Enhancing implant performance: 20% reduction in Pseudomonas aeruginosa bacterial initial formation with Cu_0.75Ti_0.25O₂ coating

In healthcare facilities, bacterial infections primarily originate from patients and healthcare workers, although the environment also plays a significant role. The environment can act as a reservoir for potentially infectious micro-organisms, contributing to their transmission. As human lifestyles improve, the importance of creating and maintaining a healthy environment becomes increasingly evident. Without environmental awareness, individuals are naturally exposed to various harmful bacteria that can spread on inanimate surfaces, on equipment, and through the air.

Antimicrobial resistance poses a substantial global health threat, with six major pathogens (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Streptococcus pneumonia, and Acinetobacter baumannii) being responsible for 929 000 deaths worldwide in 2019.¹ Moreover, bacterial biofilms, which accumulate on surfaces ranging from household water pipes to medical equipment, contribute to persistent health problems and infections.^2,3 Surgical implant-related infections, particularly with the presence of biofilms, remain a challenging and catastrophic complication. In 2017, for instance, multidrug-resistant Pseudomonas aeruginosa caused an estimated 32 600 infections and 2700 deaths among hospitalized patients in the United States.

Pseudomonas aeruginosa, among various Pseudomonas species, is the most common cause of infections in humans, affecting the bloodstream, lungs, and other organs.⁴ Consequently, extensive research has been conducted on this particular pathogen and its biofilm formation on surfaces. For instance, Kim et al. demonstrated that Pseudomonas aeruginosa forms denser biofilms under microgravity conditions than those on earth.⁵ In addition, Pseudomonas aeruginosa biofilms are commonly found in chronic wounds and contribute to conditions such as chronic osteomyelitis, an infection where micro-organisms attach to dead bone.⁶ In general, it is widely accepted that organisms associated with biofilms are accountable for more than 65% of microbial infections. These organisms demonstrate significant resistance to antimicrobial agents as well as components of the host defense system, encompassing both innate and adaptive immunity.⁷ 

Due to the aforementioned challenges,⁸ effectively controlling germ growth has become a critical concern, and extensive efforts are being directed toward combating biofilm formation.^9–11 Developing methods to modify the surfaces of biomaterials or materials while preserving their mechanical properties is essential in addressing this issue. Various strategies have been employed to combat the detrimental effects of biofilm contamination, including anti-adhesion techniques, contact activation, and the release of biocidal agents.¹² However, these treatments often exhibit limited efficacy as incomplete eradication of bacteria can lead to increased resistance against cleaning agents.¹³ As a result, alternative approaches such as surface coating with anti-biofilm agents and related materials have been developed.¹⁴ Among these approaches, thin film technology has shown significant promise in enhancing antibacterial activity. Furthermore, the preparation of thin films at the molecular level can play a crucial role in determining the interaction between human biology and materials, making it an essential aspect to consider.

In biomedical applications, the choice of coating material remains a significant consideration. Polymer surfaces are widely accessible, cost-effective, and effective in combating biofilm growth.¹⁵ However, they tend to exhibit lower strength and are prone to degradation in the body’s biochemical environment.¹⁶ 

Alternatively, other compounds such as metals, alloys, or ceramics can be utilized as coatings. Various metal ions, including Zn⁺², Cu⁺², Al⁺³, Ti⁺⁴, and Ag⁺, have been extensively investigated for their antimicrobial properties.^17–19 Copper-based coatings, for instance, show promise as biocompatible materials for developing anti-biofilm coatings, effectively protecting surfaces against harmful biofilm pathogenesis.²⁰ Several studies have demonstrated the antimicrobial activity of copper or copper oxide against a wide range of bacteria.^21–23 

In addition, Ti-based coatings are frequently employed as biomaterials in implant applications. Doping copper into the TiO₂ host offers several advantages. Copper serves as an antibacterial agent, while TiO₂ helps reduce the recombination of charge carriers, which is crucial for antibacterial activity.²¹ These synergistic effects contribute to the enhanced antibacterial properties of Cu-doped TiO₂ coatings. Overall, the choice of coating material is a critical factor in biomedical applications, with both polymer and metal-based coatings offering distinct advantages in terms of antimicrobial properties and biocompatibility.^21–23 

In this article, we utilized the Pulsed Laser Deposition (PLD) technique to grow (Cu,Ti) oxide films on glass surfaces. These coated surfaces were then subjected to exposure to Pseudomonas aeruginosa, a specific bacterium of interest. Our main objective was to investigate the antimicrobial activity of the coatings against this bacterium, focusing on the influence of surface properties and varying Cu/Ti ratios. Remarkably, we observed a significant 20% reduction in biofilm formation by bacterial cells on Cu_0.75Ti_0.25O_x coated glass surfaces compared to the control glass samples in the initial steps. We attribute this reduction to the alteration of copper content present at the surface of the films. Our findings highlight the potential of these coatings to effectively inhibit biofilm formation by Pseudomonas aeruginosa, emphasizing the significance of surface modifications and the specific Cu/Ti ratio in achieving such antimicrobial effects, which can be further used for bioimplants.

In this section, various techniques and methods are employed to synthesize and characterize the Cu_1−xTi_xO₂ thin films, including evaluating the roughness, wettability, surface energy, composition, and antibacterial activity.

A series of thin films with varying thicknesses were fabricated on a 12 mm diameter glass substrate using PLD (refer to the schematic setup depicted in Fig. 1).²⁴ Prior to introduction into the vacuum chamber, the glass substrate underwent ultrasonic cleaning with ethanol, followed by rinsing in deionized water and subsequent drying with compressed air. Various compositions of Cu_1−xTi_xO₂ (CTO) were tested, specifically x values of 0.25, 0.5, and 0.75. For the preparation of (Cu,Ti)O₂ targets, a conventional solid-state process was employed, which involved mixing CuO and TiO₂ powders to the desired stoichiometry, followed by multiple firing cycles at 800 °C in an air environment. The resulting product was then pressed and sintered for 24 h at 1100 °C. During the film deposition process, a KrF excimer laser (λ = 248 nm, 3 Hz) was scanned over the target surface at a fluence of 2.45 J/cm². The substrate, positioned at a distance of 4.5 cm from the target, was maintained at a temperature of 200 °C under a base pressure of 1.1 × 10⁻⁷ mbar. To achieve different film thicknesses, the number of laser pulses was varied from 4500 to 10 000, while employing a low deposition rate of 0.0024 nm/pulse to prevent splashing or the formation of particulates on the films.²⁵ 

The structural quality of the films was assessed through x-ray reflectance (XRR) utilizing a Bruker D8 Discover diffractometer (CuKα₁, λ = 1.5405 Å) within the 2θ 0°–5° range. The film thickness was determined based on the relative position of the vibrational maxima in relation to the square of their atomic number. The topography analysis was performed using the atomic force microscopy (AFM, PicoSPM) technique, and the squared roughness (rms) representing the square root of the surface height distribution was extracted using WSxM 5.0 software for a 1 μm² area in tapping mode.²⁶ Chemical analysis of Cu_0.75Ti_0.25O₂ thin films was carried out by x-ray photoelectron spectroscopy (XPS) with an Al Kα excitation source (hν = 1486.6 eV) operated at 10 mA and 10 kV using a Specs GmbH system in an analyzer chamber with a base vacuum of 5 × 10⁻¹⁰ mbar. The XPS spectra were obtained in a large-area mode with a passband energy of 60 eV and a step size of 0.05 eV.

To facilitate biofilm formation, the stained bacteria were first collected and suspended in a specially formulated ABT minimal medium, which contained 2 g of glucose per liter and 2 g of casamino acids per liter (referred to as ABTGC). This bacterial suspension was then introduced into an agarose chamber. Prior to imaging, the bacteria were diluted to an optical density of OD600 = 1.5 and stained with Vybrant DyeCycle Green Stain (Invitrogen) for 30 min while being agitated. After an incubation period of 6 h at 37 °C, the agarose chamber was mounted on the Zeiss light-sheet microscope (Z.1). During the imaging process, the focal plane was adjusted to focus on the glass surface where the bacteria adhered and initiated biofilm formation. The entire experiment was repeated three times independently for accurate quantification analysis. For the preparation of the experimental setup, 12-mm glass coverslips, both coated and uncoated, were positioned in a 24-well flat-bottomed microplate. Next, 200 μl of the bacterial suspension was added to each well, followed by an incubation period of 24 h at 37 °C with the lid closed. Subsequently, the well contents were aspirated and washed, and a 0.1% aqueous solution of CV (Sigma-Aldrich) was added to the wells containing the biofilm. The plate was incubated at room temperature for 30 min. Afterward, the CV solution was removed, and the wells were washed three times using 200 μl of sterile water to avoid disturbing the biofilm. The plate was left to air-dry for 30 min. Finally, 200 μl of a 30% (v/v) glacial acetic acid solution was added to each well to dissolve the dye bound to the adherent biofilm, and absorbance readings were taken at 570 nm. To study the initial attachment of mPAO1, the adhesion of individual bacteria to both control and Cu_0.75Ti_0.25O₂-coated glass surfaces was visualized using a light-sheet microscope. The mPAO1 cells were incubated in a custom-made light-sheet chamber designed to simulate a three-dimensional environment conducive to natural biofilm formation. The setup involved a bacterial inoculation chamber and a Zeiss light-sheet microscope Z.1 (Carl Zeiss), as previously described by Khong et al.³² To create the bacterial inoculation chamber, a 1% liquid agarose solution was injected into a 1 ml syringe, and the syringe tip was then sealed with parafilm after removing the tip. A square styrene rod was placed on a yellow tip holder and inserted into the center of the cylindrical agarose column, forming a hollow chamber suitable for bacterial inoculation. Once the agarose solidified, the square styrene rod was removed, and a long glass strip, either coated or uncoated, was positioned against the agarose wall. The imaging chamber was then extended to the desired length (approximately half of the agarose chamber) by pushing the syringe plunger, enabling subsequent light-sheet imaging.

The surface characteristics of the deposited thin films of Cu_1−xTi_xO₂ were examined using the AFM (Atomic Force Microscopy) technique. Figures 2(a) and 2(b) present representative images of Cu_0.75Ti_0.25O₂ films with thicknesses of 20 and 24 nm, respectively. These images illustrate that the films exhibit a uniform coverage with a yellow transparent color, indicating a homogeneous deposition without any presence of pin-hole defects. Moreover, all the films display excellent adhesion to the glass substrate. The roughness measurements of both samples reveal values less than 1 nm, indicating remarkably smooth surfaces. The thicknesses of the films were determined using XRR analysis, as depicted in Fig. 2, and were found to be 11, 16, 20, and 24 nm.

Figure 3 illustrates the variation in the contact angle with respect to film thickness. For distilled water, the contact angles were measured to be 60.4°, 67.5°, 76.1°, and 81.3° for film thicknesses of 11, 16, 20, and 24 nm, respectively. Similarly, for ethylene glycol, the contact angles were found to be 42.2°, 53.7°, 55.5°, and 65.7° for the same respective film thicknesses. Figures 3(b) and 3(c) depict typical images used to measure the contact angle for water and ethylene glycol, respectively (Table I). The obtained data suggest that the thin Cu_0.75Ti_0.25O₂ films exhibit inherent hydrophilicity, as indicated by contact angles of less than 90°. The surface, serving as the point of contact between the bacteria and the material, plays a crucial role in promoting or preventing bacterial adhesion. Surface wettability, a fundamental property, governs the interactions between solid and liquid phases in biological systems as hydrophilic surfaces facilitate adhesion. By utilizing the Owens–Wendt equation and the contact angles of these films, the surface energy was calculated (see Fig. 3 and Table I).

We utilized the dispersive $(γld)$ and polar $(γlp)$ fractions to calculate the surface energy, which were found to be 21.8 and 51 mJ/m² for water and 34 and 30.4 mJ/m² for ethylene glycol, respectively, as previously reported.³¹ By considering these components, the total surface energy was determined, resulting in surface energies of 40.75, 33.98, 29.79, and 24.45 mJ/m² for Cu_0.75Ti_0.25O₂ films with thicknesses of 11, 16, 20, and 24 nm, respectively (Fig. 3). Table I clearly demonstrates that increasing the film thickness leads to modifications in both the surface energy and contact angle. In fact, the contact angle and surface energy exhibited an inverse relationship. These changes can be explained by Eq. (1), where an increase in θ leads to a decrease in the term cos(θ). As a result, the surface tension of the solid decreases. The variation in the contact angle strongly depends on surface roughness, which is influenced by factors such as the density of layers, the preparation method of thin films, and geometric factors.³³ 

The relative concentration of Cu(I) species, Cu(I), Cu(II) species, and Cu₂ and the total area of the shake-up satellite peak, S, were used to calculate their respective proportions. The calculated relative concentration of Cu(I) species was determined to be 38.03% and 39.28% on the surfaces of the 16 and 24 nm thick films, respectively. The Ti 2p spectrum exhibited two peaks, at ∼457.71 eV (2p_3/2) and 463.21 eV (2p_1/2), with a difference of ∼5.5 eV, closely matching the 5.6 eV difference for Ti(IV) species. Thus, the peak fitting confirmed the presence of only Ti(IV) species on the film surface, ruling out the presence of any Ti (0)-metallic species. This observation suggests the presence of Ti⁴⁺–O bonds within the system. The experimental Cu/Ti ratio in the samples was determined as 70.32/29.68 for the 16 nm film and 80.11/19.89 for the 24 nm film. This indicates a deviation of ∼±5% from the nominal composition of the target used during the deposition. The deviation primarily arises from two factors: the XPS experiment, which relies on proper background selection and accurate peak positioning, and the variation in experimental conditions, such as pressure, energy calibration of the laser, scan range, etc., between different depositions. These results confirm the reliability of the experimental composition of the thin films. Furthermore, the O 1s asymmetric peak could be deconvoluted into multiple peaks, where O₁ and O₂ correspond to the lattice oxygen of CuO and Cu₂O, while O₃ and O₄ represent oxygen vacancies and adsorbed oxygen, respectively.

To investigate the ability of Cu_1−xTi_xO₂ coatings with varying compositions (x = 0.25, 0.50, 0.75) to resist biofilm formation, we conducted an experiment using round glass slides, both coated and uncoated, placed in microtiter plates. The efficiency of biofilm formation was assessed using the crystal violet biofilm detection assay. mPAO1 bacteria were cultured under conditions that promote biofilm formation and incubated for 24 h at 37 °C in the microtiter plates. The bacterial cells adhering to the glass surfaces were stained and quantified to evaluate the extent of biofilm formation. Our findings consistently demonstrated that the biomass of the biofilm was significantly lower on the glass surfaces coated with Cu_1−xTi_xO₂ [specifically, (x = 0.75)] than the uncoated control surfaces [refer to Fig. 5(a)]. It is widely recognized that the initial attachment of bacteria to a surface is crucial for biofilm formation. Therefore, we tested the hypothesis that a Cu_1−xTi_xO₂ coating with x = 0.75 could hinder the initial attachment of mPAO1 bacteria while the other two compositions (x = 0.25, 0.50) did not significantly alter the attachment (see Table II). To examine the initial attachment of mPAO1, we visualized the adhesion of individual bacteria to both the control and Cu_0.75Ti_0.25O₂-coated glass surfaces using a light sheet microscope. The mPAO1 cells were incubated for 6 h in a specially prepared light sheet chamber under conditions favorable for biofilm formation. Our results revealed a significant reduction in the adhesion of mPAO1 bacterial cells to the glass surface coated with Cu_0.75Ti_0.25O₂ compared to that to the uncoated glass surface. The light sheet image illustrates the initial accumulation of fluorescently labeled mPAO1 bacterial cells (depicted in green) on both the control and Cu_0.75Ti_0.25O₂-coated glass surfaces. Each bacterial cell is visualized as a green dot on a single plane [refer to Figs. 5(b) and 5(c)].

Structural studies initially revealed that the Cu_0.75Ti_0.25O₂ films deposited on glass substrates by PLD are amorphous, as evidenced by their x-ray diffraction patterns. In addition, AFM images displayed a remarkably low surface roughness of ∼1 nm. Moreover, all the films exhibited hydrophilic properties, although the thicker ones displayed higher contact angles (81.3° for the 24 nm film compared to 60.4° for the 11 nm film). In a related study, Matlaga et al. reported that biomaterials with contact angles exceeding 65° can be classified as hydrophobic and such surfaces have the potential to inhibit cell attachment.³⁵ Conversely, surfaces with contact angles ranging from ∼40° to 80° tend to exhibit adhesive properties, promoting relatively high cell and bacterial attachment.³⁶ This corroborates our findings as the contact angles of the Cu_0.75Ti_0.25O₂ films were less than 90°, indicating their hydrophilic nature. Therefore, we can infer that the surface topography of the Cu–Ti oxide layer facilitates cell adhesion.

Overall, our comprehensive data analysis supports the conclusion that the Cu_0.75Ti_0.25O₂ films possess a hydrophilic surface, making them favorable for promoting cell attachment and potentially enhancing biomedical applications.

Based on the XPS data, our analysis revealed that the films predominantly contain Cu and Ti elements. Notably, the Cu/Ti ratio increases with film thickness, indicating the migration of copper ions toward the film surface. Furthermore, the presence of both Cu⁺ and Cu⁺² ions on the surface was confirmed, with the thicker film exhibiting a slightly higher proportion of Cu⁺ ions. This phenomenon could potentially enhance the penetration of copper ions through bacterial cell membranes, leading to the disruption of cellular processes and eventual cell death. The initial attachment of bacteria plays a critical role in biofilm formation as it establishes the foundation for inter-bacterial interactions and communication within the community.³⁷ Previous studies have attributed the toxicity of copper to the release of copper ions under wetting conditions. Our findings reinforce the significance of copper by confirming its essential presence. The interaction between copper ions on the film surface and proteins in Pseudomonas aeruginosa bacteria can be described as follows: One important mechanism involves the formation of copper complexes that generate radicals capable of inactivating viruses. In addition, copper can bind to thiol and other protein groups, leading to the disruption of enzyme structure and function, similar to other transition metals. Copper ions can also chelate with proteins by binding to amino and carboxyl groups, resulting in protein inactivation. Thus, our study highlights the presence and role of copper ions in the films. Their ability to interact with proteins in Pseudomonas aeruginosa bacteria through complex formation, radical generation, and protein binding contributes to the disruption of bacterial processes, emphasizing their potential as antimicrobial agents.

Furthermore, the antimicrobial activity of copper is closely linked to its interaction with DNA. Copper ions, specifically Cu⁺², possess the capability to form chelates with phosphate groups, resulting in the disruption of hydrogen bonds within DNA and subsequent microbial death. Copper ions exhibit a distinct affinity to DNA, enabling them to bind and disturb the helical structure of DNA and integrate within and between DNA strands. Moreover, copper can form complexes with mRNA, contributing to the degradation of viruses. This multifaceted interaction of copper with genetic material underscores its potent antimicrobial properties.^38,39

Copper’s antimicrobial effects are not limited to nucleic acids; they also extend to lipids. In the presence of copper, lipids can undergo peroxidation, leading to the formation of gaps in the cell membrane and consequent disruption of cellular integrity. XPS analysis confirmed the presence of Cu⁺ ions on the surface, which exhibited higher cytotoxicity against bacteria than Cu⁺² ions. The combination of Cu⁺ ions and OH⁻ ions (active oxygen) effectively destroyed microbial membranes and DNA by interacting with proteins that contain –CH and –SH groups. This interaction resulted in the disruption of microbial cellular components, further emphasizing copper’s antimicrobial properties.

In addition, adding Ti to a thin coating improves adherence with implant-use TiAlV alloy. The thin film and TiAlV alloy are compatible and chemically affine due to their titanium-based chemical compositions. Titanium naturally creates oxide layers, improving its adherence. The thin film’s titanium oxide layers interact with the TiAlV alloy’s titanium oxide layer to promote adhesion and bonding. Titanium aids interface atomic interdiffusion. Titanium atoms travel between the thin film and alloy, forming intermetallic compounds and strengthening their link. Titanium in the thin coating enhances surface roughness, which increases contact points and surface area, improving mechanical interlocking with the TiAlV alloy.

Furthermore, our light sheet microscope imaging setup is well-suited for studying the initial attachment and behavior of bacterial cells on various surfaces, including walls and tilted surfaces.³² In line with this, using the light sheet microscope, we demonstrated a reduction in the initial attachment of mPAO1 bacterial cells on Cu_0.75Ti_0.25O₂-coated glass surfaces compared to uncoated control surfaces. These findings align with previous reports highlighting the antimicrobial activities of copper-plated surfaces.⁴⁰ We observed similar antimicrobial activity of the Cu_0.75Ti_0.25O₂ coating in preventing initial biofilm formation on vertical surfaces. Ongoing efforts are focused on improving the performance of copper coatings through modifications in compound compositions, as reported in previous studies.^38,40 These observations further support the crucial role of copper ions in exerting toxicity against bacteria.⁴¹ 

In this study, we investigated the growth of Pseudomonas aeruginosa bacteria on thin Cu–Ti films deposited on glass coverslips. Our findings shed light on the interaction between bacterial cells and the Cu–Ti films, providing valuable insights into their antimicrobial properties. The deposited films exhibited a hydrophilic and smooth surface, which are favorable characteristics for bacterial cell attachment. XPS analyses confirmed the presence of active Cu⁺ and Cu²⁺ ions on the film surface. These ions were found to penetrate the bacterial cells, leading to the destruction of their DNA and RNA, effectively inhibiting bacterial growth.

By utilizing our light sheet microscope imaging setup, we observed a remarkable 20% reduction in bacterial growth on the Cu_0.75Ti_0.25O₂ coated surfaces compared to that on uncoated surfaces, as demonstrated by the crystal violet test. This provides evidence that the Cu_0.75Ti_0.25O₂ surfaces exhibit resistance to biofilm formation. We attribute this reduction in biofilm formation to the inhibition of the initial attachment of individual mPAO1 bacterial cells, consistent with previous reports on the antimicrobial activities of copper-plated surfaces.

While our experimental conditions may not fully replicate real-life scenarios, we believe that these findings have significant implications for various applications, particularly in the healthcare industry and in the prevention of surgical implant-related infections. Biofilm formation poses a substantial risk, and our study contributes to the understanding of strategies to mitigate this risk.

Finally, our study provides valuable insights into the antimicrobial properties of Cu–Ti films and their impact on bacterial growth and biofilm formation. These results hold promise for the development of effective approaches to combat bacterial infections, benefiting diverse fields where biofilm-related risks are a concern, such as healthcare and surgical implant applications.

The authors are thankful for the Indo-French collaboration through the financial support of the Laboratoire Franco-Indien pour le Cooperation Scientifique (LAFICS) from the CNRS and are thankful for the financial support through MERLION Project No. 5.02.18, “Interaction of Living Matter with Oxide Thin films.” M.S.R.R. is thankful for a fellowship for Invited Professors from the University of Caen, Normandie. Partial support from the Program Emergence “InCox” supported by the Region Normandie and the RIN ONCOTHERA is also acknowledged.

The authors have no conflicts to disclose.

A. Yadav: Conceptualization (equal); Investigation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). S. Pradhan: Methodology (supporting); Visualization (equal). M. Khokholva: Visualization (supporting). O. El Khaloufi: Visualization (equal). N. Z. J. Khong: Investigation (equal); Methodology (equal); Visualization (equal). S. K. Lai: Methodology (equal); Visualization (equal). A. Fouchet: Visualization (equal). A. David: Visualization (equal). U. Lüders: Formal analysis (equal); Visualization (equal). H.-Y. Li: Formal analysis (equal); Methodology (equal); Visualization (equal). M. S. R. Rao: Visualization (equal). W. Prellier: Investigation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding author upon reasonable request.