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 (OD₆₀₀ = 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 OD₆₀₀ = 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 AgNO₃ solution (250, 500, 750 mg/L). Control biofilms received 30 µL of sterile H₂O. 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.

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 (10⁶ 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.

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.

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 (O₂). 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 H₂O₂ 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.

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.

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.

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.

Thus, we conclude that direct disruption of the outer membrane is not the only mechanism by which AgNPs can inhibit bacterial growth.

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.

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 O₂ to H₂O, 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 AgNO₃ (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 Cu²⁺ in Caulobacter crescentus (Louis et al., 2023) and a negative chemotaxis in response to Ni²⁺ and Co²⁺ 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.

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 Ca²⁺ 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.

Not applicable