mg/L AgNPs (Table 2). Overall, 2259 proteins were identified and our data indicated that macroscopic C. metallidurans biofilms, although at advanced development stages, are still dynamically evolving at the proteomic level. Resident bacterial populations continuously adjusted their protein expression profile in response to the environment (Fig. 12). Our results suggest that older biofilms were less reactive to stress than younger ones in terms of the number of differentially regulated proteins. Nevertheless, many pathways were similarly regulated in all stages in response to AgNPs and others were age-specific. Table 2 Number of differentially expressed (DE), significantly upregulated and downregulated proteins in biofilmsof different ages after 24 h of exposure to 500 mg/L AgNPs versus untreated control biofilms (p < 0.05). Age # DE proteins Upregulated Downregulated 14 h 434 221 213 18 h 325 185 140 24 h 264 124 140 Figure 12: Venn diagram representing the proteome common to all biofilm stages in addition to age-specific proteomes. (A): Upregulated. (B): Downregulated. (p < 0.05) Age-independent response to AgNPs: The presence of a core framework A set of 51 proteins was upregulated in biofilms exposed to AgNPs for all tested maturation stages compared to their respective untreated controls. Likewise, a set of 45 proteins were downregulated in all stages. Many proteins were also matching in 2 maturation stages, but they will not be discussed in this analysis. The fact that a selected set of proteins is expressed according to the same pattern independently of the biofilm’s age suggests that these pathways are essential for the adaptation of C. metallidurans to the stress elicited by AgNPs. Proteins involved in iron metabolism and siderophores biosynthesis/uptake (Supplementary table 1), such as N-(2-amino-2-carboxyethyl)-L-glutamate synthase SbnA, siderophore synthetase component protein Rmet_1113 and siderophore biosynthesis protein Rmet_1112 were upregulated in all biofilm ages, same for biopolymer transport protein ExbB and biopolymer transport protein Rmet_2279. Bacterial siderophores are meant for iron scavenging, however it was demonstrated that some can bind other metals and complexes with copper, zinc and cobalt have been reported in E. coli (Chaturvedi, Kaveri S.; Hung, Chia S.; Crowley Jan R.; Stapleton, Ann E.; Henderson, 2013; Le Brun et al., 1996). To date, alcaligin E is the only siderophore reported for C. metallidurans CH34. Surprisingly, although bacteria-mediated AgNP synthesis through Ag + reduction has attracted considerable research interest, data on the interactions between siderophores and silver ions is currently almost inexistent. For instance, in experiments with extracts from P. fluorescens the formation of silver-siderophores complexes did not occur (Bhattacharya, 2011). In P. aeruginosa PAO1, 24 h exposure to 15 mg/L AgNPs had a negative impact on the total siderophore production (Kumar et al., 2022). Thus, the exact relationship between bacterial siderophores and silver is yet to be uncovered. We speculate that the upregulation of siderophore synthesis in C. metallidurans biofilms exposed to AgNPs may be due either to a strategy for silver chelation or because of a disturbed iron homeostasis. Our results showed that expression of the periplasmic HmuT (Rmet _5376) was upregulated while the heme-binding HmuY-like Rmet_5374 was downregulated. Both are involved in heme uptake and utilization, but they have distinct characteristics and functions within the heme acquisition systems. The hemin-binding periplasmic protein HmuT binds heme in the periplasm and delivers it to ABC transporters for cytoplasmic uptake. HmuY was identified as an outer membrane protein in Porphyromonas gingivalis (Wójtowicz et al., 2009), which is exposed to the extracellular environment and specialized in exogenous heme acquisition. This suggests that heme-scavenging pathways are potentially disrupted by direct contact with Ag + or AgNPs. Periplasmic proteins are better protected from metal toxicity and the increased expression represents therefore a potential compensation mechanism. The re-adjustment in the expression of redox-involved proteins following biofilm exposure to AgNPs extended to the vital pathway of cell respiration and the electron transport chain (ETC). The ubiquinol-cytochrome c reductase iron-sulfur subunit PetA and the cytochrome c oxidase subunit 2 CoxB were downregulated. Ubiquinol-cytochrome c reductase (complex III) transfers electrons from ubiquinol to cytochrome c, while cytochrome c oxidase (complex IV) is the last enzyme in the electron transfer chain. It receives electrons from cytochrome c and transfers them to molecular oxygen, reducing it to water. Thus, the inhibition of PetA and CoxB indicated an alteration in the functioning of the traditional electron transfer pathway. On the other hand, the hydrogenase maturation factors HypD2 (Rmet_1539) and HypB2 (Rmet_1536), ubiquinol oxidase subunit 2 (Rmet_0948) and cytochrome o ubiquinol oxidase subunit I (Rmet_0949) were upregulated. HypD2 and HypB2 are involved in the maturation of [NiFe] hydrogenases, which oxidize H₂ to produce electrons (Greene et al., 2015). It was demonstrated that [NiFe] hydrogenases transfer electrons to several quinones including ubiquinone, reducing it to ubiquinol (Radu et al., 2014). In turn, ubiquinol is a substrate for both ubiquinol oxidase and cytochrome o ubiquinol oxidase, which shuttle electrons to the respiratory chain. Cytochrome o ubquinol oxidase is capable of acting as a terminal oxidase reducing molecular oxygen to water and generating a proton motive force and membrane potential (Kita et al., 1982). C. metallidurans CH34 is known for its ability to grow lithoautotrophically using molecular hydrogen as electron donor in order to fuel its ATP synthesis (Mergeay et al., 1985). Taken together, these results suggest that C. metallidurans biofilms exposed to AgNPs activated alternative effectors to support cell respiration. In fact, exposure to silver and silver nanomaterials has been shown to disrupt cell respiration and to elicit re-adjustments in the expression of involved proteins. In E. coli , silver ions are reported to induce a collapse of the proton motive force through perturbation of electron transfer. In addition, the uncoupling from oxidative phosphorylation results in an interrupted reduction of O 2 to H 2 O, an accumulation of electrons and the buildup of ROS (Holt and Bard, 2005). In Saccharomyces cerevisiae , exposure to AgNPs was followed by a reduced performance of the ETC (Márquez et al., 2018). In Geobacter sulfurreducens components of the ETC were upregulated after 24 h of anaerobic exposure to AgNO 3 (Liu et al., 2024). Several Ag-sensitive sites in the ETC have been suggested, such as the position between b-cytochromes and cytochrome a2 in addition to NADH and SDH regions (Afiqah et al., 2016). Moreover, exposure to AgNPs acted on the expression of numerous proteins containing iron-sulfur clusters, critical sites for redox active enzymes. It was demonstrated that silver ions can substitute iron in Fe-S clusters and inactivate the involved enzyme (Martic et al., 2013). The putative iron-sulfur cluster insertion protein ErpA was significantly induced in our biofilms exposed to AgNPs, suggesting that the bacteria are responding to Ag-induced damage by increasing the production of proteins that assist in the assembly or repair of Fe-S clusters. Another important pathway solicited in the C. metallidurans biofilm population after exposure to AgNPs was the chemotaxis system with the upregulation of the methyl-accepting chemotaxis sensory transducers Rmet_5250 and Rmet_3683, the chemotaxis protein CheA (Rmet_3689) and flagellin FliC2. Chemotaxis sensory transducers detect changes in the concentration of specific chemicals in the environment and help bacteria move toward or away from these stimuli. CheA is a histidine kinase that is inhibited by attractants and assumed to be activated by repellents (Bren and Eisenbach, 2000), it also controls flagellar rotational direction switches. Flagellin FliC2 is a structural protein of the bacterial flagellum and the assembly of FliC2 filaments acts as an extracellular helical propeller required for bacterial movement. Thus, in our case, chemical changes in the environment caused by AgNPs seem to be sensed. Negative chemotaxis due to metals exposure has been observed in other bacteria, such as chemotaxis away from Cu 2+ in Caulobacter crescentus (Louis et al., 2023) and a negative chemotaxis in response to Ni 2+ and Co 2+ in E. coli (Tso and Adler, 1974). Two proteins directly involved in metal detoxification were iteratively induced in C. metallidurans biofilms, the P-type ATPase CupA (Rmet_3524) and its associated copper chaperone CupC (Rmet_3525). These proteins are typically involved in copper homeostasis, however, expression of cupA is also upregulated with other metals such as Pb and Zn (Monchy et al., 2006). Therefore, their significant induction after exposure to AgNPs strongly suggests their involvement in silver binding and expulsion. Phasin (PHA-granule associated protein; Rmet_5124) and the phasin domain-containing protein PhaP (Rmet_1200) are associated with polyhydroxyalkanoate (PHA) granules, which are carbon storage materials in many bacteria. In E. coli , PhaP expression had a protective effect increasing the tolerance of the cells to superoxide stress and decreasing the expression of stress-related genes such as ibpA , dnaK, groEL and groES (Almeida et al., 2011). In C. necator , Cu-PHA intracellular precipitates were reported after exposure to copper stress and this was suggested as the mechanism behind the protective role of PHA in these conditions (Novackova et al., 2022). Their upregulation in this experiment suggests their potential involvement in the cellular strategy to manage stress induced by AgNPs. For the downregulated pathways, we report a sustained decreased expression of several extra-cytoplasmic solute receptors, i.e. Rmet_5616, Rmet_3900, Rmet_3076, Rmet_3086 and Rmet_4286, in addition to ABC transporters and other membrane related proteins suggesting a decreased membrane permeability. An overview of the age-independent proteomic response is presented in Fig. 13. Figure 13: Schematic summary of the age-independent proteomic response of C. metallidurans CH34 biofilms to 24 h exposure to 500 mg/L AgNPs. Green arrows refer to an upregulated expression while a red arrow refers to a downregulation. Age-related response to AgNPs We identified a significant number of differentially expressed proteins that were specific to each biofilm age. The 14 h-old biofilms exposed to 500 mg/L AgNPs were characterized by an important upregulation of multiple transcription regulators (IscR, FnrL, Rmet_0611, Rmet_3639 and Rmet_3673). In addition, pathways from the core response such as translation were further reinforced by the expression of supplementary ribosomal subunits, implying active protein synthesis. There was also increased expression of additional receptors for the pathway of iron uptake through siderophores. Moreover, in these biofilms one of the most downregulated protein was a Ca 2+ sensor (Rmet_3380). Previous studies have shown that silver nanoparticles can disrupt calcium homeostasis in E. coli , K. pneumoniae and Enterobacter roggenkampii with negative consequences on cell division, metabolism, signaling and viability (Lee et al., 2014; Molina-Hernandez et al., 2021). Unexpectedly, the SilA protein was specifically downregulated in 14 h-old biofilms exposed to AgNPs. This is an inner membrane protein part of a complex responsible for the transport of silver ions out of the cell. It was demonstrated that the deletion of silA from the plasmid pMG101 in E. coli J53 resulted in a complete loss of resistance to silver (Randall et al., 2014). The 18 h-old biofilms exposed to AgNPs were characterized by the upregulation of enzymes part of the regulatory network controlling nitrogen metabolism and anaerobic respiration (NarH, NirS, NosZ) and the reinforcement of ETC through increased expression of NADH:ubiquinone oxidoreductase subunit M (Rmet_0939), NADH-quinone oxidoreductase subunit A (Rmet_0927), the iron-sulfur cluster binding protein Rmet_5897 and the iron-sulfur cluster assembly scaffold protein IscU (Rmet_1026). The 24 h-old biofilms were specifically characterized by the reinforcement of detoxification and repairs systems through the increased abundance of copper and silver tricomponent efflux pump CusA, peroxiredoxin/glutaredoxin thioredoxin reductase Prx5, the DNA repair protein RadA and the isoaspartyl dipeptidase AnsB. Membrane bound proteins and proteins involved in membrane maintenance were also induced such as 3-oxoacyl-(Acyl-carrier-protein) synthase II Rmet_4389, membrane protein Rmet_1462, lipopolysaccharide heptosyltransferase 1 rfaC, hopanoid biosynthesis associated radical SAM protein hpnH while flavin and glutathione related enzymes where inhibited (flavodoxin, riboflavin biosynthesis protein RibD, hydroxyacylglutathione hydrolase gloB, glutaredoxin grxC). Finally, we noticed an upregulation of transposases in 14 h- and 24 h-old biofilms after exposure to AgNPs. The enhanced expression of transposases could suggest an increased activity of transposons, leading to greater genetic diversity. This could be an adaptive response, allowing bacteria to explore new genetic combinations that might confer survival advantages under stress conditions, a phenomenon known as stress-induced mutagenesis. Conclusion This study provides new insights into the complex interactions between the metal-resistant bacterium C. metallidurans CH34 and antimicrobial silver nanoparticles (AgNPs), revealing both population-level and molecular-scale responses. While low concentrations of AgNPs effectively inhibited growth in low-density cultures, high-density populations exhibited increased tolerance, albeit with impaired biofilm formation. Mature macroscopic biofilms demonstrated the highest resistance levels even at increased AgNPs concentrations, highlighting the protective role of biofilm structure and development stage. Interestingly, proteomic results uncovered a multifaceted molecular adaptation to silver nanoparticles. We report for the first time the presence of a core proteomic response to AgNPs independently of the biofilm age. This response involved an increased induction of pathways for iron metabolism and siderophores synthesis and uptake, the activation of chemotaxis systems, metal detoxification mechanisms in addition to re-adjusted expression of ETC effectors, promoting alternative pathways for electron trafficking. These findings enhance our understanding of bacterial resilience to nanoparticle-based antimicrobials and underscore the importance of considering microbial community density and developmental state when evaluating AgNP efficacy. Future studies should explore how these adaptive responses impact long-term ecological dynamics or influence the design of more effective nanoparticle-based antimicrobials. Declarations Funding declaration This work was supported by the Fund for Collective Fundamental Research (FRFC) grant to D.C.G (CDR J.0071.21), the European Space Agency (ESA-PRODEX) and the Belgian Science Policy (Belspo) through the BIOFILMS project (C4000129318, C4000137308), and the Tunisian Ministry of Higher Education and Scientific Research. Ethics declaration Not applicable Conflicts of interest There are no conflicts of interest to declare. References Afiqah, R.N., Paital, B., Kumar, S., Majeed, A.B.A., Tripathy, M., 2016. AgNO3 dependant modulation of glucose mediated respiration kinetics in Escherichia coli at different pH and temperature. J. Mol. Recognit. 29, 544–554. https://doi.org/10.1002/jmr.2554 Almeida, de A., Catone, M. V., Rhodius, V.A., Gross, C.A., Pettinari, M.J., 2011. Unexpected stress-Reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in Escherichia coli . Appl. Environ. Microbiol. 77, 6622–6629. https://doi.org/10.1128/AEM.05469-11 Ameen, F., AlYahya, S., Govarthanan, M., ALjahdali, N., Al-Enazi, N., Alsamhary, K., Alshehri, W.A., Alwakeel, S.S., Alharbi, S.A., 2020. Soil bacteria Cupriavidus sp. mediates the extracellular synthesis of antibacterial silver nanoparticles. J. Mol. Struct. 1202, 127233. https://doi.org/10.1016/j.molstruc.2019.127233 Ayhan, F.Y., Karaarslan, U., Gülderen, M., Özbek, Ö.A., Akaslan Kara, A., Güner Özenen, G., Ağın, H., 2024. A New Potential Threat for Nosocomial Infections: Cupriavidus metallidurans as a Cause of Bacteremia in Children. J. Dr Behcet Uz Child. s Hosp. 14, 65–68. https://doi.org/10.4274/jbuch.galenos.2024.35492 Bao, S., Wang, H., Zhang, W., Xie, Z., Fang, T., 2016. An investigation into the effects of silver nanoparticles on natural microbial communities in two freshwater sediments. Environ. Pollut. 219, 696–704. https://doi.org/10.1016/j.envpol.2016.06.071 Bhattacharya, A., 2011. Siderophore mediated metal uptake by Pseudomonas fluorescens and its comparison to Iron (III) chelation. Ceylon J. Sci. (Biological Sci. 39, 147–155. https://doi.org/10.4038/cjsbs.v39i2.3001 Bisht, K., 2023. Temperature-specific adaptations and genetic requirements in a biofilm formed by Pseudomonas aeruginosa . Front. Microbiol. 13, 1–13. https://doi.org/10.3389/fmicb.2022.1032520 Bondarenko, O.M., Sihtmäe, M., Kuzmičiova, J., Ragelienė, L., Kahru, A., Daugelavičius, R., Bondarenko, O.M., Sihtmäe, M., Kuzmičiova, J., Ragelienė, L., 2018. Plasma membrane is the target of rapid antibacterial action of silver nanoparticles in Escherichia coli and Pseudomonas aeruginosa Plasma membrane is the target of rapid antibacterial action of silver nanoparticles in Escherichia coli and Pseudomonas aeru . https://doi.org/10.2147/IJN.S177163 Bren, A., Eisenbach, M., 2000. How signals are heard during bacterial chemotaxis: Protein-protein interactions in sensory signal propagation. J. Bacteriol. 182, 6865–6873. https://doi.org/10.1128/JB.182.24.6865-6873.2000 Burkowska-But, A., Sionkowski, G., Walczak, M., 2014. Influence of stabilizers on the antimicrobial properties of silver nanoparticles introduced into natural water. J. Environ. Sci. (China) 26, 542–549. https://doi.org/10.1016/S1001-0742(13)60451-9 Cámara, M., Green, W., Macphee, C.E., Rakowska, P.D., Slater-jefferies, J., Steventon, K., Webb, J.S., 2022. Economic significance of bio films : a multidisciplinary and cross-sectoral challenge 1–8. https://doi.org/10.1038/s41522-022-00306-y Carlo Luca Romano, Delia Romano, 2017. A Modern approach to biofilm related orthopaedic implant infection, Advances in Experimental Medicine and Biology. Chaturvedi, Kaveri S.; Hung, Chia S.; Crowley Jan R.; Stapleton, Ann E.; Henderson, J.P., 2013. Pathogens During Infection. Nat. Chem. Biol. 8, 731–736. https://doi.org/10.1038/nchembio.1020.The Chen, R., Choudhary, P., Schurr, R.N., Bhattacharya, P., Brown, J.M., Chun Ke, P., 2012. Interaction of lipid vesicle with silver nanoparticle-serum albumin protein corona. Appl. Phys. Lett. 100, 3–6. https://doi.org/10.1063/1.3672035 Cheng, H.J., Wang, H., Zhang, J.Z., 2020. Phytofabrication of Silver Nanoparticles Using Three Flower Extracts and Their Antibacterial Activities Against Pathogen Ralstonia solanacearum Strain YY06 of Bacterial Wilt. Front. Microbiol. 11. https://doi.org/10.3389/fmicb.2020.02110 D’Inzeo, T., Santangelo, R., Fiori, B., De Angelis, G., Conte, V., Giaquinto, A., Palucci, I., Scoppettuolo, G., Di Florio, V., Giani, T., Sanguinetti, M., Rossolini, G.M., Spanu, T., 2015. Catheter-related bacteremia by Cupriavidus metallidurans . Diagn. Microbiol. Infect. Dis. 81, 9–12. https://doi.org/10.1016/j.diagmicrobio.2014.09.015 de Carvalho, C.C.C.R., 2018. Marine biofilms: A successful microbial strategy with economic implications. Front. Mar. Sci. 5, 1–11. https://doi.org/10.3389/fmars.2018.00126 de Duve, C., 1959. The function of intracellular hydrolases. Exp. Cell Res. 7, 169–182. https://doi.org/10.1016/0014-4827(59)90241-1 Deutsch, E.W., Bandeira, N., Perez-Riverol, Y., Sharma, V., Carver, J.J., Mendoza, L., Kundu, D.J., Wang, S., Bandla, C., Kamatchinathan, S., Hewapathirana, S., Pullman, B.S., Wertz, J., Sun, Z., Kawano, S., Okuda, S., Watanabe, Y., Maclean, B., Maccoss, M.J., Zhu, Y., Ishihama, Y., Vizcaíno, J.A., 2023. The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. 51, D1539–D1548. https://doi.org/10.1093/nar/gkac1040 Donelli, G., 2015. Biofilm-based Healthcare-associated Infections. Advances in Experimental Medicine and Biology. Ellena, G., Fahrion, J., Gupta, S., Dussap, C.G., Mazzoli, A., Leys, N., Mastroleo, F., 2024. Development and implementation of a simulated microgravity setup for edible cyanobacteria. npj Microgravity 10, 1–14. https://doi.org/10.1038/s41526-024-00436-x Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633. https://doi.org/10.1038/nrmicro2415 Garcés, M., Magnani, N.D., Pecorelli, A., Calabró, V., Marchini, T., Cáceres, L., Pambianchi, E., Galdoporpora, J., Vico, T., Salgueiro, J., Zubillaga, M., Moretton, M.A., Desimone, M.F., Alvarez, S., Valacchi, G., Evelson, P., 2021. Alterations in oxygen metabolism are associated to lung toxicity triggered by silver nanoparticles exposure. Free Radic. Biol. Med. 166, 324–336. https://doi.org/10.1016/j.freeradbiomed.2021.02.008 Greene, B.L., Wu, C.H., McTernan, P.M., Adams, M.W.W., Dyer, R.B., 2015. Proton-Coupled Electron Transfer Dynamics in the Catalytic Mechanism of a [NiFe]-Hydrogenase. J. Am. Chem. Soc. 137, 4558–4566. https://doi.org/10.1021/jacs.5b01791 Grewe, C.B., Pulz, O., 2012. The Biotechnology of Cyanobacteria II: Their Diverisity in Space and Time, Ecology of Cyanobacteria. Guo, Y., Yang, M., Xie, R.C., Compton, R.G., 2021. The oxygen reduction reaction at silver electrodes in high chloride media and the implications for silver nanoparticle toxicity. Chem. Sci. 12, 397–406. https://doi.org/10.1039/d0sc04295a Holt, K.B., Bard, A.J., 2005. Interaction of silver(I) ions with the respiratory chain of Escherichia coli : An electrochemical and scanning electrochemical microscopy study of the antimicrobial mechanism of micromolar Ag. Biochemistry 44, 13214–13223. https://doi.org/10.1021/bi0508542 Inbakandan, D., Kumar, C., Abraham, L.S., Kirubagaran, R., Venkatesan, R., Khan, S.A., 2013. Silver nanoparticles with anti microfouling effect: A study against marine biofilm forming bacteria. Colloids Surfaces B Biointerfaces 111, 636–643. https://doi.org/10.1016/j.colsurfb.2013.06.048 Jakubovics, N.S., Goodman, S.D., Mashburn-Warren, L., Stafford, G.P., Cieplik, F., 2021. The dental plaque biofilm matrix. Periodontol. 2000 86, 32–56. https://doi.org/10.1111/prd.12361 Kita, K., Kasahara, M., Anraku, Y., 1982. Formation of a membrane potential by reconstituted liposomes made with cytochrome b562-o complex, a terminal oxidase of Escherichia coli K12. J. Biol. Chem. 257, 7933–7935. https://doi.org/10.1016/s0021-9258(18)34273-x Koh, K.S., Lam, K.W., Alhede, M., Queck, S.Y., Labbate, M., Kjelleberg, S., Rice, S.A., 2007. Phenotypic diversification and adaptation of Serratia marcescens MG1 biofilm-derived morphotypes. J. Bacteriol. 189, 119–130. https://doi.org/10.1128/JB.00930-06 Kora, A.J., Arunachalam, J., 2011. Assessment of antibacterial activity of silver nanoparticles on Pseudomonas aeruginosa and its mechanism of action. World J. Microbiol. Biotechnol. 27, 1209–1216. https://doi.org/10.1007/s11274-010-0569-2 Kumar, S., Paliya, B.S., Singh, B.N., 2022. Superior inhibition of virulence and biofilm formation of Pseudomonas aeruginosa PAO1 by phyto-synthesized silver nanoparticles through anti-quorum sensing activity. Microb. Pathog. 170, 105678. https://doi.org/10.1016/j.micpath.2022.105678 Kurvet, I., Kahru, A., Bondarenko, O., Ivask, A., Ka, A., 2013. Particle-Cell Contact Enhances Antibacterial Activity of Silver Nanoparticles 8. https://doi.org/10.1371/journal.pone.0064060 Langevin, S., Vincelette, J., Bekal, S., Gaudreau, C., 2011. First case of invasive human infection caused by Cupriavidus metallidurans . J. Clin. Microbiol. 49, 744–745. https://doi.org/10.1128/JCM.01947-10 Le Brun, N.E., Keech, A.M., Mauk, M.R., Mauk, A.G., Andrews, S.C., Thomson, A.J., Moore, G.R., 1996. Charge compensated binding of divalent metals to bacterioferritin: H+ release associated with cobalt(II) and zinc(II) binding at dinuclear metal sites. FEBS Lett. 397, 159–163. https://doi.org/10.1016/S0014-5793(96)01172-6 Lee, W., Kim, K.J., Lee, D.G., 2014. A novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli . BioMetals 27, 1191–1201. https://doi.org/10.1007/s10534-014-9782-z Liao, S., Zhang, Y., Pan, X., Zhu, F., Jiang, C., Liu, Q., Cheng, Z., Dai, G., Wu, G., Wang, L., Chen, L., 2019. Antibacterial activity and mechanism of silver nanoparticles against multidrug-resistant Pseudomonas aeruginosa. Int. J. Nanomedicine 14, 1469–1487. https://doi.org/10.2147/IJN.S191340 Liu, C., Guo, D., Wen, S., Dang, Y., Sun, D., Li, P., 2024. Transcriptomic insights unveil the crucial roles of cytochromes, NADH, and pili in Ag(I) reduction by Geobacter sulfurreducens . Chemosphere 358, 142174. https://doi.org/10.1016/j.chemosphere.2024.142174 Liu, G., Zhang, Y., Knibbe, W.J., Feng, C., Liu, W., Medema, G., van der Meer, W., 2017. Potential impacts of changing supply-water quality on drinking water distribution: A review. Water Res. 116, 135–148. https://doi.org/10.1016/j.watres.2017.03.031 Louis, G., Cherry, P., Michaux, C., Rahuel-Clermont, S., Dieu, M., Tilquin, F., Maertens, L., Van Houdt, R., Renard, P., Perpete, E., Matroule, J.Y., 2023. A cytoplasmic chemoreceptor and reactive oxygen species mediate bacterial chemotaxis to copper. J. Biol. Chem. 299, 1–11. https://doi.org/10.1016/j.jbc.2023.105207 Márquez, I.G., Ghiyasvand, M., Massarsky, A., Babu, M., Samanfar, B., Omidi, K., Moon, T.W., Smith, M.L., Golshani, A., 2018. Zinc oxide and silver nanoparticles toxicity in the baker’s yeast, Saccharomyces cerevisiae. PLoS One 13, 1–19. https://doi.org/10.1371/journal.pone.0193111 Martic, M., Jakab-Simon, I.N., Haahr, L.T., Hagen, W.R., Christensen, H.E.M., 2013. Heterometallic [AgFe3S4] ferredoxin variants: Synthesis, characterization, and the first crystal structure of an engineered heterometallic iron-sulfur protein. J. Biol. Inorg. Chem. 18, 261–276. https://doi.org/10.1007/s00775-012-0971-3 Martin, C.S., Maximino, M.D., Martins, J.F.V.A., Pazin, W.M., Constantino, C.J.L., 2024. Exploring the effects of silver, silica-coated silver, and gold nanoparticles on lipid vesicles: Insights from LUVs and GUVs. J. Mol. Liq. 406. https://doi.org/10.1016/j.molliq.2024.125081 Matoulková, D., Kosar, K., Slabý, M., Sigler, K., 2012. Occurrence and species distribution of strictly anaerobic bacterium Pectinatus in brewery bottling halls. J. Am. Soc. Brew. Chem. 70, 262–267. https://doi.org/10.1094/ASBCJ-2012-0910-01 Mazur, P., Skiba-Kurek, I., Mrowiec, P., Karczewska, E., Drożdż, R., 2020. Synergistic ros-associated antimicrobial activity of silver nanoparticles and gentamicin against Staphylococcus epidermidis. Int. J. Nanomedicine 15, 3551–3562. https://doi.org/10.2147/IJN.S246484 Merbt, S.N., Bernal, S., Proia, L., Martí, E., Casamayor, E.O., 2017. Photoinhibition on natural ammonia oxidizers biofilm populations and implications for nitrogen uptake in stream biofilms. Limnol. Oceanogr. 62, 364–375. https://doi.org/10.1002/lno.10436 Mergeay, M., Nies, D., Schlegel, H.G., Gerits, J., Charles, P., Van Gijsegem, F., 1985. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 162, 328–334. https://doi.org/10.1128/jb.162.1.328-334.1985 Metcalf, D.G., Bowler, P.G., 2013. Biofilm delays wound healing: A review of the evidence. Burn. Trauma 1, 5–12. https://doi.org/10.4103/2321-3868.113329 Molina-Hernandez, J.B., Aceto, A., Bucciarelli, T., Paludi, D., Valbonetti, L., Zilli, K., Scotti, L., Chaves-López, C., 2021. The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death. Sci. Rep. 11, 1–13. https://doi.org/10.1038/s41598-021-00545-7 Monchy, S., Vallaeys, T., Bossus, A., Mergeay, M., 2006. Metal transport ATPase genes from Cupriavidus metallidurans CH34: A transcriptomic approach. Int. J. Environ. Anal. Chem. 86, 677–692. https://doi.org/10.1080/03067310600583824 Novackova, I., Hrabalova, V., Slaninova, E., Sedlacek, P., Samek, O., Koller, M., Krzyzanek, V., Hrubanova, K., Mrazova, K., Nebesarova, J., Obruca, S., 2022. The role of polyhydroxyalkanoates in adaptation of Cupriavidus necator to osmotic pressure and high concentration of copper ions. Int. J. Biol. Macromol. 206, 977–989. https://doi.org/10.1016/j.ijbiomac.2022.03.102 Perez-Riverol, Y., Bai, J., Bandla, C., García-Seisdedos, D., Hewapathirana, S., Kamatchinathan, S., Kundu, D.J., Prakash, A., Frericks-Zipper, A., Eisenacher, M., Walzer, M., Wang, S., Brazma, A., Vizcaíno, J.A., 2022. The PRIDE database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552. https://doi.org/10.1093/nar/gkab1038 Przemieniecki, S.W., Oćwieja, M., Ciesielski, S., Halecki, W., Matras, E., Gorczyca, A., 2022. Chemical Structure of Stabilizing Layers of Negatively Charged Silver Nanoparticles as an Effector of Shifts in Soil Bacterial Microbiome under Short-Term Exposure. Int. J. Environ. Res. Public Health 19. https://doi.org/10.3390/ijerph192114438 Radu, V., Frielingsdorf, S., Evans, S.D., Lenz, O., Jeuken, L.J.C., 2014. Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha . J. Am. Chem. Soc. 136, 8512–8515. https://doi.org/10.1021/ja503138p Radzig, M.A., Nadtochenko, V.A., Koksharova, O.A., Kiwi, J., Lipasova, V.A., Khmel, I.A., 2013. Antibacterial effects of silver nanoparticles on gram-negative bacteria: Influence on the growth and biofilms formation, mechanisms of action. Colloids Surfaces B Biointerfaces 102, 300–306. https://doi.org/10.1016/j.colsurfb.2012.07.039 Randall, C.P., Gupta, A., Jackson, N., Busse, D., O’Neill, A.J., 2014. Silver resistance in Gram-negative bacteria: A dissection of endogenous and exogenous mechanisms. J. Antimicrob. Chemother. 70, 1037–1046. https://doi.org/10.1093/jac/dku523 Rodríguez-Rojas, A., Kim, J.J., Johnston, P.R., Makarova, O., Eravci, M., Weise, C., Hengge, R., Rolff, J., 2020. Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress, PLoS Genetics. https://doi.org/10.1371/journal.pgen.1008649 Ryder, V.J., Chopra, I., O’Neill, A.J., 2012. Increased Mutability of Staphylococci in Biofilms as a Consequence of Oxidative Stress. PLoS One 7. https://doi.org/10.1371/journal.pone.0047695 Schacht, V.J., Neumann, L. V., Sandhi, S.K., Chen, L., Henning, T., Klar, P.J., Theophel, K., Schnell, S., Bunge, M., 2013. Effects of silver nanoparticles on microbial growth dynamics. J. Appl. Microbiol. 114, 25–35. https://doi.org/10.1111/jam.12000 Schneider, G., 2017. Antimicrobial silver nanoparticles - Regulatory situation in the European Union. Mater. Today Proc. 4, S200–S207. https://doi.org/10.1016/j.matpr.2017.09.187 Seong, M., Lee, D.G., 2017. Silver Nanoparticles Against Salmonella enterica Serotype Typhimurium: Role of Inner Membrane Dysfunction. Curr. Microbiol. 74, 661–670. https://doi.org/10.1007/s00284-017-1235-9 Sillen, W.M.A., Thijs, S., Abbamondi, G.R., Janssen, J., Weyens, N., White, J.C., Vangronsveld, J., 2015. Effects of silver nanoparticles on soil microorganisms and maize biomass are linked in the rhizosphere. Soil Biol. Biochem. 91, 14–22. https://doi.org/10.1016/j.soilbio.2015.08.019 Sim, W., Barnard, R.T., Blaskovich, M.A.T., Ziora, Z.M., 2018. Antimicrobial silver in medicinal and consumer applications: A patent review of the past decade (2007–2017). Antibiotics 7, 1–15. https://doi.org/10.3390/antibiotics7040093 Souza-Egipsy, V., González-Toril, E., Zettler, E., Amaral-Zettler, L., Aguilera, A., Amils, R., 2008. Prokaryotic community structure in algal photosynthetic biofilms from extreme acidic streams in Río Tinto (Huelva, Spain). Int. Microbiol. 11, 251–260. https://doi.org/10.2436/20.1501.01.69 Staley, B.J.T., Wiley, A.R., York, N., 1963. Biodiversity of Microbial Life: Foundation of Earth’s Biosphere. Acta Paediatr. 52, 534–534. https://doi.org/10.1111/j.1651-2227.1963.tb03813.x Stewart, P.S., Franklin, M.J., 2008. Physiological heterogeneity in biofilms. Nat. Rev. Microbiol. 6, 199–210. https://doi.org/10.1038/nrmicro1838 Stone, M., Emelko, M.B., Droppo, I.G., Silins, U., 2011. Biostabilization and erodibility of cohesive sediment deposits in wildfire-affected streams. Water Res. 45, 521–534. https://doi.org/10.1016/j.watres.2010.09.016 Thuptimdang, P., Limpiyakorn, T., McEvoy, J., Prüß, B.M., Khan, E., 2015. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity. J. Hazard. Mater. 290, 127–133. https://doi.org/10.1016/j.jhazmat.2015.02.073 Tso, W.W., Adler, J., 1974. Negative chemotaxis in Escherichia coli. J. Bacteriol. 118, 560–576. https://doi.org/10.1128/jb.118.2.560-576.1974 Valdiglesias, V., 2022. Cytotoxicity and Genotoxicity of Nanomaterials. Nanomaterials 12, 279–290. https://doi.org/10.3390/nano12040634 Wójtowicz, H., Guevara, T., Tallant, C., Olczak, M., Sroka, A., Potempa, J., Solà, M., Olczak, T., Gomis-Rüth, F.X., 2009. Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis. PLoS Pathog. 5. https://doi.org/10.1371/journal.ppat.1000419 Wu, K., Li, H., Cui, X., Feng, R., Chen, W., Jiang, Y., Tang, C., Wang, Yaohai, Wang, Yan, 2022. Mutagenesis and Resistance Development of Bacteria. Antimicrob. Agents Chemother. 66. Xiao, X., He, E.J., Lu, X.R., Wu, L.J., Fan, Y.Y., Yu, H.Q., 2021. Evaluation of antibacterial activities of silver nanoparticles on culturability and cell viability of Escherichia coli . Sci. Total Environ. 794, 148765. https://doi.org/10.1016/j.scitotenv.2021.148765 Zaitsev, S.Y., Zaitsev, I.S., Milaeva, I. V., 2021. Structural changes of the lipid model systems in the presence of enzymes or silver nanoparticles. Biointerface Res. Appl. Chem. 11, 15068–15074. https://doi.org/10.33263/BRIAC116.1506815074 Ziege, R., Tsirigoni, A.M., Large, B., Serra, D.O., Blank, K.G., Hengge, R., Fratzl, P., Bidan, C.M., 2021. Adaptation of Escherichia coli Biofilm Growth, Morphology, and Mechanical Properties to Substrate Water Content. ACS Biomater. Sci. Eng. 7, 5315–5325. https://doi.org/10.1021/acsbiomaterials.1c00927 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 22 Nov, 2025 Submission checks completed at journal 20 Nov, 2025 First submitted to journal 10 Nov, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:00:56","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":229060,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/99b3c13218c9f41c0e773968.html"},{"id":98931495,"identity":"e6044fb3-15bc-4546-9f44-988c1075d2f2","added_by":"auto","created_at":"2025-12-24 09:00:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106711,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup for the exposure of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 macroscopic biofilms to AgNPs on AgNPs-loaded MM284 agar plates. 1) Exposure of upper layers cells via AgNPs droplets. 2) Pre-formed macroscopic biofilm. 3) Porous Supor membrane. 4) AgNPs loaded MM284 agar. 5) Exposure of bottom layers cells through upwards diffusion of silver material.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/9416e1a8f817038eb82d6ed5.png"},{"id":99310673,"identity":"baec3581-1520-4fa2-a6e7-4e67b9b13fe6","added_by":"auto","created_at":"2025-12-31 16:13:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30923,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AgNP exposure on the growth of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 in MM284 (a) and on the viable count in stationary phase liquid cultures after 24 h (b). The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/86d35b350529f48910cbdaff.png"},{"id":99310436,"identity":"2f59d9d9-0d08-4fb3-8a22-d107a1a7def1","added_by":"auto","created_at":"2025-12-31 16:12:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58863,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 12.5 mg/L AgNP exposure on (a) hydrolytic activity scored by FDA assay and (b) oxidative stress levels in stationary phase liquid cultures of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 scored by DCFH-DA assay (expressed as percentage of fluorescent cells). The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/76f2bed07ea40b33c0ea5f64.png"},{"id":99310983,"identity":"79f5879f-6ad7-4533-ab2b-69ce67e5e264","added_by":"auto","created_at":"2025-12-31 16:13:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":188409,"visible":true,"origin":"","legend":"\u003cp\u003eCrystal violet quantification of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 biofilm formation on polypropylene after 24 h of exposure to 4 mg/L of AgNPs. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/646f577b0d9ad350a4d3df76.png"},{"id":98931504,"identity":"60b13814-e12d-43db-aa02-ff80abb0e663","added_by":"auto","created_at":"2025-12-24 09:00:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":417600,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrograph of a macroscopic \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 biofilm grown for 48 h on a Supor membrane. Magnification = 8000 X.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/a83458148eca7e05d17ce3f6.jpeg"},{"id":99310254,"identity":"1208e679-da90-445f-afb8-1523eb174f40","added_by":"auto","created_at":"2025-12-31 16:12:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30558,"visible":true,"origin":"","legend":"\u003cp\u003eViability of 24 h-old \u003cem\u003eC. metallidurans \u003c/em\u003eCH34 biofilms exposed for 24 h to AgNPs or AgNO\u003csub\u003e3\u003c/sub\u003e. The average values of at least three independent experiments with standard deviations are shown. Asterisks above the error bars indicate significant differences (p \u0026lt; 0.05) based on one way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/d88bf02bd5bf051c58797df2.png"},{"id":98931518,"identity":"f557f1dc-c151-419b-91c7-0e1a1a4dd688","added_by":"auto","created_at":"2025-12-24 09:00:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17849,"visible":true,"origin":"","legend":"\u003cp\u003eCell survival in biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e exposed to AgNPs at different maturation stages. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/5969d702e9d5bab76b25d208.png"},{"id":98931530,"identity":"2ba68648-37ef-44e9-a26f-23eb7a4cb7f0","added_by":"auto","created_at":"2025-12-24 09:00:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":25565,"visible":true,"origin":"","legend":"\u003cp\u003eAge-dependent evolution of cells population in biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/aba2c160254dccbdba45c5dc.png"},{"id":99310247,"identity":"9e52c14e-85fc-4a49-b323-d63bb392747b","added_by":"auto","created_at":"2025-12-31 16:12:20","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":135938,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative stress measurement scored by DCFH-DA fluorescence assay in biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 after 24 h exposure to AgNPs. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/a1e3c739576cf2f87869938b.jpeg"},{"id":99310955,"identity":"2dbbfeae-6b11-48eb-803d-2017b6952d2c","added_by":"auto","created_at":"2025-12-31 16:13:36","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":250791,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis of Baclight RedoxSensor™ Green fluorescence in \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms and planktonic cells exposed to AgNPs (mg/L).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/5fb205dc961a8ead4ae460bc.png"},{"id":98931509,"identity":"af13ac07-a7d4-4d85-82ea-c8c63c0080a7","added_by":"auto","created_at":"2025-12-24 09:00:54","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1920811,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative scanning electron microscope images of biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34. Untreated 14 h-old biofilm (A) and untreated 24 h-old biofilm (C) versus 14 h-old biofilm exposed to 750 mg/L AgNPs (B) and 24 h-old biofilm exposed to 750 mg/L AgNPs (D). Magnification = 30000 X.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/5b77177d817d3039e95b3e67.png"},{"id":98931515,"identity":"039ac282-6001-4ff6-8234-1214f31b3188","added_by":"auto","created_at":"2025-12-24 09:00:55","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":82877,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram representing the proteome common to all biofilm stages in addition to age-specific proteomes. (A): Upregulated. (B): Downregulated. (p \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/3c0524c27c3a281f6dae199a.png"},{"id":98931513,"identity":"ed13c503-9d1e-4f33-8e23-d7dcbf2975a0","added_by":"auto","created_at":"2025-12-24 09:00:55","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":116846,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic summary of the age-independent proteomic response of \u003cem\u003eC. metallidurans\u003c/em\u003eCH34 biofilms to 24 h exposure to 500 mg/L AgNPs. Green arrows refer to an upregulated expression while a red arrow refers to a downregulation.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/f53ece9461c545761a5cab56.png"},{"id":99787805,"identity":"bdb20f43-a778-4176-9f56-06b31a932d58","added_by":"auto","created_at":"2026-01-08 12:36:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4095894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/2ff2c2d6-d047-4450-978b-65018633292e.pdf"},{"id":98931514,"identity":"0eef0e8e-00a7-4455-a82d-aa3069007c58","added_by":"auto","created_at":"2025-12-24 09:00:55","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3636157,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8079940/v1/c67901f86eaab05b88b48a1f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The dynamic response of Cupriavidus metallidurans CH34 cells and biofilms to silver nanoparticles and the importance of biofilm maturation stage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnvironments where a source of humidity is present are prone to microbial proliferation in the form of biofilms. Biofilms are microbial communities encased in or glued together by a self-made matrix composed mainly of water and extracellular polymeric substances (EPS). These substances include polysaccharides, proteins and nucleic acids (Flemming and Wingender, 2010). Biofilm formation is key for persistence during unfavorable conditions; it retains moisture and shields resident cells from physical and chemical stress. It is acknowledged that the resistance of biofilm cells to stressful conditions is orders of magnitude higher than free cells. At early stages, biofilms are rarely visible without staining techniques but given enough time and nutrients, these organized microbial communities can develop into macroscopically visible structures. The thicker the biofilm, the more resilient it is as thickness acts as a protective physical barrier. Macroscopic biofilms occur naturally in humid environments and are important for the balance of ecosystems. They can be encountered in rivers and streams (Merbt et al., 2017; Souza-Egipsy et al., 2008; Stone et al., 2011) or as oceanic microbial mats, which are considered the largest biofilm structures on the planet (Staley et al., 1963). However, macroscopic biofilms can also develop on marine equipment (de Carvalho, 2018), buildings and monuments (Grewe and Pulz, 2012), water distribution infrastructures (Liu et al., 2017), industrial equipment (Matoulková et al., 2012) and more critically on teeth (Jakubovics et al., 2021), wounds (Metcalf and Bowler, 2013) and medical devices (Carlo Luca Romano and Delia Romano, 2017; Donelli, 2015).\u003c/p\u003e\n\u003cp\u003eIn the built environment, uncontrolled biofilm formation is not only a health hazard, but also a technical challenge as well. The proliferation of biofilms can cause clogging, leakage and corrosion of expensive equipment in addition to the spoilage of goods with considerable economic consequences. The global impact of biofilms on the economy was estimated to 3,967 billion dollars (Cámara et al., 2022). Although biofilm formation is attracting a growing interest in the scientific community, many fundamental aspects of this microbial lifestyle such as communication, stress adaptation or genetic and phenotypic variability remain enigmatic. In addition, these biological structures grow differently depending on their environment (i.e., nutrient flow, shear forces, humidity level) and dynamically adapt on many levels throughout time: types and numbers of resident cells, chemical gradients (Stewart and Franklin, 2008), matrix architecture and composition (Bisht, 2023; Koh et al., 2007; Ziege et al., 2021). Moreover, less is known about these dynamics within macroscopic biofilms due to their complexity. This is very challenging in an industrial context where microbial contamination must be strictly controlled and the use of antibiotics or chemical sanitation is not an option (solvents, quaternary ammonium compounds, oxidizers).\u003c/p\u003e\n\u003cp\u003eTherefore, a knowledge gap in understanding fundamental aspects of macroscopic biofilms behavior exists. In this work, we investigate the response of macroscopic biofilms of \u003cem\u003eCupriavidus metallidurans\u003c/em\u003e to silver nanoparticles (AgNPs), which are a widely employed sanitation substance (Sim et al., 2018). More specifically Agpure W10 silver nanoparticles will be used, as they are well-characterized and listed by the OECD as reference material (NM-300K) for nanotoxicology and environmental safety assessments (Schneider, 2017). AgNPs elicit a multifactor stress, being at the same time an antimicrobial, a nanomaterial and a metal. Several mechanisms behind the direct antibacterial properties of AgNPs have been hypothesized but have not been proven to full extent. For instance, the antimicrobial properties of AgNPs have been attributed mainly to a chemical origin through the release of free silver ions and the proximal delivery of chemisorbed Ag\u003csup\u003e+\u003c/sup\u003e a phenomenon also described as “nanoparticles-enhanced silver ions stress” (Kurvet et al., 2013). Variations in the AgNP properties, such as shape, size, coating and administered concentration, affect these described mechanisms and formulating a general definition about AgNPs mechanism of action is therefore challenging. The differences may be further exaggerated by cell density because of higher cell numbers, less dissolved oxygen, higher EPS content, higher content in antioxidant metabolites and cell debris capable of trapping AgNPs and Ag\u003csup\u003e+\u003c/sup\u003e ions or neutralizing oxidative stress.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. metallidurans\u003c/em\u003e is a metal-resistant species adapted to oligotrophic environments such as water distribution systems and has some strains caused medical concerns in healthcare environments (Ayhan et al., 2024; D’Inzeo et al., 2015; Langevin et al., 2011). Furthermore, closely related species have been reported to produce extracellular AgNPs (Ameen et al., 2020). We explored the interaction between silver nanoparticles and \u003cem\u003eC.\u0026nbsp;metallidurans\u003c/em\u003e type strain CH34 liquid cultures and macroscopic biofilms at different maturation stages via assessing growth and survival, the cellular response, morphology and its whole proteome to elucidate molecular mechanisms behind interactions with silver nanomaterials.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eViability and adhesion of planktonic cells exposed to a single dose of AgNPs in a liquid media\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eC. metallidurans\u003c/em\u003e CH34 was grown in Tris-buffered MM284 (pH 7) supplemented with 0.2% gluconate for 48 h at 30\u0026deg;C. Next, cell density was adjusted with fresh medium to OD\u003csub\u003e600\u003c/sub\u003e \u0026asymp; 0.1 (exponential phase cells) or OD\u003csub\u003e600\u003c/sub\u003e \u0026asymp; 1 (stationary phase cells) and exposed at 30\u0026deg;C (180 rpm shaking) to increasing concentrations of AgNPs (AgPure W10, mean particle size = 15 nm, RAS materials + technologies, Germany). For the exponential phase cells, optical density (600 nm) was measured at 2 h, 3 h, 5 h and 24 h. For the stationary phase cells, viable counts were performed after 24 h of exposure.\u003c/p\u003e\n\u003cp\u003eAdhesion on polypropylene tubes was also assessed for the stationary phase cultures. For this, the culture was discarded and the test tubes were gently rinsed 3 times with deionized water and left to dry at room temperature. Adhered biomass was stained by 0.1 % crystal violet (CV) (0.1 g of CV in 2 mL 95% ethanol and 98 mL of deionized water) for 15 minutes. Afterwards, the CV solution was removed and the tubes were rinsed 3 times with deionized water. The amount of CV was quantified by solubilizing in 4 mL of 95% ethanol and measuring absorbance at 600 nm.\u003c/p\u003e\n\u003ch2\u003eViability and adhesion of planktonic cells exposed to AgNP-loaded solid media\u003c/h2\u003e\n\u003cp\u003eSolid agar media loaded with AgNPs was prepared by adding AgNP suspensions, according to the concentrations to be tested, to MM284 agar just before pouring the plates To test viability, a viable count was performed by spotting a tenfold serial dilution of a \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003eCH34 stationary phase culture (OD\u003csub\u003e600\u003c/sub\u003e = 1) on AgNP-loaded MM284 agar and MM284 agar (control), incubating at 30\u0026deg;C for 48 h in the dark and counting colonies. To test the effect of AgNPs on biofilm growth, sterile Supor membranes (\u0026Oslash; \u0026asymp; 0.2 \u0026micro;m) were placed on the surface of AgNP-loaded MM284 agar and MM284 agar (control). Membranes were then inoculated with 30 \u0026micro;L from an OD\u003csub\u003e600\u003c/sub\u003e = 1 culture and plates were incubated in the dark at 30\u0026deg;C for 24 h. Next, biofilms were collected by removing the membranes from the agar, placing them in sterile Eppendorf tubes with 1 mL of filter-sterilized phosphate-buffered saline (PBS) and vortexing at maximum speed for at least 30 seconds until all visible biomass was homogenously resuspended. Cell enumeration by total viable count was performed by spotting 10 \u0026micro;L of a serial 10-fold dilution in sterile PBS on MM284 agar and counting colonies after 24 h at 30\u0026deg;C.\u003c/p\u003e\n\u003ch2\u003eCell viability in macroscopic biofilm exposed to a single dose of AgNPs and AgNO\u003csub\u003e3\u003c/sub\u003e on solid media\u003c/h2\u003e\n\u003cp\u003eBiofilms were grown on LB agar for 24h then transferred to petri dishes with only agar (to prevent potential cell recovery). They were gently covered by 30 \u0026micro;L of increasing concentrations of AgNPs suspension or AgNO\u003csub\u003e3\u003c/sub\u003e solution (250, 500, 750 mg/L). Control biofilms received 30 \u0026micro;L of sterile H\u003csub\u003e2\u003c/sub\u003eO. After additional 24 h in the dark at 30\u0026deg;C, biofilms were collected and resuspended as previously described and cell survival was determined via viable count.\u003c/p\u003e\n\u003ch2\u003eEffect of biofilm development stage on susceptibility to silver nanoparticles\u003c/h2\u003e\n\u003cp\u003eBiofilms were grown on Supor membranes placed on LB agar plates for 6, 9, 14, 18 and 24 h as previously described, then transferred to AgNP-loaded MM284 agar plates or to MM284 agar plates for controls. In addition, to ensure total exposure, biofilms on AgNP-loaded agar plates were also covered with 30 \u0026micro;L of AgNP suspension (with corresponding concentration) (Fig. 1). Controls were covered with 30 \u0026micro;L of sterile deionized water. After 24 h of incubation in the dark at 30\u0026deg;C, membranes were placed in 1 mL PBS, biofilms were resuspended by vortex for at least 30 seconds and cells enumerated by viable count.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the role of the biofilm matrix on the susceptibility to AgNPs, 1 mL of an OD\u003csub\u003e600\u003c/sub\u003e \u0026asymp; 1 culture of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 was centrifuged, and the resulting pellet was resuspended in 30 \u0026micro;L PBS to obtain a cell number equivalent to a 24 h-old biofilm population. This suspension was transferred to Supor membranes placed on AgNP-loaded MM284 agar plates or on MM284 agar plates for controls. After drying for 5 minutes, 30 \u0026micro;L of AgNP suspension (with the corresponding concentration) was added. Controls received 30 \u0026micro;L of sterile deionized water. All samples were incubated in the dark at 30\u0026deg;C. After 24 h, biomass on the membranes was collected and enumerated by viable count.\u003c/p\u003e\n\u003cp\u003eFigure 1: Experimental setup for the exposure of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 macroscopic biofilms to AgNPs on AgNPs-loaded MM284 agar plates. 1) Exposure of upper layers cells via AgNPs droplets. 2) Pre-formed macroscopic biofilm. 3) Porous Supor membrane. 4) AgNPs loaded MM284 agar. 5) Exposure of bottom layers cells through upwards diffusion of silver material.\u003c/p\u003e\n\u003ch2\u003eAssessment of total hydrolytic activity and oxidative stress in liquid cultures\u003c/h2\u003e\n\u003cp\u003eCells were grown in MM284 until reaching an OD\u003csub\u003e600\u003c/sub\u003e of \u0026asymp; 1 and subsequently exposed to 12.5 mg/L of AgNPs for 24 h. For total hydrolytic activity, one mL of cell suspension was washed and re-suspended in 900 \u0026micro;L PBS, to which 100\u0026micro;L of fluorescein diacetate (FDA; 200 \u0026micro;M in dimethyl sulfoxide, Sigma) was added. For assessing oxidative stress, one mL of cell suspension was washed and re-suspended in 900 \u0026micro;L PBS, to which 100 \u0026micro;L of 200 \u0026micro;M of dichlorodihydrofluorescein diacetate (H\u003csub\u003e2\u003c/sub\u003eDCFDA called also DCFH-DA)\u0026nbsp;in dimethyl sulfoxide were added. These mixtures were incubated in the dark at 30\u0026deg;C with agitation (180 rpm) for 1 h and analyzed through flow cytometry using the FITC filter (530 nm filter, 488 nm laser; Novocyte flow cytometer, Agilent) and a flow rate 66 \u0026micro;L/min. Unwanted signal was filtered by setting thresholds for FSC-H at 750 and SSC-H \u0026nbsp; at 1000. The percentage of fluorescent cells in the population was counted (threshold was based on unstained control).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssessment of reductase activity, electron chain function and oxidative stress in macroscopic biofilms exposed to AgNP-loaded agar\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBiofilms on Supor membranes were grown for 14 h and 18 h and then exposed to MM284 agar plates loaded with different concentrations of AgNPs as previously described. After 24 h in the dark at 30\u0026deg;C, biofilms were resuspended and a serial dilution in PBS was prepared. Reductase activity and electron chain function are both revealed by the fluorescence of the Baclight RedoxSensor\u0026trade; Green reagent (Thermo Fisher Scientific, Geel, Belgium). One \u0026micro;L of stock solution was diluted in 99 \u0026micro;L PBS. Two \u0026micro;L was used for 200\u0026micro;L of sample and incubated for 30 min. Next, samples were analyzed with a flow cytometer (Novocyte, Agilent) at a flow rate 66 \u0026micro;L/min and fluorescence was detected with an FITC filter (530 nm filter, 488 nm laser). Unwanted signal was filtered by setting thresholds for FSC-H at 750 and SSC-H \u0026nbsp;at 1000. For the assessment of oxidative stress levels through 2\u0026prime;,7\u0026prime;-dichlorofluorescein diacetate, the resuspended biofilms were processed as described for planktonic cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eScanning electron microscopy\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCell morphology and biofilm architecture were analyzed by scanning electron microscopy (SEM) using a Field Emission Scanning Microscope (Jeol JSM-7200F). Samples were prepared by directly fixing the biofilms grown on Supor membranes in a twice-refreshed fixation buffer (3.125 % (w/v) glutaraldehyde in 0.131 M sodium cacodylate, Sigma-Aldrich). After fixation, the specimens were washed three times with 0.15 M sodium cacodylate solution. Next, they were dehydrated in an ascending ethanol series (30, 50, 70, 90, 95, and 2 times 100% (w/v), Sigma-Aldrich) for 10 minutes in each solution. The dehydrated samples were dried in pure hexamethyldisilane (Merck Millipore) for 30 minutes then air-dried and coated with gold in an ion sputter coater (Jeol, JFC-1100E). Samples were observed at least at 10 different positions and images were collected.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProteomic analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProtein extraction and quantification\u003c/p\u003e\n\u003cp\u003eFor the proteomic analysis, the effect of AgNPs on biofilms of \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003eCH34 was investigated at the concentration of 500 mg/L versus controls. Membrane-grown biofilms were placed in 1 mL filter-sterile PBS and the biomass was resuspended by vortex for at least 30 seconds. Biofilm suspensions were then centrifuged at 9000 rpm for 15 minutes at 4\u0026deg;C. Supernatants were discarded and pellets were washed with cold and filter sterilized PBS then centrifuged again. The resulting pellets were later dissolved in 250 \u0026micro;L lysis buffer (2% SDS in 50mM Ammonium Bicarbonate). The mixtures were vortexed for 3 x 10 seconds until pellets were dissolved. These solutions were transferred to 1.5 mL tubes and incubated for 5 minutes at 95\u0026deg;C. After incubation, tubes were cooled on ice for 5 minutes, followed by a brief centrifugation. Samples were lysed using ultrasonication (4 x 10 s, amplitude 40%, 1 cycle, in between 1 min on ice) (Imlab, Boutersem, Belgium). After sonication, samples were centrifuged for 20 minutes at 20,817g (14,000 rpm) and 4\u0026deg;C. Finally, supernatants were transferred to fresh tubes. The protein quantification was carried out using the BCA assay (Merck Life Science BV, Hoeilaart, Belgium) following the manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMass spectrometry analysis of protein samples\u003c/p\u003e\n\u003cp\u003eExtracted proteins were further processed for LC-MS/MS analysis as previously described (Ellena et al., 2024). Briefly, LC-MS/MS analysis was performed using a nanoElute UHPLC (Bruker Daltonics, Bremen, Germany) connected to a QTOF-MS instrument (Impact II, Bruker Daltonics, Germany) via a CaptiveSpray nanoflow electrospray source (Bruker Daltonics, Bremen, Germany). In total, 2 \u0026micro;g of tryptic digest (in solvent A) was injected onto a trapping column setup (300 \u0026micro;m x 5 mm, C18 PepMap 300, 5 \u0026micro;m, 100 \u0026Aring;; Bruker Daltonics, Bremen, Germany). Subsequently, peptides were separated using a C18 Reprosil AQ, 1.9 \u0026micro;m, 120 \u0026Aring;, 0.075 x 150 mm column operated at 40\u0026deg;C (Bruker Daltonics, Bremen, Germany) at a flow rate of 0.2 \u0026micro;L/min. Gradient conditions were: 2\u0026ndash;35% solvent B for 100 min; 35\u0026ndash;95% solvent B for 10 min; and 95% solvent B held for 10 min (solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile). In all the full-scan measurements, a lock-mass (m/z 1221.9906, Hexakis (1H, 1H, 4H-hexafluorobutyloxy)phosphazine) (Bruker Daltonics, Bremen, Germany) was used as internal calibrator. The mass spectrometry proteomics data was submitted to the ProteomeXchange Consortium78 via the PRIDE79 partner repository (Deutsch et al., 2023; Perez-Riverol et al., 2022) with the dataset identifier PXD060314 and 10.6019/PXD060314.\u003c/p\u003e\n\u003cp\u003eRaw data processing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll raw mass spectrometry spectra files were processed using MaxQuant software version 2.0.1.0 (Max-Plank Institute of Biochemistry, Department of Proteomics and Signal Transduction, Munich, Germany) and the proteins were identified with the built-in Andromeda search engine (Cox and Mann, 2008; Tyanova et al., 2016a). The database searches were performed against a database containing all \u003cem\u003eCupriavidus metallidurans\u003c/em\u003e (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34) (\u003cem\u003eRalstonia metallidurans\u003c/em\u003e) (Taxonomy ID: 266264). UniProt protein sequences (downloaded from ftp.uniprot.org on 2024-04-23). Default MaxQuant parameter settings were used: cysteine carbamidomethylation as fixed modification, and methionine oxidation and Protein N-terminal acetylation, as variable modification; False-discovery rate (FDR) cutoffs set to 1% on peptide, protein and site decoy level; trypsin as a digestion enzyme; 7 amino acids as minimum peptide length.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNormalization and Differential Expression Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe resulting data from MaxQuant with minimum 2 unique peptides was retained and was processed to remove reverse hits and contaminants from the data, then transformed intensities to log2 for further analysis. To remove the technical variation among replicates, median normalization on log2-transformed intensities was performed using an in-house python script (Kammers et al., 2015; Tyanova et al., 2016b). Missing values in the data were replaced by value from a normal distribution with settings width =0.3 and downshift =1.8. Protein differential expression was calculated using LIMMA R package (Kammers et al., 2015), based on the empirical Bayes moderated test-statistics. The expression was considered significant at a p-value \u0026lt;0.05. Proteins with a fold-change (FC) greater than 1.5 and smaller than 0.66 (log2FC \u0026gt; 0.5849 and \u0026lt; -0.5849) were considered for interpretation.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003eEffect of AgNP exposure on the viability and metabolism of planktonic cells\u003c/h2\u003e\n\u003cp\u003eTo have a better understanding of the impact of AgNPs on \u003cem\u003eC. metallidurans\u003c/em\u003e CH34, we started by studying the effect of a single dose of this nanomaterial on planktonic cells in the exponential (assessment of growth) (Fig. 2a) and stationary phase (viability assay) (Fig. 2b). Exponential phase cultures (OD ≈ 0.1) were extremely sensitive to AgNPs and growth in liquid media was rapidly inhibited by 0.25 mg/L AgNPs. Although a comparison is not straightforward, this is much less than the minimal inhibitory concentration (MIC) of 50-nm AgNPs for \u003cem\u003eP. aeruginosa\u003c/em\u003e that was 2 mg/L (Kora and Arunachalam, 2011) and the MIC of biogenic AgNPs (with a size between 24 and 43 nm) for\u003cem\u003e\u0026nbsp;Ralstonia solanacearum\u003c/em\u003e YY06 that was 10 mg/L (Cheng et al., 2020). For \u003cem\u003eCupriavidus necator\u003c/em\u003e H16, the MIC of Ag pure W10 silver nanoparticles (size \u0026lt; 15 nm) was 60 mg/L (Schacht et al., 2013). However, as indicated, results should be compared with caution as all these studies were conducted in complex organic medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor stationary phase cultures, a 3-log reduction in CFU/mL was observed from 4 mg/L to 12.5 mg/L and full inhibition at 25 mg/L AgNPs. Not many comparable results are described in literature. Nevertheless, 2 mg/L of nano-Ag (average size 3 nm) killed 99 % of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 in 6 h (10\u003csup\u003e6\u003c/sup\u003e CFU/mL cultivated in LB) (Lee et al., 2014). For reference, Agpure W10 concentrations for antimicrobial testing recommended by the manufacturer ranged between 50 and 1000 mg/kg.\u003c/p\u003e\n\u003cp\u003eFigure 2: Effect of AgNP exposure on the growth of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 in MM284 (a) and on the viable count in stationary phase liquid cultures after 24 h (b). The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e\n\u003cp\u003eNext, \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003ecultures exposed to 12.5 mg/L were used to assess the time-dependent effect of AgNPs on hydrolytic activity and oxidative stress through FDA and DCFH-DA assays, respectively. Hydrolytic activity reflects the ability of cells to break down complex molecules using enzymes of the hydrolases family (de Duve, 1959), serving as a key indicator of cell metabolic activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAssessment of the hydrolytic activity through the intracellular transformation of the pre-fluorophore FDA into fluorescein essentially by esterases revealed that cultures exposed to 12.5 mg/L AgNPs had an increased metabolic activity at 4 h of exposure (53.48 ± 4.18 %) compared to 1 h (10.01 ± 0.49 %) and 0 h (8.61 ± 2.47 %). At 24 h, the hydrolytic activity was high and 87.51 ± 0.56 % of the bacterial populations were fluorescent (Fig 3a).\u003c/p\u003e\n\u003cp\u003eStudies assessing FDA hydrolysis in bacterial monocultures exposed to silver nanoparticles are scarce, but relevant insights can be drawn from research on the effects of silver and silver nanomaterials on hydrolytic activity in soil, lake and sediment samples. For example, increased FDA hydrolysis was observed in sediments exposed to AgNPs at a concentration of 1000 mg/kg dry weight, whereas bulk silver at the same conditions showed no significant effect (Bao et al., 2016). Another study reported increased esterase activity in soil microbiota exposed to silver ions at 10 mg/kg of soil (Przemieniecki et al., 2022). Conversely, FDA hydrolysis decreased in lake water samples spiked with at least 1 mg/L of AgNPs-PVP, while AgNPs-citrate had no significant effect (Burkowska-But et al., 2014). A similar decrease in FDA hydrolysis was observed in soil treated with nanosilver at 1000 mg/kg (Shin et al., 2012) and in the maize rhizosphere exposed to 100 mg/kg of AgNPs (Sillen et al., 2015). These findings highlight the variable impact of AgNPs on hydrolytic activity depending on the environmental context and nanoparticle type. Our results showed that an increase of hydrolytic activity was only observed starting from 4 h after exposure, therefore sampling time may also influence the outcomes and depending on the species, the metabolic reaction time to AgNPs could be different.\u003c/p\u003e\n\u003cp\u003eFigure 3: Effect of 12.5 mg/L AgNP exposure on (a) hydrolytic activity scored by FDA assay and (b) oxidative stress levels in stationary phase liquid cultures of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 scored by DCFH-DA assay (expressed as percentage of fluorescent cells). The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e\n\u003cp\u003eTo investigate oxidative stress levels after exposure to AgNPs, we performed a time-dependent measurement of the intracellular transformation of the pre-fluorophore DCFH-DA in stationary phase cultures of\u003cem\u003e\u0026nbsp;C. metallidurans\u003c/em\u003e CH34 exposed to 12.5 mg/L AgNPs versus non-exposed controls. Despite high metabolic activity in all NP-exposed replicates, oxidative stress was low at 1 h and 4 h (Fig. 3b). However, after 24 h of exposure oxidative stress increased significantly in AgNP-exposed cells. In addition, we noticed a large divergence in the 24-h replicates, where 3 replicates exhibited high fluorescence levels and 3 replicates had lower fluorescence, which resulted in a high standard deviation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was demonstrated that AgNPs increased oxidative stress levels (DCFH-DA measurements) in \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella enterica\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003e(Liao et al., 2019; Seong and Lee, 2017; Xiao et al., 2021) and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (luminol-based measurements) (Mazur et al., 2020). AgNPs can catalyze the generation of reactive oxygen species (ROS) after adsorption of molecular oxygen (O\u003csub\u003e2\u003c/sub\u003e).\u0026nbsp;In fact, adsorbed oxygen can interact with the conduction band electrons of the AgNPs, leading to a transfer of electrons from the nanoparticles to the oxygen molecules. Thus, the AgNP surface is not only highly attractive to oxygen but also facilitates electron transfer reactions. It amplifies the reduction of O₂ and consequently the production of the superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH), this phenomenon is called surface catalysis\u0026nbsp;(Guo et al., 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAgNPs can also disturb the electron transport chain, leading to an amplification of the naturally occurring electron leakage, which in turn can cause an increase in the reduction of oxygen to superoxide anions (Valdiglesias, 2022). Moreover, AgNPs bind to glutathione, a major antioxidant, and deplete the cellular reserve, thereby reducing the cell’s capacity to neutralize ROS (Molina-Hernandez et al., 2021). AgNPs can activate NADPH oxidase enzymes on cell membranes, which catalyze the production of superoxide anions from oxygen (Garcés et al., 2021). In addition, silver ions released from AgNPs can promote Fenton-like reactions in the presence of H₂O₂, generating highly reactive hydroxyl radicals.\u003c/p\u003e\n\u003cp\u003eWhen ROS levels are elevated they can cause DNA damage, which in turn increases bacterial mutation rates. It was demonstrated that the exposure of \u003cem\u003eE. coli\u003c/em\u003e to high concentrations of AgNPs (480 mg/L) resulted in the formation of mutant colonies that acquired resistance through\u0026nbsp;single-nucleotide polymorphism (SNP) affecting genes involved in silver transport and in osmoregulation (Wu et al., 2022). Experiments where \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eMG1655 was exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e also resulted in SNP mutations characterizing oxidative stress-surviving populations (Rodríguez-Rojas et al., 2020).\u003c/p\u003e\n\u003cp\u003eAdded to this, it was observed that mutation rates in biofilms are higher compared to planktonic cells, sometimes by up to 60-fold. They were also linked to endogenous oxidative stress (Ryder et al., 2012).\u003c/p\u003e\n\u003cp\u003eTherefore, exposure to AgNPs may have increased the mutagenic pressure in \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms facilitating the emergence of stress-resistant variants. This could explain why half of the replicates showed low levels of oxidative stress after 24 h compared to the initially identical replicates receiving the same treatment.\u003c/p\u003e\n\u003cp\u003eFigure 4: Crystal violet quantification of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 biofilm formation on polypropylene after 24 h of exposure to 4 mg/L of AgNPs. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e\n\u003cp\u003eOur results showed that exposure to 4 mg/L AgNPs did not stop the growth but did inhibit biofilm formation of \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003e(Fig. 4). Silver nanoparticles can prevent biofilm formation through a combination of physical, chemical and biological mechanisms. They disrupt initial adhesion by possibly acting both on the cells (surface charge, membrane damage, interference with quorum sensing) and the substrate (surface charge, adsorption). In other bacteria, the effect of AgNPs on biofilm formation is better studied in clinically relevant species than in environmental ones. For the latter, in 12h-old \u003cem\u003eP. putida\u003c/em\u003e KT2440 biofilms, cell survival was totally inhibited by 25.86 mg/L of AgNPs (40-60 nm) and further biomass development was repressed in older ones (Thuptimdang et al., 2015). Exposure for 24 h to concentrations of AgNPs (8.3 ± 1.9 nm) higher than 16 mg/L completely inhibited biofilm formation by \u003cem\u003eSerratia proteamaculans\u003c/em\u003e 94 (Radzig et al., 2013). A study by Inbakandan et al. (2013) showed that exposure to 50 mg/L of AgNPs (15-34 nm) for 4 days inhibited biofilm formation in sixteen marine bacterial species isolated from the hull of a ship (Inbakandan et al., 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, low-density cultures of \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003ewere extremely sensitive to AgNPs when exposed in mineral media, while high density cultures could tolerate higher concentrations of the antimicrobial. AgNPs at 12.5 mg/L rapidly induced a significant metabolic activity in \u003cem\u003eC. metallidurans\u003c/em\u003e cells while oxidative stress levels remained low for hours. Although these cultures were not critically affected by 4 mg/L AgNPs, at this concentration their biofilm formation capacity was compromised.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffect of AgNP exposure on pre-formed macroscopic biofilms\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFirst, biofilm formation on Supor membranes was tested (Fig 1 and 2 Supplementary material). The diffusion of nutrients from the LB agar plates through the Supor membrane allowed \u003cem\u003eC. metallidurans\u003c/em\u003e to produce visible biofilms onto the porous synthetic surface in hours. At 48 h of maturation, these biofilms were at least 3-7 µm thick (after dehydration) and densely packed with cells (Fig. 5).\u003c/p\u003e\n\u003cp\u003eBased on the previous results where stationary phase cultures were inhibited by 25 mg/L AgNPs, we estimated that a higher AgNP concentration was needed to affect these macroscopic biofilms. Therefore, 250, 500 and 750 mg/L AgNPs were used. In addition, the effect of AgNPs was compared to AgNO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eFigure 5: SEM micrograph of a macroscopic \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 biofilm grown for 48 h on a Supor membrane. Magnification = 8000 X.\u003c/p\u003e\n\u003cp\u003eFigure 6: Viability of 24 h-old \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003eCH34 biofilms exposed for 24 h to AgNPs or AgNO\u003csub\u003e3\u003c/sub\u003e. The average values of at least three independent experiments with standard deviations are shown. Asterisks above the error bars indicate significant differences (p \u0026lt; 0.05) based on one way ANOVA.\u003c/p\u003e\n\u003cp\u003eSurprisingly, exposure for 24 h to AgNP concentrations as high as 750 mg/L had no effect on the viable population in 24 h-old macroscopic biofilms. AgNO\u003csub\u003e3\u003c/sub\u003e at 750 mg/L caused a significant decrease in CFU/mL (Fig 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe setup based on AgNP-loaded MM284 agar was used to investigate the effect of this nanomaterial on pre-formed macroscopic biofilms at different growth stages (6, 9, 14, 18 and 24 h) (Fig. 7). In 6 h- and 9 h-old biofilms, a complete inactivation was observed, while cells in 14 h-old biofilms resisted 250 mg/L AgNPs and cells in 18 h- and 24 h-old biofilms survived the highest concentration. Further analysis showed that young biofilms had significantly less protein content than older ones (Fig 4. Supplementary material) and less resident cells (Fig. 8). We also noticed that the viable population in these biofilms reached a maximum at 14 h and stabilized onwards. It is possible to assume that the resistance to high concentrations of AgNPs may be due to the high cell number (more cells means more binding and inactivation of AgNPs) or to higher EPS content, or to cells metabolic state (stationary/dormant).\u003c/p\u003e\n\u003cp\u003eFigure 7: Cell survival in biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e exposed to AgNPs at different maturation stages.\u0026nbsp;The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e\n\u003cp\u003eFigure 8:\u0026nbsp;Age-dependent\u0026nbsp;evolution of cells population in biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e\n\u003cp\u003eTo test if the cell density was responsible for the resistance to 250 mg/L AgNPs observed in 14 h-old biofilms, equivalent biomass of planktonic cells (≈ 10\u003csup\u003e9\u0026nbsp;\u003c/sup\u003eCFU/mL) was deposited on Supor membranes placed on AgNP-loaded MM284 agar. After 24 h of exposure, the viable count showed no survival in the biomass exposed to AgNPs (Supplementary Figure 3). Therefore, the resistance phenotype observed in 14 h-, 18 h- and 24 h-old biofilms could not be explained by cell density.\u003c/p\u003e\n\u003ch2\u003eOxidative stress and cellular respiration\u003c/h2\u003e\n\u003cp\u003eResults demonstrated that 24 h exposure to AgNPs induced a significant increase in oxidative stress levels in the cell population within macroscopic biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 (Fig. 9). Stress levels increased when the AgNP concentration was increased from 250 mg/L to 750 mg/L. Interestingly, 18 h-old biofilms exposed to 750 mg/L AgNPs showed higher oxidative stress levels (45.01 ± 6.74 % fluorescent cells) than 14 h-old biofilms (30.72 ± 2.60 % fluorescent cells) although they showed a better survival (Fig. 7). From the redox sensor-based analysis (Fig. 10), it appears that the 24 h exposure to 250 mg/L AgNPs did not affect the redox balance in both 14 h- and 18 h-old biofilms as their fluorescence response overlays with that of the untreated biofilms. However, tripling the AgNP concentration did induce a change in the redox profile and in the fluorescence corresponding to the reduction of the sensor. Thus, these biofilms demonstrated a more reducing environment, especially in the 14 h samples. A reducing environment helps in scavenging ROS and other free radicals, minimizing oxidative damage to cellular components. Moreover, exposure to silver materials has been linked to uncoupling of the electron transport chain (ETC) and accumulation of reducing power (NADH) (Holt and Bard, 2005). Taken together, these results indicate that at 250 mg/L AgNPs 14 h- and 18 h-old biofilms experienced comparable oxidative stress levels and comparable reductase activity. In both biofilms, oxidative stress levels and reductase activity increased when the AgNPs concentration also increased, with the highest oxidative stress levels scored for the 18 h-old biofilms and the highest reductase activity observed in the 14 h samples, respectively.\u003c/p\u003e\n\u003cp\u003eFigure 9: Oxidative stress measurement scored by DCFH-DA fluorescence assay in biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 after 24 h exposure to AgNPs. The average values of at least three independent experiments with standard deviations are shown.\u003c/p\u003e\n\u003cp\u003eFigure 10: Flow cytometry analysis of Baclight RedoxSensor™ Green fluorescence in \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms and planktonic cells exposed to AgNPs (mg/L).\u003c/p\u003e\n\u003ch2\u003eCellular morphology in macroscopic biofilms\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eC. metallidurans\u003c/em\u003e cells exposed to AgNPs appeared similar to untreated cells and no external damages or deformations could be observed even when clusters of nanoparticles were adherent to bacteria (Fig. 11). The biological effects of AgNPs strongly depend on their physicochemical properties such as size, shape capping agents and dissolution rate. It was suggested that variations in these parameters could induce different cellular outcomes. For example, AgNPs slowly releasing bactericidal Ag\u003csup\u003e+\u003c/sup\u003e should induce oxidative stress and morphological anomalies, while for rapidly dissolving AgNPs such phenomena may not be observed, putatively due to the fast cell killing (Bondarenko et al., 2018). In Gram-negative bacteria, the cell envelope is the first component that enters in contact with external stressors. This envelope is divided into three main compartments: the outer membrane (OM) composed of a hydrophilic external layer of lipopolysaccharides (LPS) plus a hydrophobic inner layer of phosphatidylethanolamines (PE), a hydrophilic periplasmic space containing proteins and peptidoglycans and a hydrophobic inner membrane (IM) composed in majority of PE. In literature, AgNPs have been associated with cell membrane damage and the distortion of cell morphology in gram-negative bacteria. Studies on lipid vesicles exposed to AgNPs demonstrated changes in their fluidity (Chen et al., 2012). Rupture of lipid vesicles and nanodroplets was reported as well (Martin et al., 2024; Zaitsev et al., 2021). Hence, the toxic effect of these nanomaterials has been linked to mechanisms affecting the cellular membrane. Our results showed that although AgNPs significantly reduced cell survival and increased oxidative stress in exposed biofilms, external cell morphology of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 was not affected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 11: Representative scanning electron microscope images of biofilms of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34. Untreated 14 h-old biofilm (A) and untreated 24 h-old biofilm (C) versus 14 h-old biofilm exposed to 750 mg/L AgNPs (B) and 24 h-old biofilm exposed to 750 mg/L AgNPs (D). Magnification = 30000 X.\u003c/p\u003e\n\u003cp\u003eThus, we conclude that direct disruption of the outer membrane is not the only mechanism by which AgNPs can inhibit bacterial growth.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eProteomic response to AgNPs in macroscopic biofilms of\u0026nbsp;C. metallidurans\u0026nbsp;CH34\u003c/h2\u003e\n\u003cp\u003eAs demonstrated, \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms showed a different survival rate and metabolic response to AgNPs depending on their maturation stage. To gain a better understanding of the molecular pathways behind the observed phenomena, we analyzed the proteome of biofilms of different ages after 24 h of exposure to 500 mg/L AgNPs (Table 2). Overall, 2259 proteins were identified and our data indicated that macroscopic \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms, although at advanced development stages, are still dynamically evolving at the proteomic level. Resident bacterial populations continuously adjusted their protein expression profile in response to the environment (Fig. 12). Our results suggest that older biofilms were less reactive to stress than younger ones in terms of the number of differentially regulated proteins. Nevertheless, many pathways were similarly regulated in all stages in response to AgNPs and others were age-specific.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"377\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003eTable 2 Number of differentially expressed (DE), significantly upregulated and downregulated proteins in biofilmsof different ages after 24 h of exposure to 500 mg/L AgNPs versus untreated control biofilms (p \u0026lt; 0.05).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e# DE proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eUpregulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDownregulated\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e24 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eFigure 12: Venn diagram representing the proteome common to all biofilm stages in addition to age-specific proteomes. (A): Upregulated. (B): Downregulated. (p \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003eAge-independent response to AgNPs: The presence of a core framework\u003c/p\u003e\n\u003cp\u003eA set of 51 proteins was upregulated in biofilms exposed to AgNPs for all tested maturation stages compared to their respective untreated controls. Likewise, a set of 45 proteins were downregulated in all stages. Many proteins were also matching in 2 maturation stages, but they will not be discussed in this analysis. The fact that a selected set of proteins is expressed according to the same pattern independently of the biofilm’s age suggests that these pathways are essential for the adaptation of \u003cem\u003eC. metallidurans\u003c/em\u003e to the stress elicited by AgNPs. Proteins involved in iron metabolism and siderophores biosynthesis/uptake (Supplementary table 1), such as N-(2-amino-2-carboxyethyl)-L-glutamate synthase SbnA, siderophore synthetase component protein Rmet_1113 and siderophore biosynthesis protein Rmet_1112 were upregulated in all biofilm ages, same for biopolymer transport protein ExbB and biopolymer transport protein Rmet_2279. Bacterial siderophores are meant for iron scavenging, however it was demonstrated that some can bind other metals and complexes with copper, zinc and cobalt have been reported in \u003cem\u003eE. coli\u003c/em\u003e (Chaturvedi, Kaveri S.; Hung, Chia S.; Crowley Jan R.; Stapleton, Ann E.; Henderson, 2013; Le Brun et al., 1996). To date, alcaligin E is the only siderophore reported for \u003cem\u003eC. metallidurans\u0026nbsp;\u003c/em\u003eCH34. Surprisingly, although bacteria-mediated AgNP synthesis through Ag\u003csup\u003e+\u003c/sup\u003e reduction has attracted considerable research interest, data on the interactions between siderophores and silver ions is currently almost inexistent. For instance, in experiments with extracts from \u003cem\u003eP. fluorescens\u003c/em\u003e the formation of silver-siderophores complexes did not occur (Bhattacharya, 2011). In \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, 24 h exposure to 15 mg/L AgNPs had a negative impact on the total siderophore production (Kumar et al., 2022). Thus, the exact relationship between bacterial siderophores and silver is yet to be uncovered. We speculate that the upregulation of siderophore synthesis in \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms exposed to AgNPs may be due either to a strategy for silver chelation or because of a disturbed iron homeostasis.\u003c/p\u003e\n\u003cp\u003eOur results showed that expression of the periplasmic HmuT (Rmet _5376) was upregulated while the heme-binding HmuY-like Rmet_5374 was downregulated. Both are involved in heme uptake and utilization, but they have distinct characteristics and functions within the heme acquisition systems. The hemin-binding periplasmic protein HmuT binds heme in the periplasm and delivers it to ABC transporters for cytoplasmic uptake. HmuY was identified as an outer membrane protein in \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (Wójtowicz et al., 2009), which is exposed to the extracellular environment and specialized in exogenous heme acquisition. This suggests that heme-scavenging pathways are potentially disrupted by direct contact with Ag\u003csup\u003e+\u003c/sup\u003e or AgNPs. Periplasmic proteins are better protected from metal toxicity and the increased expression represents therefore a potential compensation mechanism. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe re-adjustment in the expression of redox-involved proteins following biofilm exposure to AgNPs extended to the vital pathway of cell respiration and the electron transport chain (ETC). The ubiquinol-cytochrome c reductase iron-sulfur subunit PetA and the cytochrome c oxidase subunit 2 CoxB were downregulated. Ubiquinol-cytochrome c reductase (complex III) transfers electrons from ubiquinol to cytochrome c, while cytochrome c oxidase (complex IV) is the last enzyme in the electron transfer chain. It receives electrons from cytochrome c and transfers them to molecular oxygen, reducing it to water. Thus, the inhibition of PetA and CoxB indicated an alteration in the functioning of the traditional electron transfer pathway. On the other hand, the hydrogenase maturation factors HypD2 (Rmet_1539) and HypB2 (Rmet_1536), ubiquinol oxidase subunit 2 (Rmet_0948) and cytochrome o ubiquinol oxidase subunit I (Rmet_0949) were upregulated. HypD2 and HypB2 are involved in the maturation of [NiFe]\u0026nbsp;hydrogenases, which oxidize H₂ to produce electrons\u0026nbsp;(Greene et al., 2015).\u0026nbsp;It was demonstrated that [NiFe] hydrogenases transfer electrons to several quinones including ubiquinone, reducing it to ubiquinol\u0026nbsp;(Radu et al., 2014). In turn, ubiquinol is a substrate for both ubiquinol oxidase and cytochrome o ubiquinol oxidase, which shuttle electrons to the respiratory chain. Cytochrome o ubquinol oxidase is capable of acting as a terminal oxidase reducing molecular oxygen to water and generating a proton motive force and membrane potential\u0026nbsp;(Kita et al., 1982). \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 is known for its ability to grow lithoautotrophically using molecular hydrogen as electron donor in order to fuel its ATP synthesis\u0026nbsp;(Mergeay et al., 1985).\u0026nbsp;Taken together, these results suggest that \u003cem\u003eC.\u0026nbsp;metallidurans\u003c/em\u003e biofilms exposed to AgNPs activated alternative effectors to support cell respiration. In fact, exposure to silver and silver nanomaterials has been shown to disrupt cell respiration and to elicit re-adjustments in the expression of involved proteins. In \u003cem\u003eE. coli\u003c/em\u003e, silver ions are reported to induce a collapse of the proton motive force through perturbation of electron transfer. In addition, the uncoupling from oxidative phosphorylation results in an interrupted reduction of O\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO, an accumulation of electrons and the buildup of ROS\u0026nbsp;(Holt and Bard, 2005). In \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, exposure to AgNPs was followed by a reduced performance of the ETC\u0026nbsp;(Márquez et al., 2018). In \u003cem\u003eGeobacter sulfurreducens\u003c/em\u003e components of the ETC were upregulated after 24 h of anaerobic exposure to AgNO\u003csub\u003e3\u003c/sub\u003e (Liu et al., 2024). Several Ag-sensitive sites in the ETC have been suggested, such as the position between b-cytochromes and cytochrome a2 in addition to NADH and SDH regions\u0026nbsp;(Afiqah et al., 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, exposure to AgNPs acted on the expression of numerous proteins containing iron-sulfur clusters, critical sites for redox active enzymes. It was demonstrated that silver ions can substitute iron in Fe-S clusters and inactivate the involved enzyme (Martic et al., 2013). The putative iron-sulfur cluster insertion protein ErpA was significantly induced in our biofilms exposed to AgNPs, suggesting that the bacteria are responding to Ag-induced damage by increasing the production of proteins that assist in the assembly or repair of Fe-S clusters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother important pathway solicited in the \u003cem\u003eC. metallidurans\u003c/em\u003e biofilm population after exposure to AgNPs was the chemotaxis system with the upregulation of the methyl-accepting chemotaxis sensory transducers Rmet_5250 and Rmet_3683, the chemotaxis protein CheA (Rmet_3689) and flagellin FliC2. Chemotaxis sensory transducers detect changes in the concentration of specific chemicals in the environment and help bacteria move toward or away from these stimuli. CheA is a histidine kinase that is inhibited by attractants and assumed to be activated by repellents (Bren and Eisenbach, 2000), it also controls flagellar rotational direction switches. Flagellin FliC2 is a structural protein of the bacterial flagellum and the assembly of FliC2 filaments acts as an extracellular helical propeller required for bacterial movement. Thus, in our case, chemical changes in the environment caused by AgNPs seem to be sensed. Negative chemotaxis due to metals exposure has been observed in other bacteria, such as chemotaxis away from Cu\u003csup\u003e2+\u003c/sup\u003e in \u003cem\u003eCaulobacter crescentus\u003c/em\u003e (Louis et al., 2023) and a negative chemotaxis in response to Ni\u003csup\u003e2+\u003c/sup\u003e and Co\u003csup\u003e2+\u003c/sup\u003e in \u003cem\u003eE. coli\u003c/em\u003e (Tso and Adler, 1974).\u003c/p\u003e\n\u003cp\u003eTwo proteins directly involved in metal detoxification were iteratively induced in \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms, the P-type ATPase CupA (Rmet_3524) and its associated copper chaperone CupC (Rmet_3525). These proteins are typically involved in copper homeostasis, however, expression of \u003cem\u003ecupA\u003c/em\u003e is also upregulated with other metals such as Pb and Zn (Monchy et al., 2006). Therefore, their significant induction after exposure to AgNPs strongly suggests their involvement in silver binding and expulsion.\u003c/p\u003e\n\u003cp\u003ePhasin (PHA-granule associated protein; Rmet_5124) and the phasin domain-containing protein PhaP (Rmet_1200) are associated with polyhydroxyalkanoate (PHA) granules, which are carbon storage materials in many bacteria. In \u003cem\u003eE. coli\u003c/em\u003e, PhaP expression had a protective effect increasing the tolerance of the cells to superoxide stress and decreasing the expression of stress-related genes such as \u003cem\u003eibpA\u003c/em\u003e, dnaK, \u003cem\u003egroEL\u003c/em\u003e and \u003cem\u003egroES\u003c/em\u003e (Almeida et al., 2011). In \u003cem\u003eC. necator\u003c/em\u003e, Cu-PHA intracellular precipitates were reported after exposure to copper stress and this was suggested as the mechanism behind the protective role of PHA in these conditions (Novackova et al., 2022). Their upregulation in this experiment suggests their potential involvement in the cellular strategy to manage stress induced by AgNPs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the downregulated pathways, we report a sustained decreased expression of several extra-cytoplasmic solute receptors, i.e. Rmet_5616, Rmet_3900, Rmet_3076, Rmet_3086 and Rmet_4286, in addition to ABC transporters and other membrane related proteins suggesting a decreased membrane permeability.\u003c/p\u003e\n\u003cp\u003eAn overview of the age-independent proteomic response is presented in Fig. 13.\u003c/p\u003e\n\u003cp\u003eFigure 13: Schematic summary of the age-independent proteomic response of \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 biofilms to 24 h exposure to 500 mg/L AgNPs. Green arrows refer to an upregulated expression while a red arrow refers to a downregulation.\u003c/p\u003e\n\u003cp\u003eAge-related response to AgNPs\u003c/p\u003e\n\u003cp\u003eWe identified a significant number of differentially expressed proteins that were specific to each biofilm age. The 14 h-old biofilms exposed to 500 mg/L AgNPs were characterized by an important upregulation of multiple transcription regulators (IscR,\u0026nbsp;FnrL, Rmet_0611, Rmet_3639 and Rmet_3673). In addition, pathways from the core response such as translation were further reinforced by the expression of supplementary ribosomal subunits, implying active protein synthesis. There was also increased expression of additional receptors for the pathway of iron uptake through siderophores. Moreover, in these biofilms one of the most downregulated protein was a Ca\u003csup\u003e2+\u003c/sup\u003e sensor (Rmet_3380). Previous studies have shown that silver nanoparticles can disrupt calcium homeostasis in \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eEnterobacter roggenkampii\u003c/em\u003e with negative consequences on cell division, metabolism, signaling and viability (Lee et al., 2014; Molina-Hernandez et al., 2021). Unexpectedly, the SilA protein was specifically downregulated in 14 h-old biofilms exposed to AgNPs. This is an inner membrane protein part of a complex responsible for the transport of silver ions out of the cell. It was demonstrated that the deletion of \u003cem\u003esilA\u003c/em\u003e from the plasmid pMG101 in \u003cem\u003eE. coli\u003c/em\u003e J53 resulted in a complete loss of resistance to silver (Randall et al., 2014).\u003c/p\u003e\n\u003cp\u003eThe 18 h-old biofilms exposed to AgNPs were characterized by the upregulation of enzymes part of the regulatory network controlling nitrogen metabolism and anaerobic respiration (NarH, NirS, NosZ) and the reinforcement of ETC through increased expression of NADH:ubiquinone oxidoreductase subunit M (Rmet_0939), NADH-quinone oxidoreductase subunit A (Rmet_0927), the iron-sulfur cluster binding protein Rmet_5897 and the iron-sulfur cluster assembly scaffold protein IscU (Rmet_1026).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 24 h-old biofilms were specifically characterized by the reinforcement of detoxification and repairs systems through the increased abundance of copper and silver tricomponent efflux pump CusA, peroxiredoxin/glutaredoxin thioredoxin reductase Prx5, the DNA repair protein RadA and the isoaspartyl dipeptidase AnsB. Membrane bound proteins and proteins involved in membrane maintenance were also induced such as 3-oxoacyl-(Acyl-carrier-protein) synthase II Rmet_4389, membrane protein Rmet_1462, lipopolysaccharide heptosyltransferase 1 rfaC, hopanoid biosynthesis associated radical SAM protein hpnH while flavin and glutathione related enzymes where inhibited (flavodoxin, riboflavin biosynthesis protein RibD, hydroxyacylglutathione hydrolase gloB, glutaredoxin grxC).\u003c/p\u003e\n\u003cp\u003eFinally, we noticed an upregulation of transposases in 14 h- and 24 h-old biofilms after exposure to AgNPs. The enhanced expression of transposases could suggest an increased activity of transposons, leading to greater genetic diversity. This could be an adaptive response, allowing bacteria to explore new genetic combinations that might confer survival advantages under stress conditions, a phenomenon known as stress-induced mutagenesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides new insights into the complex interactions between the metal-resistant bacterium \u003cem\u003eC. metallidurans\u003c/em\u003e CH34 and antimicrobial silver nanoparticles (AgNPs), revealing both population-level and molecular-scale responses. While low concentrations of AgNPs effectively inhibited growth in low-density cultures, high-density populations exhibited increased tolerance, albeit with impaired biofilm formation. Mature macroscopic biofilms demonstrated the highest resistance levels even at increased AgNPs concentrations, highlighting the protective role of biofilm structure and development stage.\u003c/p\u003e\n\u003cp\u003eInterestingly, proteomic results uncovered a multifaceted molecular adaptation to silver nanoparticles. We report for the first time the presence of a core proteomic response to AgNPs independently of the biofilm age. This response involved an increased induction of pathways for iron metabolism and siderophores synthesis and uptake, the activation of chemotaxis systems, metal detoxification mechanisms in addition to re-adjusted expression of ETC effectors, promoting alternative pathways for electron trafficking.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings enhance our understanding of bacterial resilience to nanoparticle-based antimicrobials and underscore the importance of considering microbial community density and developmental state when evaluating AgNP efficacy. Future studies should explore how these adaptive responses impact long-term ecological dynamics or influence the design of more effective nanoparticle-based antimicrobials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Fund for Collective Fundamental Research (FRFC) grant to D.C.G (CDR J.0071.21), the European Space Agency (ESA-PRODEX) and the Belgian Science Policy (Belspo) through the BIOFILMS project (C4000129318, C4000137308), and the Tunisian Ministry of Higher Education and Scientific Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAfiqah, R.N., Paital, B., Kumar, S., Majeed, A.B.A., Tripathy, M., 2016. AgNO3 dependant modulation of glucose mediated respiration kinetics in \u003cem\u003eEscherichia coli\u003c/em\u003e at different pH and temperature. J. Mol. Recognit. 29, 544\u0026ndash;554. https://doi.org/10.1002/jmr.2554\u003c/li\u003e\n \u003cli\u003eAlmeida, \u0026nbsp;de A., Catone, M. V., Rhodius, V.A., Gross, C.A., Pettinari, M.J., 2011. Unexpected stress-Reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in \u003cem\u003eEscherichia coli\u003c/em\u003e. Appl. Environ. Microbiol. 77, 6622\u0026ndash;6629. https://doi.org/10.1128/AEM.05469-11\u003c/li\u003e\n \u003cli\u003eAmeen, F., AlYahya, S., Govarthanan, M., ALjahdali, N., Al-Enazi, N., Alsamhary, K., Alshehri, W.A., Alwakeel, S.S., Alharbi, S.A., 2020. Soil bacteria\u003cem\u003e\u0026nbsp;Cupriavidus\u003c/em\u003e sp. mediates the extracellular synthesis of antibacterial silver nanoparticles. J. Mol. Struct. 1202, 127233. https://doi.org/10.1016/j.molstruc.2019.127233\u003c/li\u003e\n \u003cli\u003eAyhan, F.Y., Karaarslan, U., G\u0026uuml;lderen, M., \u0026Ouml;zbek, \u0026Ouml;.A., Akaslan Kara, A., G\u0026uuml;ner \u0026Ouml;zenen, G., Ağın, H., 2024. A New Potential Threat for Nosocomial Infections: \u003cem\u003eCupriavidus metallidurans\u0026nbsp;\u003c/em\u003eas a Cause of Bacteremia in Children. J. Dr Behcet Uz Child. s Hosp. 14, 65\u0026ndash;68. https://doi.org/10.4274/jbuch.galenos.2024.35492\u003c/li\u003e\n \u003cli\u003eBao, S., Wang, H., Zhang, W., Xie, Z., Fang, T., 2016. An investigation into the effects of silver nanoparticles on natural microbial communities in two freshwater sediments. Environ. Pollut. 219, 696\u0026ndash;704. https://doi.org/10.1016/j.envpol.2016.06.071\u003c/li\u003e\n \u003cli\u003eBhattacharya, A., 2011. Siderophore mediated metal uptake by \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e and its comparison to Iron (III) chelation. Ceylon J. Sci. (Biological Sci. 39, 147\u0026ndash;155. https://doi.org/10.4038/cjsbs.v39i2.3001\u003c/li\u003e\n \u003cli\u003eBisht, K., 2023. Temperature-specific adaptations and genetic requirements in a biofilm formed by\u003cem\u003e\u0026nbsp;Pseudomonas aeruginosa\u003c/em\u003e. Front. Microbiol. 13, 1\u0026ndash;13. https://doi.org/10.3389/fmicb.2022.1032520\u003c/li\u003e\n \u003cli\u003eBondarenko, O.M., Sihtm\u0026auml;e, M., Kuzmičiova, J., Ragelienė, L., Kahru, A., Daugelavičius, R., Bondarenko, O.M., Sihtm\u0026auml;e, M., Kuzmičiova, J., Ragelienė, L., 2018. Plasma membrane is the target of rapid antibacterial action of silver nanoparticles in \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e Plasma membrane is the target of rapid antibacterial action of silver nanoparticles in \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePseudomonas aeru\u003c/em\u003e. https://doi.org/10.2147/IJN.S177163\u003c/li\u003e\n \u003cli\u003eBren, A., Eisenbach, M., 2000. How signals are heard during bacterial chemotaxis: Protein-protein interactions in sensory signal propagation. J. Bacteriol. 182, 6865\u0026ndash;6873. https://doi.org/10.1128/JB.182.24.6865-6873.2000\u003c/li\u003e\n \u003cli\u003eBurkowska-But, A., Sionkowski, G., Walczak, M., 2014. Influence of stabilizers on the antimicrobial properties of silver nanoparticles introduced into natural water. J. Environ. Sci. (China) 26, 542\u0026ndash;549. https://doi.org/10.1016/S1001-0742(13)60451-9\u003c/li\u003e\n \u003cli\u003eC\u0026aacute;mara, M., Green, W., Macphee, C.E., Rakowska, P.D., Slater-jefferies, J., Steventon, K., Webb, J.S., 2022. Economic significance of bio films : a multidisciplinary and cross-sectoral challenge 1\u0026ndash;8. https://doi.org/10.1038/s41522-022-00306-y\u003c/li\u003e\n \u003cli\u003eCarlo Luca Romano, Delia Romano, 2017. A Modern approach to biofilm related orthopaedic implant infection, Advances in Experimental Medicine and Biology.\u003c/li\u003e\n \u003cli\u003eChaturvedi, Kaveri S.; Hung, Chia S.; Crowley Jan R.; Stapleton, Ann E.; Henderson, J.P., 2013. Pathogens During Infection. Nat. Chem. Biol. 8, 731\u0026ndash;736. https://doi.org/10.1038/nchembio.1020.The\u003c/li\u003e\n \u003cli\u003eChen, R., Choudhary, P., Schurr, R.N., Bhattacharya, P., Brown, J.M., Chun Ke, P., 2012. Interaction of lipid vesicle with silver nanoparticle-serum albumin protein corona. Appl. Phys. Lett. 100, 3\u0026ndash;6. https://doi.org/10.1063/1.3672035\u003c/li\u003e\n \u003cli\u003eCheng, H.J., Wang, H., Zhang, J.Z., 2020. Phytofabrication of Silver Nanoparticles Using Three Flower Extracts and Their Antibacterial Activities Against Pathogen \u003cem\u003eRalstonia\u003c/em\u003e \u003cem\u003esolanacearum\u003c/em\u003e Strain YY06 of Bacterial Wilt. Front. Microbiol. 11. https://doi.org/10.3389/fmicb.2020.02110\u003c/li\u003e\n \u003cli\u003eD\u0026rsquo;Inzeo, T., Santangelo, R., Fiori, B., De Angelis, G., Conte, V., Giaquinto, A., Palucci, I., Scoppettuolo, G., Di Florio, V., Giani, T., Sanguinetti, M., Rossolini, G.M., Spanu, T., 2015. Catheter-related bacteremia by \u003cem\u003eCupriavidus\u003c/em\u003e \u003cem\u003emetallidurans\u003c/em\u003e. Diagn. Microbiol. Infect. Dis. 81, 9\u0026ndash;12. https://doi.org/10.1016/j.diagmicrobio.2014.09.015\u003c/li\u003e\n \u003cli\u003ede Carvalho, C.C.C.R., 2018. Marine biofilms: A successful microbial strategy with economic implications. Front. Mar. Sci. 5, 1\u0026ndash;11. https://doi.org/10.3389/fmars.2018.00126\u003c/li\u003e\n \u003cli\u003ede Duve, C., 1959. The function of intracellular hydrolases. Exp. Cell Res. 7, 169\u0026ndash;182. https://doi.org/10.1016/0014-4827(59)90241-1\u003c/li\u003e\n \u003cli\u003eDeutsch, E.W., Bandeira, N., Perez-Riverol, Y., Sharma, V., Carver, J.J., Mendoza, L., Kundu, D.J., Wang, S., Bandla, C., Kamatchinathan, S., Hewapathirana, S., Pullman, B.S., Wertz, J., Sun, Z., Kawano, S., Okuda, S., Watanabe, Y., Maclean, B., Maccoss, M.J., Zhu, Y., Ishihama, Y., Vizca\u0026iacute;no, J.A., 2023. The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. 51, D1539\u0026ndash;D1548. https://doi.org/10.1093/nar/gkac1040\u003c/li\u003e\n \u003cli\u003eDonelli, G., 2015. Biofilm-based Healthcare-associated Infections. Advances in Experimental Medicine and Biology.\u003c/li\u003e\n \u003cli\u003eEllena, G., Fahrion, J., Gupta, S., Dussap, C.G., Mazzoli, A., Leys, N., Mastroleo, F., 2024. Development and implementation of a simulated microgravity setup for edible cyanobacteria. npj Microgravity 10, 1\u0026ndash;14. https://doi.org/10.1038/s41526-024-00436-x\u003c/li\u003e\n \u003cli\u003eFlemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623\u0026ndash;633. https://doi.org/10.1038/nrmicro2415\u003c/li\u003e\n \u003cli\u003eGarc\u0026eacute;s, M., Magnani, N.D., Pecorelli, A., Calabr\u0026oacute;, V., Marchini, T., C\u0026aacute;ceres, L., Pambianchi, E., Galdoporpora, J., Vico, T., Salgueiro, J., Zubillaga, M., Moretton, M.A., Desimone, M.F., Alvarez, S., Valacchi, G., Evelson, P., 2021. Alterations in oxygen metabolism are associated to lung toxicity triggered by silver nanoparticles exposure. Free Radic. Biol. Med. 166, 324\u0026ndash;336. https://doi.org/10.1016/j.freeradbiomed.2021.02.008\u003c/li\u003e\n \u003cli\u003eGreene, B.L., Wu, C.H., McTernan, P.M., Adams, M.W.W., Dyer, R.B., 2015. Proton-Coupled Electron Transfer Dynamics in the Catalytic Mechanism of a [NiFe]-Hydrogenase. J. Am. Chem. Soc. 137, 4558\u0026ndash;4566. https://doi.org/10.1021/jacs.5b01791\u003c/li\u003e\n \u003cli\u003eGrewe, C.B., Pulz, O., 2012. The Biotechnology of Cyanobacteria II: Their Diverisity in Space and Time, Ecology of Cyanobacteria.\u003c/li\u003e\n \u003cli\u003eGuo, Y., Yang, M., Xie, R.C., Compton, R.G., 2021. The oxygen reduction reaction at silver electrodes in high chloride media and the implications for silver nanoparticle toxicity. Chem. Sci. 12, 397\u0026ndash;406. https://doi.org/10.1039/d0sc04295a\u003c/li\u003e\n \u003cli\u003eHolt, K.B., Bard, A.J., 2005. Interaction of silver(I) ions with the respiratory chain of \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e: An electrochemical and scanning electrochemical microscopy study of the antimicrobial mechanism of micromolar Ag. Biochemistry 44, 13214\u0026ndash;13223. https://doi.org/10.1021/bi0508542\u003c/li\u003e\n \u003cli\u003eInbakandan, D., Kumar, C., Abraham, L.S., Kirubagaran, R., Venkatesan, R., Khan, S.A., 2013. Silver nanoparticles with anti microfouling effect: A study against marine biofilm forming bacteria. Colloids Surfaces B Biointerfaces 111, 636\u0026ndash;643. https://doi.org/10.1016/j.colsurfb.2013.06.048\u003c/li\u003e\n \u003cli\u003eJakubovics, N.S., Goodman, S.D., Mashburn-Warren, L., Stafford, G.P., Cieplik, F., 2021. The dental plaque biofilm matrix. Periodontol. 2000 86, 32\u0026ndash;56. https://doi.org/10.1111/prd.12361\u003c/li\u003e\n \u003cli\u003eKita, K., Kasahara, M., Anraku, Y., 1982. Formation of a membrane potential by reconstituted liposomes made with cytochrome b562-o complex, a terminal oxidase of \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e K12. J. Biol. Chem. 257, 7933\u0026ndash;7935. https://doi.org/10.1016/s0021-9258(18)34273-x\u003c/li\u003e\n \u003cli\u003eKoh, K.S., Lam, K.W., Alhede, M., Queck, S.Y., Labbate, M., Kjelleberg, S., Rice, S.A., 2007. Phenotypic diversification and adaptation of \u003cem\u003eSerratia marcescens\u003c/em\u003e MG1 biofilm-derived morphotypes. J. Bacteriol. 189, 119\u0026ndash;130. https://doi.org/10.1128/JB.00930-06\u003c/li\u003e\n \u003cli\u003eKora, A.J., Arunachalam, J., 2011. Assessment of antibacterial activity of silver nanoparticles on \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and its mechanism of action. World J. Microbiol. Biotechnol. 27, 1209\u0026ndash;1216. https://doi.org/10.1007/s11274-010-0569-2\u003c/li\u003e\n \u003cli\u003eKumar, S., Paliya, B.S., Singh, B.N., 2022. Superior inhibition of virulence and biofilm formation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 by phyto-synthesized silver nanoparticles through anti-quorum sensing activity. Microb. Pathog. 170, 105678. https://doi.org/10.1016/j.micpath.2022.105678\u003c/li\u003e\n \u003cli\u003eKurvet, I., Kahru, A., Bondarenko, O., Ivask, A., Ka, A., 2013. Particle-Cell Contact Enhances Antibacterial Activity of Silver Nanoparticles 8. https://doi.org/10.1371/journal.pone.0064060\u003c/li\u003e\n \u003cli\u003eLangevin, S., Vincelette, J., Bekal, S., Gaudreau, C., 2011. First case of invasive human infection caused by \u003cem\u003eCupriavidus metallidurans\u003c/em\u003e. J. Clin. Microbiol. 49, 744\u0026ndash;745. https://doi.org/10.1128/JCM.01947-10\u003c/li\u003e\n \u003cli\u003eLe Brun, N.E., Keech, A.M., Mauk, M.R., Mauk, A.G., Andrews, S.C., Thomson, A.J., Moore, G.R., 1996. Charge compensated binding of divalent metals to bacterioferritin: H+ release associated with cobalt(II) and zinc(II) binding at dinuclear metal sites. FEBS Lett. 397, 159\u0026ndash;163. https://doi.org/10.1016/S0014-5793(96)01172-6\u003c/li\u003e\n \u003cli\u003eLee, W., Kim, K.J., Lee, D.G., 2014. A novel mechanism for the antibacterial effect of silver nanoparticles on \u003cem\u003eEscherichia coli\u003c/em\u003e. BioMetals 27, 1191\u0026ndash;1201. https://doi.org/10.1007/s10534-014-9782-z\u003c/li\u003e\n \u003cli\u003eLiao, S., Zhang, Y., Pan, X., Zhu, F., Jiang, C., Liu, Q., Cheng, Z., Dai, G., Wu, G., Wang, L., Chen, L., 2019. Antibacterial activity and mechanism of silver nanoparticles against multidrug-resistant \u003cem\u003ePseudomonas aeruginosa.\u003c/em\u003e Int. J. Nanomedicine 14, 1469\u0026ndash;1487. https://doi.org/10.2147/IJN.S191340\u003c/li\u003e\n \u003cli\u003eLiu, C., Guo, D., Wen, S., Dang, Y., Sun, D., Li, P., 2024. Transcriptomic insights unveil the crucial roles of cytochromes, NADH, and pili in Ag(I) reduction by \u003cem\u003eGeobacter sulfurreducens\u003c/em\u003e. Chemosphere 358, 142174. https://doi.org/10.1016/j.chemosphere.2024.142174\u003c/li\u003e\n \u003cli\u003eLiu, G., Zhang, Y., Knibbe, W.J., Feng, C., Liu, W., Medema, G., van der Meer, W., 2017. Potential impacts of changing supply-water quality on drinking water distribution: A review. Water Res. 116, 135\u0026ndash;148. https://doi.org/10.1016/j.watres.2017.03.031\u003c/li\u003e\n \u003cli\u003eLouis, G., Cherry, P., Michaux, C., Rahuel-Clermont, S., Dieu, M., Tilquin, F., Maertens, L., Van Houdt, R., Renard, P., Perpete, E., Matroule, J.Y., 2023. A cytoplasmic chemoreceptor and reactive oxygen species mediate bacterial chemotaxis to copper. J. Biol. Chem. 299, 1\u0026ndash;11. https://doi.org/10.1016/j.jbc.2023.105207\u003c/li\u003e\n \u003cli\u003eM\u0026aacute;rquez, I.G., Ghiyasvand, M., Massarsky, A., Babu, M., Samanfar, B., Omidi, K., Moon, T.W., Smith, M.L., Golshani, A., 2018. Zinc oxide and silver nanoparticles toxicity in the baker\u0026rsquo;s yeast, \u003cem\u003eSaccharomyces cerevisiae.\u003c/em\u003e PLoS One 13, 1\u0026ndash;19. https://doi.org/10.1371/journal.pone.0193111\u003c/li\u003e\n \u003cli\u003eMartic, M., Jakab-Simon, I.N., Haahr, L.T., Hagen, W.R., Christensen, H.E.M., 2013. Heterometallic [AgFe3S4] ferredoxin variants: Synthesis, characterization, and the first crystal structure of an engineered heterometallic iron-sulfur protein. J. Biol. Inorg. Chem. 18, 261\u0026ndash;276. https://doi.org/10.1007/s00775-012-0971-3\u003c/li\u003e\n \u003cli\u003eMartin, C.S., Maximino, M.D., Martins, J.F.V.A., Pazin, W.M., Constantino, C.J.L., 2024. Exploring the effects of silver, silica-coated silver, and gold nanoparticles on lipid vesicles: Insights from LUVs and GUVs. J. Mol. Liq. 406. https://doi.org/10.1016/j.molliq.2024.125081\u003c/li\u003e\n \u003cli\u003eMatoulkov\u0026aacute;, D., Kosar, K., Slab\u0026yacute;, M., Sigler, K., 2012. Occurrence and species distribution of strictly anaerobic bacterium \u003cem\u003ePectinatus\u003c/em\u003e in brewery bottling halls. J. Am. Soc. Brew. Chem. 70, 262\u0026ndash;267. https://doi.org/10.1094/ASBCJ-2012-0910-01\u003c/li\u003e\n \u003cli\u003eMazur, P., Skiba-Kurek, I., Mrowiec, P., Karczewska, E., Drożdż, R., 2020. Synergistic ros-associated antimicrobial activity of silver nanoparticles and gentamicin against \u003cem\u003eStaphylococcus epidermidis.\u003c/em\u003e Int. J. Nanomedicine 15, 3551\u0026ndash;3562. https://doi.org/10.2147/IJN.S246484\u003c/li\u003e\n \u003cli\u003eMerbt, S.N., Bernal, S., Proia, L., Mart\u0026iacute;, E., Casamayor, E.O., 2017. Photoinhibition on natural ammonia oxidizers biofilm populations and implications for nitrogen uptake in stream biofilms. Limnol. Oceanogr. 62, 364\u0026ndash;375. https://doi.org/10.1002/lno.10436\u003c/li\u003e\n \u003cli\u003eMergeay, M., Nies, D., Schlegel, H.G., Gerits, J., Charles, P., Van Gijsegem, F., 1985. \u003cem\u003eAlcaligenes eutrophus\u003c/em\u003e CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 162, 328\u0026ndash;334. https://doi.org/10.1128/jb.162.1.328-334.1985\u003c/li\u003e\n \u003cli\u003eMetcalf, D.G., Bowler, P.G., 2013. Biofilm delays wound healing: A review of the evidence. Burn. Trauma 1, 5\u0026ndash;12. https://doi.org/10.4103/2321-3868.113329\u003c/li\u003e\n \u003cli\u003eMolina-Hernandez, J.B., Aceto, A., Bucciarelli, T., Paludi, D., Valbonetti, L., Zilli, K., Scotti, L., Chaves-L\u0026oacute;pez, C., 2021. The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death. Sci. Rep. 11, 1\u0026ndash;13. https://doi.org/10.1038/s41598-021-00545-7\u003c/li\u003e\n \u003cli\u003eMonchy, S., Vallaeys, T., Bossus, A., Mergeay, M., 2006. Metal transport ATPase genes from \u003cem\u003eCupriavidus metallidurans\u0026nbsp;\u003c/em\u003eCH34: A transcriptomic approach. Int. J. Environ. Anal. Chem. 86, 677\u0026ndash;692. https://doi.org/10.1080/03067310600583824\u003c/li\u003e\n \u003cli\u003eNovackova, I., Hrabalova, V., Slaninova, E., Sedlacek, P., Samek, O., Koller, M., Krzyzanek, V., Hrubanova, K., Mrazova, K., Nebesarova, J., Obruca, S., 2022. The role of polyhydroxyalkanoates in adaptation of \u003cem\u003eCupriavidus necator\u003c/em\u003e to osmotic pressure and high concentration of copper ions. Int. J. Biol. Macromol. 206, 977\u0026ndash;989. https://doi.org/10.1016/j.ijbiomac.2022.03.102\u003c/li\u003e\n \u003cli\u003ePerez-Riverol, Y., Bai, J., Bandla, C., Garc\u0026iacute;a-Seisdedos, D., Hewapathirana, S., Kamatchinathan, S., Kundu, D.J., Prakash, A., Frericks-Zipper, A., Eisenacher, M., Walzer, M., Wang, S., Brazma, A., Vizca\u0026iacute;no, J.A., 2022. The PRIDE database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543\u0026ndash;D552. https://doi.org/10.1093/nar/gkab1038\u003c/li\u003e\n \u003cli\u003ePrzemieniecki, S.W., Oćwieja, M., Ciesielski, S., Halecki, W., Matras, E., Gorczyca, A., 2022. Chemical Structure of Stabilizing Layers of Negatively Charged Silver Nanoparticles as an Effector of Shifts in Soil Bacterial Microbiome under Short-Term Exposure. Int. J. Environ. Res. Public Health 19. https://doi.org/10.3390/ijerph192114438\u003c/li\u003e\n \u003cli\u003eRadu, V., Frielingsdorf, S., Evans, S.D., Lenz, O., Jeuken, L.J.C., 2014. Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of \u003cem\u003eRalstonia eutropha\u003c/em\u003e. J. Am. Chem. Soc. 136, 8512\u0026ndash;8515. https://doi.org/10.1021/ja503138p\u003c/li\u003e\n \u003cli\u003eRadzig, M.A., Nadtochenko, V.A., Koksharova, O.A., Kiwi, J., Lipasova, V.A., Khmel, I.A., 2013. Antibacterial effects of silver nanoparticles on gram-negative bacteria: Influence on the growth and biofilms formation, mechanisms of action. Colloids Surfaces B Biointerfaces 102, 300\u0026ndash;306. https://doi.org/10.1016/j.colsurfb.2012.07.039\u003c/li\u003e\n \u003cli\u003eRandall, C.P., Gupta, A., Jackson, N., Busse, D., O\u0026rsquo;Neill, A.J., 2014. Silver resistance in Gram-negative bacteria: A dissection of endogenous and exogenous mechanisms. J. Antimicrob. Chemother. 70, 1037\u0026ndash;1046. https://doi.org/10.1093/jac/dku523\u003c/li\u003e\n \u003cli\u003eRodr\u0026iacute;guez-Rojas, A., Kim, J.J., Johnston, P.R., Makarova, O., Eravci, M., Weise, C., Hengge, R., Rolff, J., 2020. Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress, PLoS Genetics. https://doi.org/10.1371/journal.pgen.1008649\u003c/li\u003e\n \u003cli\u003eRyder, V.J., Chopra, I., O\u0026rsquo;Neill, A.J., 2012. Increased Mutability of \u003cem\u003eStaphylococci\u003c/em\u003e in Biofilms as a Consequence of Oxidative Stress. PLoS One 7. https://doi.org/10.1371/journal.pone.0047695\u003c/li\u003e\n \u003cli\u003eSchacht, V.J., Neumann, L. V., Sandhi, S.K., Chen, L., Henning, T., Klar, P.J., Theophel, K., Schnell, S., Bunge, M., 2013. Effects of silver nanoparticles on microbial growth dynamics. J. Appl. Microbiol. 114, 25\u0026ndash;35. https://doi.org/10.1111/jam.12000\u003c/li\u003e\n \u003cli\u003eSchneider, G., 2017. Antimicrobial silver nanoparticles - Regulatory situation in the European Union. Mater. Today Proc. 4, S200\u0026ndash;S207. https://doi.org/10.1016/j.matpr.2017.09.187\u003c/li\u003e\n \u003cli\u003eSeong, M., Lee, D.G., 2017. Silver Nanoparticles Against \u003cem\u003eSalmonella enterica\u003c/em\u003e Serotype Typhimurium: Role of Inner Membrane Dysfunction. Curr. Microbiol. 74, 661\u0026ndash;670. https://doi.org/10.1007/s00284-017-1235-9\u003c/li\u003e\n \u003cli\u003eSillen, W.M.A., Thijs, S., Abbamondi, G.R., Janssen, J., Weyens, N., White, J.C., Vangronsveld, J., 2015. Effects of silver nanoparticles on soil microorganisms and maize biomass are linked in the rhizosphere. Soil Biol. Biochem. 91, 14\u0026ndash;22. https://doi.org/10.1016/j.soilbio.2015.08.019\u003c/li\u003e\n \u003cli\u003eSim, W., Barnard, R.T., Blaskovich, M.A.T., Ziora, Z.M., 2018. Antimicrobial silver in medicinal and consumer applications: A patent review of the past decade (2007\u0026ndash;2017). Antibiotics 7, 1\u0026ndash;15. https://doi.org/10.3390/antibiotics7040093\u003c/li\u003e\n \u003cli\u003eSouza-Egipsy, V., Gonz\u0026aacute;lez-Toril, E., Zettler, E., Amaral-Zettler, L., Aguilera, A., Amils, R., 2008. Prokaryotic community structure in algal photosynthetic biofilms from extreme acidic streams in R\u0026iacute;o Tinto (Huelva, Spain). Int. Microbiol. 11, 251\u0026ndash;260. https://doi.org/10.2436/20.1501.01.69\u003c/li\u003e\n \u003cli\u003eStaley, B.J.T., Wiley, A.R., York, N., 1963. Biodiversity of Microbial Life: Foundation of Earth\u0026rsquo;s Biosphere. Acta Paediatr. 52, 534\u0026ndash;534. https://doi.org/10.1111/j.1651-2227.1963.tb03813.x\u003c/li\u003e\n \u003cli\u003eStewart, P.S., Franklin, M.J., 2008. Physiological heterogeneity in biofilms. Nat. Rev. Microbiol. 6, 199\u0026ndash;210. https://doi.org/10.1038/nrmicro1838\u003c/li\u003e\n \u003cli\u003eStone, M., Emelko, M.B., Droppo, I.G., Silins, U., 2011. Biostabilization and erodibility of cohesive sediment deposits in wildfire-affected streams. Water Res. 45, 521\u0026ndash;534. https://doi.org/10.1016/j.watres.2010.09.016\u003c/li\u003e\n \u003cli\u003eThuptimdang, P., Limpiyakorn, T., McEvoy, J., Pr\u0026uuml;\u0026szlig;, B.M., Khan, E., 2015. Effect of silver nanoparticles on \u003cem\u003ePseudomonas putida\u0026nbsp;\u003c/em\u003ebiofilms at different stages of maturity. J. Hazard. Mater. 290, 127\u0026ndash;133. https://doi.org/10.1016/j.jhazmat.2015.02.073\u003c/li\u003e\n \u003cli\u003eTso, W.W., Adler, J., 1974. Negative chemotaxis in \u003cem\u003eEscherichia coli.\u003c/em\u003e J. Bacteriol. 118, 560\u0026ndash;576. https://doi.org/10.1128/jb.118.2.560-576.1974\u003c/li\u003e\n \u003cli\u003eValdiglesias, V., 2022. Cytotoxicity and Genotoxicity of Nanomaterials. Nanomaterials 12, 279\u0026ndash;290. https://doi.org/10.3390/nano12040634\u003c/li\u003e\n \u003cli\u003eW\u0026oacute;jtowicz, H., Guevara, T., Tallant, C., Olczak, M., Sroka, A., Potempa, J., Sol\u0026agrave;, M., Olczak, T., Gomis-R\u0026uuml;th, F.X., 2009. Unique structure and stability of HmuY, a novel heme-binding protein of \u003cem\u003ePorphyromonas gingivalis.\u0026nbsp;\u003c/em\u003ePLoS Pathog. 5. https://doi.org/10.1371/journal.ppat.1000419\u003c/li\u003e\n \u003cli\u003eWu, K., Li, H., Cui, X., Feng, R., Chen, W., Jiang, Y., Tang, C., Wang, Yaohai, Wang, Yan, 2022. Mutagenesis and Resistance Development of Bacteria. Antimicrob. Agents Chemother. 66.\u003c/li\u003e\n \u003cli\u003eXiao, X., He, E.J., Lu, X.R., Wu, L.J., Fan, Y.Y., Yu, H.Q., 2021. Evaluation of antibacterial activities of silver nanoparticles on culturability and cell viability of \u003cem\u003eEscherichia coli\u003c/em\u003e. Sci. Total Environ. 794, 148765. https://doi.org/10.1016/j.scitotenv.2021.148765\u003c/li\u003e\n \u003cli\u003eZaitsev, S.Y., Zaitsev, I.S., Milaeva, I. V., 2021. Structural changes of the lipid model systems in the presence of enzymes or silver nanoparticles. Biointerface Res. Appl. Chem. 11, 15068\u0026ndash;15074. https://doi.org/10.33263/BRIAC116.1506815074\u003c/li\u003e\n \u003cli\u003eZiege, R., Tsirigoni, A.M., Large, B., Serra, D.O., Blank, K.G., Hengge, R., Fratzl, P., Bidan, C.M., 2021. Adaptation of \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eBiofilm Growth, Morphology, and Mechanical Properties to Substrate Water Content. ACS Biomater. Sci. Eng. 7, 5315\u0026ndash;5325. https://doi.org/10.1021/acsbiomaterials.1c00927\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biofilm, nanoparticles, proteomics, bacteria, metal resistance","lastPublishedDoi":"10.21203/rs.3.rs-8079940/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8079940/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiofilms are in many sectors a persistent technical challenge that can lead to reduced efficiency in industrial processes, increased maintenance costs, and potential product contamination. Furthermore, biofilms can contribute to the development of antimicrobial resistance, posing health risks in medical and food production environments, as well as reducing the effectiveness of cleaning and sterilization efforts. Therefore, we investigated the impact of antimicrobial silver nanoparticles (AgNPs) on macroscopically visible biofilms of \u003cem\u003eCupriavidus metallidurans\u003c/em\u003e CH34, a metal-resistant and oligotrophic bacterium. We accelerated the time-consuming process of naturally forming macroscopic biofilms via a laboratory setup using porous synthetic surfaces. This setup simulates growth dynamics on air-exposed and nutrient-leaking substrates such as rocks, wood, polymeric coatings, joints and dripping pipes. Remarkably, 24-hour matured biofilms were unaffected by AgNPs at concentrations as high as 750 mg/L. Biofilm resilience to AgNPs was age-dependent and although densely populated, young biofilms (6\u0026ndash;9 hours) were inactivated by AgNPs while older ones (14\u0026ndash;24 hours) exhibited resistance in the same conditions. Additional experiments suggested that the resistance in older biofilms was not attributed to cell density, but potentially to other factors like extracellular polymeric substances (EPS) or their metabolic state. AgNPs induced oxidative stress in exposed biofilms but did not result in microscopically observable morphological changes in the biofilm-resident cells, indicating that inhibition mechanisms did not involve outer membrane disruption. The proteomic analysis of macroscopic biofilms exposed to AgNPs revealed an age-independent protein set that was consistently upregulated/down regulated across all analyzed biofilm stages as well as age-specific pathways. These findings suggest complex adaptive molecular mechanisms in \u003cem\u003eC. metallidurans\u003c/em\u003e biofilms that confer resistance to AgNPs, involving oxidative stress management, iron metabolism, and electron transport chain adjustments.\u003c/p\u003e","manuscriptTitle":"The dynamic response of Cupriavidus metallidurans CH34 cells and biofilms to silver nanoparticles and the importance of biofilm maturation stage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-24 09:00:50","doi":"10.21203/rs.3.rs-8079940/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2025-11-22T05:57:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-20T12:35:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-11-10T18:25:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9e108c8a-3fde-4e3d-9914-542d2154b8af","owner":[],"postedDate":"December 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T10:23:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-24 09:00:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8079940","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8079940","identity":"rs-8079940","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}