Superhydrophobic Copper‐Composite Surfaces Exert Antibacterial Effects against Gram‐Negative and ‐Positive Bacteria

Copper shows a high promise in developing biomedical materials with antibacterial effect. The antibacterial effect can be enhanced by nanostructured surfaces with superhydrophobic properties, which reduce the solid contact area available for bacterial adhesion and adherent growth. Here, three structured surfaces are fabricated to test the combined effect of copper and superhydrophobicity for antibacterial effects. One of the samples is superhydrophobic but does not contain copper, one contains copper but is not superhydrophobic, and the third is both superhydrophobic and contained copper. The antibiofilm and bactericidal effects of these samples are tested against medically important Gram‐positive and ‐negative bacteria including Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and Pseudomonas aeruginosa (P. aeruginosa). The findings indicate that copper alone without superhydrophobicity, while decreasing the cell viability in most of the tested species, supports remarkably more biomass compared to the reference sample. The superhydrophobic and copper bearing samples, while allowing adherent growth to take place, provide the greatest bactericidal effect against two P. aeruginosa strains, and both the antibiofilm and/or bactericidal effects against S. aureus and S. epidermidis. Thus, this study reports that nanostructured materials, combining superhydrophobicity with copper, can be the method of choice to neutralize pathogens with different cell‐wall structures and surface components mediating adherent growth.


Introduction
Bacterial attachment and colonization can cause major problems and even device failure in a wide range of fields, including marine, [1] medical, and healthcare [2] industries.A lot of research has been directed toward design and fabrication of antibacterial surfaces.Metals, like copper (Cu), [3] hafnium (Hf), [4] silver (Ag), [5] and gold (Au), [6] have been shown to possess antibacterial properties.Metal oxide nanoparticles, such as copper oxide (CuO), [7] hafnium oxide (HfO 2 ), [8] zinc oxide (ZnO), [9] and titanium dioxide (TiO 2 ), [10] can directly bind and interact with bacterial membranes due to their high surface-to-volume ratio. [11][14][15] The antimicrobial effect of copper is wellestablished, and ongoing research is being addressed to develop copper-coated hard and soft surfaces to reduce/prevent microbial contamination and, subsequently, reduction of hospital acquired infections. [16]Surface geometry plays a vital role in eliminating or reducing bacterial adhesion.It has been shown that various surface morphologies of hierarchical structures significantly affect the bactericidal activity of copper surfaces. [17]uperhydrophobic (SHB) surfaces can possibly prevent bacterial adhesion and biofilm formation due to trapped air, called the plastron, between the bacteria and the surface.The antibacterial effect of superhydrophobic surfaces mainly derives from the reduction of contact area between the surface and the bacterial contamination, which means bacterial adhesion occurs in a much smaller area of SHB surfaces. [18]Superhydrophobic surfaces are characterized by high water contact angles (CA > 150°) and low roll-off angles or sliding angles (SA < 10°) [19,20] for droplets in the microliter range.A superhydrophobic surface can be fabricated by coating a structured surface with a low surface energy material or a coating or by introducing roughness in inherently hydrophobic materials.Fabrication of dual micro-nanoscale hierarchical structures can be done by, e.g., etching, deposition, and replication methods. [21][24][25][26] Cai et al. [22] showed that the antibacterial activity of superhydrophobic films obtained from deposition of polydopamine nanoparticles, Ag nanoparticles, and hydrophobic coating was tested against the Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus.They indicated that superhydrophobicity leads to reduction of bacterial adhesion due to release of Ag + ions from silver nanoparticles.Superhydrophobicity was obtained by a thin fluoropolymer coating, which compromises long term performance of these surfaces.
Copper nanoparticle deposition was used on polydopamine coated wood to obtain superhydrophobicity and antibacterial abilities. [23]It was shown that a bacteriostatic ring was formed around a superhydrophobic wood sample against E. coli and S. aureus.Tripathy et al. [24] reported high aspect ratio superhydrophobic surfaces coated with a thin layer of silver or copper.It was shown that the thin layer of metal coating improved the killing of bacteria compared to uncoated surface.Jiang et al. [25] showed that copper nanowires embedded into polydimethylsiloxane (PDMS) with the purpose of extending the dissolution rate of copper, offering long-term antifouling behavior.
A combination of low surface energy coating and bactericidal agent to fabricate metal-sputtered superhydrophobic surfaces was reported by Kefallinou et al. [26] The resulting material exhibits antiadhesive and bacterial properties; however, the antibacterial activity was tested only against Gram-negative Synechococcus sp.PCC7942, and no information was provided on durability of superhydrophobicity.The mechanical durability of SHB surfaces has a direct effect on antibacterial performance.The durability of superhydrophobic surfaces is often affected by mechanical loads, such as abrasion, due to the presence of vulnerable micro/nanostructures.The destruction of hierarchical structures results in a reduction of antiwetting properties and consequently, bacterial colonization on the damaged/wetted surfaces. [18,27]Copper hydroxide nanowires embedded in PDMS have been shown to exert antibacterial effect against Gram-negative (E. coli and Klebsiella pneumoniae) and Gram-positive (S. aureus) bacterial strains.This material maintained its superhydrophobicity when twisted, abraded, and tape peeled. [27,28]Our previous work on superhydrophobic Cu-PDMS composites has shown similar superior durability compared with most nanostructured superhydrophobic surfaces. [29]n this work, bacterial adhesion and biofilm formation onto our superhydrophobic Cu-PDMS composite material was evaluated using following three clinically relevant biofilm-forming bacterial species with different cell-wall structures and composition: Gram-positive S. aureus ATCC25923 and S. epidermidis-Staphylococcus ep RP62A, and two Gram-negative (P.aeruginosa) strains (ATCC15442 and ATCC9027).We compared the performance of the combined superhydrophobic and copper containing surfaces to superhydrophobic-only and copper-only surfaces against the indicated bacterial species incubated under conditions allowing adherent growth.Our findings demonstrated the highest promise for a superhydrophobic Cu-PDMS composite in conferring the greatest antibiofilm effect against both the Gramnegative and -positive species, and that the possible mechanismsof-actions involve either preventive and/or bactericidal activities.

Surface Morphology, Chemical Composition, and Wettability
Scanning electron microscopy (SEM) was used to study surface morphology of three structured surfaces.Figure 1 shows the digital photographs, SEM images, schematic illustrations of the cross-section, and cross-section SEMs of three structured surfaces.The micro-Cu sample is a copper-PDMS composite, and it is included in the antibacterial test as a structured surface that contains copper but is not superhydrophobic (Figure 1a).The SHB nano-HfO 2 is a HfO 2 -PDMS composite that is included in the antibacterial test as a structured surface that is superhydrophobic but does not contain copper (Figure 1b).The SHB nano-Cu is a Cu-PDMS composite that is superhydrophobic and is included in the antibacterial test as a sample that both contains copper and is superhydrophobic (Figure 1c).
The topography of superhydrophobic PDMS-backed hafnium oxide and copper surfaces is defined by etched aluminum micro and nanostructures that are copied into the deposited film (HfO 2 or Cu).The SHB nano-HfO 2 and SHB nano-Cu materials show hierarchical micro/nanoscale structures from tens of nanometers to tens of micrometers (Figure 1b,c).Schematics of micro-Cu and SHB nano-Cu in Figure 1a,c show that the top surface of both samples is copper.However, based on the contact angles, we consider it highly likely that, in both cases, there is a thin layer of loose PDMS monomers that have migrated on the surface to make the surface chemistry hydrophobic. [29]he composition of three structured surfaces was analyzed by energy dispersive spectroscopy (EDS).The EDS spectrum confirms the presence of copper in micro-Cu (Figure S1a, Supporting Information), and SHB nano-Cu (Figure S2a, Supporting Information) samples.The EDS also shows Hf peaks in SHB nano-HfO 2 material (Figure S3a, Supporting Information).The EDS mapping shows a widespread and uniform presence of Cu and Hf throughout the surface of SHB nano-Cu (Figure S2e, Supporting Information) and SHB nano-HfO 2 (Figure S3e, Supporting Information), respectively.Silicon (Si) and oxygen (O) peaks in spectra originate from PDMS.Contact and sliding angles were measured to study the wetting behavior of water droplets on all materials.Figure 2 shows the contact angles and sliding angles of the three structured samples and the reference planar surfaces.Planar polystyrene (PS, positive control sample), shows hydrophilicity, while planar PDMS (reference samples) and micro-Cu exhibit hydrophobicity.HfO 2 -PDMS and Cu-PDMS composite materials show excellent superhydrophobic propertiems with a high advancing and receding contact angles (ACA and RCA, above 160°) and low sliding angles (below 10°).The possible damaging effects of the ultraviolet (UV) light irradiation on the materials during sterilization/disinfection were assessed by comparing wetting properties of materials before and after the UV treatment (Figure 2a,b).The results indicate only slight change in contact angles and sliding angles for all tested samples and confirmed that the superhydrophobic samples remain so after sterilization.

Antibacterial Activity
The antibiofilm effects, bacterial adhesion, and biofilm formation, of the three different structured surfaces (micro-Cu, SHB nano-HfO 2 , and SHB nano-Cu) were tested against three clinically relevant bacterial species, involving Gram-positives S. aureus ATCC25923 (hereafter Sa_25923) and S. epidermidis (Se_RP62A), as well as Gram-negatives P. aeruginosa ATCC15442 (Pa_15442) and P. aeruginosa ATCC9027 (Pa_9027).PS having hydrophilic surface was used as the positive control of biofilm formation [30][31][32][33] and PDMS as a planar material control, since all three structured samples are composites containing PDMS.PDMS can be expected to be a nontoxic but a low surface energy, hydrophobic and a low bioadhesive surface. [34,35]

Antibacterial Activity against Gram-Positive Staphylococcus Species
Figure 3a shows the biofilm growth (adherent biomass) and the viability (number of viable cells as CFUs mL −1 ) of Sa_25923 on all materials after two days of cultivation at +37 °C under aerobic conditions.We observed nearly ten times more Sa biomass (p = 0.009) on the planar PS in comparison to PDMS, which was accompanied by a ≈4-fold (p = 0.005) decrease in the number of viable cells on PDMS compared to PS.Compared to planar PDMS, Sa cells showed significantly more efficient adherent growth on the three structured samples; both SHB nano-HfO 2 and SHB nano-Cu had ≈5-7 times (p < 0.005) and micro-Cu ≈20 times (p < 0.001) more adherent biomass compared to the planar PDMS.In stark contrast, the number of viable cells detected on these materials indicated the lowest number of colonyforming units (CFUs) for Sa on the SHB nano-Cu, which was ≈4 times lower (p < 0.009) compared to that detected for the planar PDMS.Comparison of the number of viable cells on micro-Cu, SHB nano-HfO 2 , and SHB nano-Cu indicates > 5-fold decrease in CFUs mL −1 on SHB nano-Cu and micro-Cu in comparison to SHB nano-HfO 2 (p < 0.005), indicating the importance of both Cu and SHB.Thus, while Sa formed quantitatively more biomass/biofilm on both micro-Cu and SHB nano-Cu compared to PDMS, the combination of hydrophobicity with copper is needed to exert both antibiofilm and/or bactericidal effect against this Staphylococcus species/strain.
Figure 3b shows both the adherent growth/biomass and viability of Se on the same materials incubated under identical conditions.We detected quantitatively the same amount of Sebiomass and the number of viable cells from PS and micro-Cu, clearly exceeding those detected on the other samples.The  detected biofilm mass was ≈13-fold lower (p < 0.0007) on PDMS compared to PS.Also, the number of viable cells on PMDS was decreased ≈2 times (p < 0.02) compared to those on PS.Of the structured samples, we observed that the adherent biomass on SHB nano-Cu was ≈8 times reduced (p < 0.01) compared to the biomass detected on PS, whereas no significant difference was detected for the biomasses on SHB nano-Cu and PDMS.On the other hand, the number of viable cells on SHB nano-Cu was reduced by 5 times (p < 0.005) in comparison to PDMS.Micro-Cu had ≈7 times more biomass (p = 0.002) compared to that on SHB nano-Cu.While copper alone was able to exert a clear bactericidal effect against S. aureus, the number of viable cells on this material was ≈2 times higher compared to PDMS and ≈11 times higher (p < 0.005) compared to SHB nano-Cu.Since copper alone did not decrease the biofilm formation (unlike PDMS) nor affected the number of viable cells (unlike PDMS), our findings suggest that the combined effect of both hydrophobicity and copper is needed to interfere with the initial stages of biofilm formation instead of killing already adherent cells.

Antibacterial Activity against Gram-Negative P. aeruginosa
Figure 3c shows the remaining biomass and number of viable Pa_15442 cells on the same five materials after two days of aerobic incubation at +37 °C.Pa_15442 cells displayed the lowest biofilm biomasses on PS, PDMS and SHB nano-Cu, and the greatest biomasses on micro-Cu and SHB nano-HfO 2 , exceeding two to three times (p < 0.05) the quantity detected on SHB nano-Cu.The number of viable cells was lowest on micro-Cu and SHB nano-Cu, whereas > 12 times (p < 0.05) more viable cells were detected on SHB nano-HfO 2 .The number of viable cells on SHB nano-Cu was decreased by seven-and five times (p < 0.05) in comparison to hydrophilic PS and hydrophobic PDMS, respectively, indicating that combining superhydrophobicity with copper confers a clear bactericidal effect against this P. aeruginosa strain.On the other hand, copper alone is not enough to reduce adherence/biofilm formation of this P. aeruginosa strain.
Figure 3d shows the indicated materials with Pa_9027 cells incubated under the same conditions as the other bacterial species.Pa_9027 formed the greatest biomass on SHB nano-HfO 2 , reaching nearly three times (p < 0.05) the quantity detected on PDMS.In addition, quantitatively more biomass was also detected on micro-Cu and SHB nano-Cu in comparison to PDMS.Notably, while we detected varying biomasses on the indicated materials, the number of viable cells remained nearly unchanged, indicating other factors than cell number affecting the biomass.However, the lowest viability was still detected on SHB nano-Cu; number of viable cells decreased on this material by ≈1.5 times (p = 0.03) compared to PDMS, which was also lower in comparison to the other tested materials.Thus, while these results indicated quantitatively more biomass attached to SHB nano-Cu in comparison to PDMS, we suggest that combining superhydrophobicity with copper is also effective against this P. aeruginosa strain.

Discussion
In the present study, we tested antibiofilm and antibacterial effect of structured materials combining superhydrophobicity with  Cu or HfO 2 , using planar PDMS as the material control, since all three structured samples are composites containing this polymeric organosilicon.Representatives of three clinically important bacterial species, including both Gram-negative and -positive organisms were studied here; two P. aeruginosa strains, one S. aureus strain, and one S. epidermidis strain.These strains represent bacteria with differing cell wall characteristics but sharing an ability for adherent/biofilm growth on hydrophilic PS, a typical surface material used in assessing bacterial biofilm formation. [36]iofilm is defined as an aggregate of microbial cells embedded within a self-produced matrix of extracellular polymeric substances (proteins, polysaccharides, and nucleic acids), which help the cells to adhere to a surface and in which the factors exposed at the bacterial cell surface are the key players. [37] Studies have shown that van der Waals, structural, electrostatic, and steric forces define the tendency of bacteria to attach/bind to the abiotic surfaces. [38]In addition, the composition of the culture medium for bacteria plays an important role in this process by affecting the surface forces between the bacterial cell and the substrate. [39]In the present study, we used TSB to grow each bacteria to maintain the initial composition of free ions (originating from NaCl, K 2 HPO 4 ) at the same level to compare the ability of the selected biofilm model organisms to bind and initiate adherent growth (biofilm formation) on negatively charged/hydrophilic PS, hydrophobic PDMS, and the three PDMS composite materials with/without copper and/or superhydrophobicity.In general, superhydrophobic surfaces are considered to repel majority of the Gram-negative bacteria, while Gram-positive species show only moderate adherence to such surfaces. [40]In addition, more hydrophobic bacteria tend to adhere more strongly to hydrophobic surfaces and those with hydrophilic cell surfaces to hydrophilic materials. [41]Here, our findings demonstrated opposite, as the selected Sa_25923 and Se_RP62A strains with reported hydrophobic cell surfaces [42][43][44][45] adhered ≈7-10 times more efficiently to PS in comparison to planar PDMS.Although these findings suggests that PDMS as material can prevent the adherence of the tested staphylococci, we cannot exclude the possibility that the tested staphylococci have switched between hydrophobic and hydrophilic phenotypes in response to environmental changes, as demonstrated for other bacteria. [41]This may include, e.g., synthesis of extracellular vesicles (EVs) increasing cell surface hydrophobicity, [46] which has been shown to be more efficient in S. aureus compared to S. epidermidis. [47]nother likely explanation could be the attractive forces occurring within the liquid layer due to polar moieties/charges on the bacterial cell attracted by PS.For example, the expression of an alkaline polysaccharide intercellular adhesin-PIA (poly--1,6-N-acetylglucosamine)-in high abundances at the cell surface of Se_RP62A [48] mediates binding to negatively charged hydrophilic surfaces. [49]S. aureus, instead, has shown to export highly alkaline virulence factors and cytoplasmic ribosomal proteins (r-proteins) to the cell surface stimulating electrostatic interactions with anionic components between the cells. [50]Additional studies on S. aureus have further demonstrated that a large group of normally cytoplasmic proteins associate with the bacterial surface upon the drop in pH (naturally occurring during biofilm formation), and that negatively charged extracellular DNA (eDNA) acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH. [51]hese findings could also explain adhesion and adherent growth of Sa_25923 on PS.Our previous proteomic study supports this hypothesis by showing that r-proteins were the dominating protein group on the cell surfaces of this particular strain, [52] implying that increased anionic charge could have conferred increased adherence to PS and viable cell growth thereafter.Despite the mechanisms of adhesion used and the charged bacterial cell surfaces, both Sa_25923 and Se_RP62A showed the lowest adherence and adherent growth on planar PDMS and superhydrophobic Cu-PDMS (SHB nano-Cu), implying that antiwettability and reduced contact area of the abiotic material must have provided significant antiadhesive effects against the selected staphylococci.
In the case of Gram-negative pseudomonads, our findings indicated that also strain-dependent differences should be taken into account to when designing materials against these bacteria.For example, Pa_9027 cell surfaces are more hydrophilic compared to those of Pa_15442, while Pa_15442 is more hydrophobic in comparison to Pa_9027, and in which the more efficient production of hydrophilic rhamnolipids (RLs) by Pa_9027 was suggested to be the underlining factor. [53]RLs play an important role during the first steps of biofilm formation, [54] which supports our finding showing nearly three times more efficient adhesion to PS with Pa_9027 in comparison to Pa_15442.Despite these differences, both P. aeruginosa strains demonstrated moderate adhesion/adherent growth on PDMS, which was more efficient than that demonstrated by staphylococci on the same material.57][58] Other possible explanations could include the production of hydrophobic EVs, which in Pa_9027 has been shown to occur efficiently under stressful conditions. [39]e show that modifying the physicochemical/electrostatic properties of PDMS (micro-Cu, SHB nano-HfO 2 , and SHB nano-Cu) resulted in both species-and strain-dependent variations on adhesion efficiency on the fabricated composites.In all cases, the bacterial biomasses on PDMS with copper exceeded the biomasses detected for the bacteria on only PDMS.We explain this by the attractive forces occurring between the bacterial cell surfaces and the positively charged metal coated PDMS.For example, staphylococci are efficient in production of lipoteichoic acids, a group of negatively charged cell wall polymers needed in biofilm formation. [59,60]In the case of P. aeruginosa, studies have indicated that efficient production of negatively charged eDNA is responsible for acidifying the bacterial cell surfaces, [61] which besides acting as a passive shield could also act as a highly efficient divalent metal cation chelator. [62]In addition, Pa_9027 lacks two central regulators with predicted roles in controlling the bacterial surface charge; LasR, a quorum sensing regulator defining the optimal RL expression [63,64] and AlgU, a negative regulator of alternative sigma factor (SigE) resulting in constitutive expression of alginate (Alg), a negatively charged polysaccharide. [65]Thus, these findings on staphylococci and pseudomonads together with the genetic features specific to Pa_9027 support the idea that electrostatic interactions contributed to the bacterial adhesion to PDMS with copper (micro-Cu and SHB nano-Cu).HfO 2 -based nanoparticles have been reported to show antibacterial activity against certain Gram-negative bacteria, such as Klebsiella pneumoniae and S. aureus. [66]Here, SHB nano-HfO 2 showed moderate to high binding affinity toward each bacteria, with Pa_15442 and Pa_9027 showing the greatest adherence.Other studies have indicated the ability of HfO 2 nanopores/nanofilms to bind proteins and DNA, [8,67] implying that the fabricated HfO 2 -PDMS exhibited positively charged surface under the experimental conditions used and thus could have stimulated the bacterial adherence to this material.
The crystal violet-based staining method does not distinguish dead cells from alive ones and this stain also binds to proteins, eDNA and polysaccharides. [68,69]Therefore, to assess the antibacterial effect, we determined the number of viable cells from the bacteria adhered to the tested materials.Here, the lowest number of viable cells for each bacteria were detected on micro-Cu and SHB nano-Cu.Three out of the four strains, Sa_25923, Se_RP621A, and Pa_15442 showed significantly reduced adherence to SHB nano-Cu in comparison to micro-Cu.From the tested bacteria, only S. aureus and Pa_15442 demonstrated significantly reduced viability also on micro-Cu, which was not seen with Se_RP62A or Pa_9027.Studies on another S. aureus strain (SH1000) have shown that copper at high concentrations represses the biofilm formation. [70]Based on this report and our findings we suggest that increasing the hydrophobicity in the presence of copper results in enhanced cell killing, as significantly less viable cells were seen on SHB nano-Cu in comparison to SHB nano-HfO 2 .][73] Thus, we suggest that the copper ions could have triggered more efficient ROS-mediated cell killing of Sa_25923 on micro-Cu and SHB nano-Cu, and that increasing the surface hydrophobicity of the material maximizes the antibacterial effect against this bacterium.In the case of Se_RP62A, our findings imply that combining superhydrophobicity with copper is needed to provide synergistic antibacterial action against the bacterium, since copper alone had no viability-decreasing effect against this strain.Moreover, increasing hydrophobicity in the presence of copper decreased both the adherent biofilm biomass and cell viability of Se_RP62A, which was not seen with S. aureus.It could be that the Cu-nanowire structures associated with SHB nano-Cu were able to penetrate into the bound S. epidermidis cells, thereby lysing the cells as reported previously for E. coli. [17]n the case of the pseudomonads, our findings indicated that copper alone (micro-Cu) could have conferred a strong bactericidal effect against Pa_15442 and that increased surface hydrophobicity enhanced the antibiofilm effect against this species, as evidenced by the lowest biomass on PDMS and lowest number of viable cells on micro-Cu and SHB nano-Cu.On the other hand, opposite findings were observed with Pa_9027, as the quantity of the detected biomasses on these materials had only a marginal effect on the number of viable cells on the same materials.It could be that Pa_9027 under tested conditions, instead of increasing the number of cells, increased the production of proteins/eDNA that becomes stained by the crystal violet.Nevertheless, our findings suggest that combination of superhydrophobicity and copper was also needed to promote bactericidal effect against Pa_9027, as this strain demonstrated the lowest adherence on PDMS and the lowest cell viability on SHB nano-Cu.
Taken together, the present study indicates that combining copper and superhydrophobicity provides a promising starting point to develop more efficient antibacterial and/or antibiofilm strategies against Gram-positive staphylococci and Gram-negative pseudomonads.Figure 4 summarizes the adhesion efficiency of the selected model organisms onto SHB nano-Cu (Figure 4a) and the resulting antibiofilm and bactericidal effects (Figure 4b) conferred by this abiotic material against each bacteria.Our findings indicate that the ability of the bacterial pathogen to modify their surface hydrophobicity and charge in response to different conditions/materials should be considered when designing new materials.In addition, the heterogeneity of the bacterial population should also be taken into account, since freely living bacteria can exist as both with hydrophilic and hydrophobic cell surfaces, allowing only part of them participating in the adhesion. [41]Thus, we conclude that incorporating an additional bacteriocide into the SHB nano-Cu could help combating bacterial biofilms with varying physicochemical surface properties to maximize their antibacterial/-biofilm effect.

Conclusion
Our results indicate that the combined superhydrophobic and copper containing surfaces are beneficial for achieving the most efficient antibiofilm effect against two Gram-positive Staphylococcus species and two Gram-negative P. aeruginosa strains compared to only superhydrophobic or only copper containing structured surfaces.It is clear from the results that PDMS, as a low surface-energy material, has a baseline antibacterial effect as both the biofilm biomass and the viability of the planar PDMS are on average much lower than on the planar more hydrophilic PS.In fact, the planar PDMS has the lowest adherent biomass for all strains.This is partly explained by the much lower surface area of the planar PDMS as compared to the structured samples.Superhydrophobicity could still well provide a short-term protection against adhesion, but here we focused only on longer term adhesion.
The results on viability showed that SHB nano-Cu sample had clearly the lowest overall viability for all tested strains.This is our strongest evidence in support of the combined effect of superhydrophobicity and copper.The effect of copper alone leads to lowered viability on two of the bacterial strains (S. aureus ATCC25923 and P. aeruginosa ATCC15442) but not on the other two bacteria (S. epidermidis RP62A and P. aeruginosa ATCC9027).The effect of superhydrophobicity alone did not lead to a clear antibacterial effect for any of the strains under the long-term conditions explored in this study.Finally, the clear differences between the four strains highlight the needs for testing multiple bacterial strains for reaching any conclusions that could be more universal than only applying to a single strain.

Experimental Section
Materials Preparation: Three different structured surfaces: micro-Cu, SHB nano-HfO 2 , and SHB nano-Cu as well as two planar reference surfaces, including polystyrene and PDMS, were fabricated and characterized.Polystyrene samples were cut out from the 24-well microtiter plates with hydrophilic surface character (FALCON; cell culture:polystyrene surface culture treated with plasma to make it electronegative and hydrophilic; Becton Dickinson).The planar PDMS (Sylgard 184 Silicone Elastomer Kit, Dow Chemical Co.) was prepared by mixture of monomer to the crosslinking agent (10:1) and then cured at 65 °C for 3 h in an oven.
Micro-Cu samples were fabricated by adding copper powder (100 mesh, Alfa Aesar) into PDMS (1:1), followed by curing at 65 °C for 3 h in an oven.Fabrication of HfO 2 -PDMS and Cu-PDMS composite materials was composed of aluminum etching, film deposition, PDMS casting on top of the film, and sacrificial etching of aluminum.The detailed description of etching approaches and copper deposition for superhydrophobic Cu-PDMS composite material were described in the previous work. [29]Atomic layer deposition of 20 nm hafnium oxide (200 cycles) at 200 °C was used for SHB nano-HfO 2 composite materials (growth per cycle of 1 Å cycle −1 ).The hafnium oxide layer was grown in a Beneq TFS-500 reactor using tetrakis(dimethylamido)hafnium (TDMA-Hf) as the metal and water as the oxygen precursor.For SHB nano-Cu, electroless plating was used for 1 h to deposit 30 μm copper at room temperature.
Surface Characterization: SEM (EBL Zeiss Supra 40) was used to study on morphology of materials.EDS (Tescan Mira) was performed to study the chemical characterization/elemental analysis of three structured surfaces with accelerating voltage of 10 kV for micro-Cu and SHB nano-Cu, and 15 kV for SHB nano-HfO 2 .Contact angle goniometry (THETA, Biolin Scientific) was applied to measure the advancing and receding contact angles with the needle in sessile droplet technique.The droplet pumping rate of 0.1 μL s −1 was used for measuring both advancing and receding contact angles.Advancing contact angles were measured from 2 to 5 μL droplet size and receding angles from 5 to 0 μL.Sliding angles were measured with an in-house built goniometer with 20 μL water droplets.Contact angles and sliding angles were measured at three different locations on the samples and the reported values are the average ± the standard deviation.
Bacterial Strains, Their Traits, and Culture Conditions: Two Grampositive coagulase-positive Staphylococcus species (S. aureus ATCC25923) and coagulase-negative S. epidermidis (RP62A/ATCC35984) and two Gram-negative P. aeruginosa strains (ATCC15442 and ATCC9027) were used as strong biofilm formers to test the antibiofilm effects of the designed materials.The selected biofilm forming bacteria included i) the Gram-positive S. aureus subsp.aureus ATCC25923 (hereafter Sa_25923), a clinical isolate (Seattle 1945) used as a standard laboratory testing control strain [74] and S. epidermidis RP62A (hereafter Se_RP62A) that is a slimeproducing strain isolated from 1979 to 1980 Memphis, Tennessee, outbreak of intravascular catheter-associated sepsis, [75,76] and ii) two Pseudomonas aeruginosa strains, including ATCC15442 (hereafter Pa_15442), representing a noninvasive and noncytotoxic environmental strain variant of the Pseudomonas genus, [77] and ATCC9027 (DSM 1128) (hereafter Pa_9027) that is a nonvirulent P. aeruginosa strain.All species/strains were purchased from the American Type Culture Collection (ATCC; Wesel, Germany) and routinely propagated on tryptic soy agar (TSA; Lab M Ltd., Heywood, UK) overnight (o/n) at +37 °C prior to biofilm formation assays.
Quantification of Adherent Cells/Biofilm Mass on Different Materials: To monitor the biofilm growth (biomass derived from both viable and dead bacteria), the bacterial colonies from TSA were suspended in prewarmed tryptic soy broth (TSB; Lab M Ltd., Heywood, UK) to achieve optical density 0.02 at 600 nm (OD 600 ), corresponding to ≈2 × 10 7 CFU mL −1 for S. aureus, 8 × 10 6 CFU mL −1 for S. epidermidis, and 1-6 × 10 6 CFU mL −1 for the P. aeruginosa strains.Then, 1.5 mL samples of the suspensions were Wells without any material represented polystyrene that was used as the reference/positive control of biofilm formation.After 48 h of incubation at 37 °C (250 rpm), nonadherent cells were removed and wells with/without the coupons were washed with 2 mL of phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, and 10 mm phosphate, pH 7.4).Washed coupons were transferred into new 24-well plate wells and both the PS control wells and the transferred coupons in new 24-wells were washed one more time with 1.5 mL PBS.After removing the PBS, the biofilms on coupons and PS were fixed with EtOH (96% v/v) for 15 min at room temperature and left to air-dry.Then, 600 μL of 0.1% crystal violet (v/v) (Sigma-Aldrich) was added to the wells with coupons and on PS for 5 min at room temperature.Coupons and PS were washed twice with deionized water and left to dry for 10 min at room temperature.The dye was solubilized in EtOH (96% v/v) for 30 min.Finally, 200 μL from each well was transferred to 96-well plate (FALCON; Tissue Culture Treated, polystyrene, Becton Dickinson) wells and the absorbance at 595 nm (A 595 ) was monitored using the PerkinElmer Victor3 multilabel plate reader.The experiment was repeated twice and each with three material coupons and the A 595 values, i.e., coupons with TSB, were subtracted from values obtained with the bacterial biofilms on the same materials.Unpaired t-test with Welch's correction was used to compare quantitative differences in bacterial biomasses on relevant materials (GraphPad Software, Prism, La Jolla, CA, USA, version 8.0).
Biofilm Viability Assays: The viability of the biofilms formed on the indicated materials was tested as follows.The Staphylococcal and Pseudomonas biofilms were allowed to form as described above.After removing the nonadherent cells and washing the materials twice with PBS, the biofilms were detached by scraping and pipetting in 500 μL of PBS.Suspended cells were serially diluted in PBS (10 −1 -10 −10 ) and 10 μL from each dilution were spotted onto TSA agar.After an overnight incubation at 37 °C, the colony forming units, CFUs mL −1 (number of bacterial colonies per mL of culture), were counted and compared to counts of colonies derived from PS-and PDMS-associated biofilms.The experiment was repeated twice and each with three material coupons.Unpaired t-test with Welch's correction was used to compare quantitative differences in viability.

Figure 1 .
Figure 1.Digital photographs (from top), the SEM images, schematic illustrations (cross-section), and the SEM cross-section of three structured samples: a) micro-Cu, b) SHB nano-HfO 2 , and c) SHB nano-Cu.Corresponding advancing contact angles of each material are shown in insets.
Authors.Advanced Materials Interfaces published by Wiley-VCH GmbH

Figure 2 .
Figure 2. The advancing contact angles, receding contact angles, and sliding angles of all tested materials: a) before and b) after the UV treatment (bars represent standard deviation).

Figure 3 .
Figure 3. Antibacterial properties of the surfaces as characterized by the adherent growth/biofilm biomass (blue) and number of viable cells (pink) of the selected Gram-positive and -negative biofilm models.Gram-positive Staphylococcus species: a) S. aureus ATCC25923 and b) S. epidermidis RP62A, and Gram-negative Pseudomonas aeruginosa strains: c) P. aeruginosa ATCC15442, and d) P. aeruginosa ATCC9027.*, **, *** p, denote the p values, in which p < 0.05 was considered statistically significant and p < 0.001 highly statistically significant.
Moreover, Adv.Mater.Interfaces 2023, 10, 2300121 2300121 (5 of 10) © 2023 The Authors.Advanced Materials Interfaces published by Wiley-VCH GmbH bacteria prefer to grow as biofilms, as this growth mode also protects the residing cells against stress.

Figure 4 .
Figure 4. Antibacterial effects conferred by SBH nano-Cu2+ against Gram-positive staphylococci and Gram-negative P. aeruginosa.a) Primary and secondary mechanisms of adhesion determine the strength of initial adherence, affect the accumulation of cells and early stages of biofilm formation on the indicated materials.b) Potential mechanisms of antibacterial effect of SHB nano-Cu against the selected bacteria.The strength of the antibacterial effect was compared to the adherence and the viability in relation to PS, PDMS, micro-Cu, and SHB nano-HfO2.EVs, extracellular vesicles; LTAs, lipoteichoic acids; eDNA, extracellular DNA; PIA, polysaccharide intercellular adhesin; RLs, rhamnolipids; Alg, alginate.*, ATCC9027 cell surfaces are more hydrophilic compared to those of ATCC15442.
Authors.Advanced Materials Interfaces published by Wiley-VCH GmbH pipetted into 24-well microtiter plate (FALCON; Tissue Culture Treated, polystyrene, Becton Dickinson) with the indicated materials (0.5 mm thick and 12.3 mm in diameter).Prior to biofilm assays each tested material was sterilized/disinfected on both sides by UV light irradiation at 254 nm (40 W with 10 cm distance to the lamp) overnight at room temperature.