Biomaterial-associated infections remain a major hindrance to the long-term utilization of most implanted or intravascular devices, including orthopedic prostheses, artificial valves, urinary tract and cardiovascular catheters, intraocular lenses and dentures [1,2]. Bacterial attachment and subsequent biofilm formation frequently results in indwelling device related infections and device failure [3,4]. In their biofilm state, bacteria are less susceptible to host defense mechanisms and systemic antibiotics, and also to detachment due to flow conditions. The presence of these biofilms can often cause an infection, often requiring the surgical removal of the infected device [5]. The mechanism of bacterial adhesion is complex, with initial reversible physicochemical interactions being followed by intricate irreversible molecular and cellular interactions. When the distance between the bacterial cell and other cells or abiotic surfaces is larger than 50 nm, the interactions between these two entities are nonspecific and are directly related to the distance and the dispersive component of free energy characteristics pertinent to these two surfaces [6]. The nature of these forces, i.e., whether they are attractive or repulsive, will either facilitate the bacterial attachment or prevent the cell from engaging into the molecular or cellular phase of adhesion. When the distance separating these surfaces becomes less than 5 nm, chemical interactions such as hydrogen bonding, ionic and dipole interactions, hydration and/or hydrophobic interactions become significant, often resulting in a more stable adhesion between the microorganism and the surface [7]. Various polymeric structures such as capsules, fimbriae, pili, and slime that can be present on the surface of the bacterial cell engage in the molecular specific irreversible reactions with the chemical features of the tissue or abiotic surface. Therefore, in order to effectively control the attachment and adhesion of the cells to the surfaces, it is imperative to understand the surface properties and the physicochemical interactions associated with the initial events of cell adhesion. An obstacle to obtaining this understanding is that the relative contributions of the molecular and physical interactions that govern bacterial adhesion to biomaterials are yet to be fully interpreted.

There is much debate as to the extent to which the nanometer scale surface topography of a solid substrate influences bacterial adhesion and their subsequent proliferation to form biofilms [8,9]. Investigations on the adhesion of Pseudomonas aeruginosa to poly(methyl methacrylate) contact lenses identified that surfaces with a root-mean-square (RMS) roughness parameter of 14 nm or above tended to experience an increased extent of micro-organism attachment [10]. Other studies on the attachment preferences of human pathogens P. fluorescens and Staphylococcus aureus have demonstrated that the topography of micro-rough titanium surfaces can influence the extent of cell attachment and growth, with cells showing propensity for growth in the trenches of the surface in long rows [11,12], whilst the attachment response of these bacteria towards smooth surfaces did not follow a distinct pattern [11,13]. A few early studies reported that surface roughness was a ‘minor factor’ in the attachment mechanism of bacteria, in that the bacteria displayed no preference for adhesion to surface features such as scratches or grooves [8,9]. However, preferential adherence of bacteria to grooved and braided surfaces was found by Scheuerman et al., who attributed this increased attachment propensity as being due to the presence of an increased surface area [13]. More specifically, in situations where the size of the surface features was comparable to the size of the bacterium, the bacteria-surface contact area is at a maximum and hence the binding potential of the bacteria was increased [14]. In cases where the surface features were significantly smaller than the bacterial size, the contact area was reduced, resulting in a reduced extend of binding [15]. The level of extracellular polymeric substance (EPS) production and bacterial cell morphology have also been shown to be affected by the surface architecture [11,16,17]. Nano-patterning of gold surfaces has been demonstrated to enhance the localized attachment of P. fluorescens in the trenches of the surfaces compared to that obtained using native gold surfaces, with cells showing limited EPS production and reduced cell size compare to those attached onto non-nano-patterned surfaces [11]. Our recent investigation on the effect of surface roughness on the attachment behavior and production of extracellular polymeric substances for the Gram-positive coccoid pathogen, S. aureus, and Gram-negative rod shaped bacterium, P. aeruginosa, have shown that both pathogens exhibit a greater propensity for adhesion to glass and polymer surfaces that are smooth on the scale of nanometers, resulting in elevated cellular metabolic activity, augmented production of extracellular polymeric substances, and increased number of bacterial cells undergoing attachment [17-19].

Numerous surface modification techniques, such as radio frequency (RF) plasma modification, surface abrasion, chemical coating, and chemical grafting can be employed to change surface properties of the substrate so as to inhibit or promote bacterial adhesion and biofilm formation [20-26]. In this context, the present study is an extension of our previous work on the development of a novel organic polymer coating for the prevention of growth of medically significant bacteria on three-dimensional solid surfaces. In this study, the effect of surface coating with terpinen-4-ol on the adhesion and proliferation tendencies of S. aureus were examined and compared to those previously reported for P. aeruginosa. The surfaces of substrates were modified by the fabrication of a polyterpenol thin film derived from terpinen-4-ol using RF plasma enhanced chemical vapor deposition. Terpinen-4-ol is a constituent of tea-tree oil and its monomer (liquid) form is known to inhibit growth of a broad range of bacteria [27-30]. Surfaces of coatings produced from terpinen-4-ol are known to be smooth on the scale of nanometers and defect free, with good adhesion to the substrate and good stability [31-34]. Since previous studies have shown S. aureus and P. aeruginosa to preferentially attach and adhere to surfaces that are smooth on the nanometer scale, this study examines the influence of surface architecture on the antifouling properties of the polyterpenol coating.

Our results showed that the presence of the polyterpenol thin films significantly altered the surface properties of the glass substrates, which resulted in a notable change in the adhesion behavior of both P. aeruginosa and S. aureus cells. The difference in surface architecture was reflected in the surface roughness parameters of polyterpenol coatings applied under different deposition power. This was attributed to the degree of monomer fragmentation resulting from the increased intensity of the plasma field. The lower deposition rate and higher substrate temperature associated with the increased RF power conditions allowed for a faster relaxation of film fragments within the growth region prior to deposition of the next layer of the film, resulting in a smoother surface [22]. This observation is clearly supported by the notable differences in roughness parameters of the two polyterpenol surfaces examined, particularly when the data from 10 μm × 10 μm scanning area sizes are compared. The higher peaks observed on the surfaces of the 10 W samples can be also explained by the presence of large fragments of terpinen-4-ol molecule that are loosely bound to the surface of the polymer matrix.

Modification of the surface properties using polyterpenol not only altered the surface architecture, but also the surface chemistry. The combined effect of these two parameters can strongly influence the extent of bacterial adhesion and proliferation while preserving the material properties inherent to the substrate, such as the mechanical properties [39]. Alterations of the functional groups present on the surface of the substrate change the surface hydrophobicity and surface energy. These parameters not only directly affect the interactions between the cells and the substrate, but also the manner in which the surface interacts with its immediate environment, such as the liquid medium [40]. Previously published S. aureus adhesion studies showed that bacterial cells tended to attach more effectively to surfaces bearing carboxylic and methyl functional groups than those containing hydroxyl functionality [41]. Other studies reported a reduction in bacterial adhesion for negatively charged poly(methyl methacrylate)/acrylic acid functional groups, whilst the presence of positively charged methyl methacrylate/dimethylaminoethyl methacrylate groups resulted in an increased level of bacterial cell attachment [14]. In this study, the modification of the surface chemistry of the substrates resulted in a significant increase in the amount of carbon available on the surface. The higher oxygen concentration observed in the case of the 25 W polymer films can be attributed to the oxidation of the top layer of the film, which is part of the post-degradation ageing reaction occurring in the film. Due to the nature of the fabrication process, films manufactured at higher input energy tend to contain a higher number of radicals compared to those deposited at lower power settings, as the lower power conditions favor the preservation of original molecular structure of the monomer precursor [31].

Comparison of the attachment preferences of S. aureus to unmodified glass surfaces and 25 W polymer coated substrates showed that the presence of the polyterpenol film resulted in a significant increase in the number of the attached bacterial cells, their size and the quantity of EPS these cells produced. P. aeruginosa was also found to attach more readily to the 25 W samples compared to the unmodified glass substrates, a behavior similar to that previously reported for P. aeruginosa on topographically dissimilar glass surfaces and on polyterpenol samples [34,42]. There is much debate and controversies concerning the extent to which surface free energy and its polar component affect bacterial adhesion [43,44]. In the case of S. aureus, the thermodynamically predicted preference of hydrophobic cells for hydrophobic substrates can partially account for the reduced cell adhesion to the more hydrophilic surface of the unmodified glass substrate, as reported elsewhere [45]. The 25 W polymer layer is smooth on the sub-nano-scale roughness level compared to unmodified substrate; therefore the current “attachment point” theory [46-47] that suggests that bacterial cells prefer microscopic surface irregularities as the starting point for their attachment as these provide shelter from unfavorable environmental influences clearly also cannot explain the attachment behavior. Our recent studies found that nano-scale surface roughness may have a notable effect on bacterial attachment and adhesion, with nano-scale fluctuations in roughness exerting a significant influence on the cellular response to certain surfaces [18,42,48]. The results presented here show that a higher number of bacterial cells were able to colonize the nano-smooth 25 W polymer coated substrates, whereas less smooth, unmodified glass substrates sustained a significantly lower level of cellular attachment. This adhesion tendency was observed for both strains used in this study, regardless of their taxonomic affiliation and their cell surface characteristics. Further studies on polyterpenol coatings fabricated at even higher RF powers showed a similar trend [49].

Glass substrates modified with a 10 W polyterpenol coating exhibited the least number of bacterial cells adhering to the surface. In its monomer form, terpinen-4-ol has been characterized as a nonspecific membrane active biocide that possesses adequate hydrophilicity to diffuse through liquid medium to the bacterial cytoplasmic membrane and hydrophobicity necessary to diffuse through the bacterial cytoplasmic membrane [28], where it accumulates causing a loss of membrane integrity [50]. Previous studies have demonstrated that it is possible to preserve certain functionalities and hence the biological activity of terpinen-4-ol in polyterpenol thin films by using relatively low deposition power conditions [33,34]. Hence, the significant reduction in the attachment of bacterial cells observed on these surfaces could be due to the preservation of chemical activity rather than the reduction in the surface irregularities or an increase in the hydrophobicity of the surface compared to the unmodified substrate.

Modification of the surface of glass substrates was achieved using radio frequency plasma enhanced chemical vapor deposition (RF-PECVD) of a thin layer of polymer coating using terpinen-4-ol (Australian Botanical Products, Ltd.) as a precursor. The detailed account of the experimental set-up and deposition procedure is outlined elsewhere [31]. Briefly, the RF energy (13.56 MHz) was delivered into a custom-designed glass deposition chamber by means of two capacitively coupled copper electrodes. Glass substrates (BioLab Scientific, Ltd.) were pre-washed in a 1:5 solution of Extran and distilled water, ultrasonically cleaned using distilled water heated to 50 °C for 30 min, rinsed with propan-2-ol and dried. Cleaned substrates were then placed into the deposition reactor, which was then evacuated to 200 mTorr, flushed with argon for 1 min to ensure that the substrates possessed an oxygen-free surface. The chamber was then evacuated to achieve a pressure of 50 mTorr. The terpinen-4-ol vapor was released gradually into the chamber and the glow was maintained by controlling the monomer flow [32]. Deposition was performed in two stages at room temperature and pressure of 200 mTorr. First, to improve the strength of adhesion between the coating and the substrate, a 100 nm layer of polyterpenol thin film was deposited using input power of 75 W. Then, a functional coating was deposited from terpinen-4-ol under 10 W or 25 W RF power for 10 min to achieve films of approximately the same thickness.

The roughness parameters of both the native and coated glass surfaces were quantitatively analyzed using an atomic force microscope (AFM) (NT-MDT) in semi-contact (tapping) mode, using (NSC05, NT-MDT) cantilevers with a spring constant of 11 N/m, tip radius of curvature of 10 nm, aspect ratio of 10:1 and resonance frequency of 150 KHz. 3-D interactive visualization and statistical approximation was used to analyze the topographic profiles of the surfaces. Scanning was performed perpendicular to the axis of the cantilever at a rate of typically 1 Hz, with the scan areas of 1 μm × 1 μm and 10 μm × 10 μm. At least five samples for the surfaces of each condition have been investigated.

In addition to the surface morphology, surface chemical characteristics were also determined. The chemical composition of the surface before and after coating was analyzed by means of X-ray photoelectron spectroscopy (XPS). XPS was performed using an Axis Ultra spectrometer (Kratos Analytical Ltd., UK), equipped with a monochromatic X-ray source (Al Kα, hν = 1,486.6 eV) operating at 150 W, as described elsewhere [34]. Using the Kratos Vision II software, peaks in the high-resolution regions of spectra were fitted with synthetic Gaussian-Lorentzian components. Surface wettability was determined from contact angle measurements using (KSV 101) employing the sessile drop method [51-54].

Prior to each experiment, a fresh bacterial suspension of OD_{600 nm} = 0.3 was prepared from P. aeruginosa ATCC 9025 and S. aureus CIP 68.5 cells grown in nutrient broth (Oxoid) at room temperature (∼22 °C) for 24 h. A portion of 100 μL of bacterial suspension was deposited onto the surface of each sample (in triplicate). Sterile nutrient broth (100 μL) was used as a negative control. After incubation for 18 h at 37 °C, all of the samples were gently washed with copious amounts deionized water and left to dry at room temperature for 45 min at 55% humidity. A FeSEM-ZEISS SUPRA 40VP was used to obtain high-resolution images of the bacterial cells. Just before imaging, all slides were gold coated in order to achieve better conductivity. Separately, concanavalin A Alexa 488 dye (Molecular Probes Inc.) was used to label the extracellular polysaccharide (EPS) produced by the bacteria while attaching to the surfaces. After incubation, all samples were washed with sterilized nanopure water and carefully stored until being analyzed at room temperature (∼22 °C). The confocal scanning laser microscope (CSLM) Olympus Fluoview FV1000 Spectroscopic Confocal System was used [34]. Excitation and emission wavelengths for concanavalin A are 495 nm and 519 nm, respectively. Also, viability of attached bacterial cells was determined using Live/Dead^® BacLight™ Viability kit (L7007, Invitrogen). The staining was carried out according to the manufacturer's protocol.

Using a First Ten Angstroms (FTA200) instrument, sessile drop contact angle measurements were performed on the lawns of bacterial cells to estimate the surface wettability of the bacterial samples. Bacterial cells were washed three times and then suspended in a buffer (OD450 = 0.4). A bacterial lawn was formed by depositing the bacterial suspension onto cellulose acetate membrane filters (Sartorius, pore diameter 0.2 μm), which were then sufficiently dried at ambient temperature prior to measurement of the contact angle to reach “plateau state”, whereby stable water contact angles can be measured over time. The contact angle of the water droplets deposited onto the bacterial lawns was recorded using a charge-coupled device (CCD) camera. Images were digitally saved and analyzed using the image analysis software (v 1.961; Folio Instruments Inc.).

For the electrophoretic mobility measured using a zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corp.), the bacterial cells were harvested by centrifugation for 5 min at 5,000 rpm after 24 h of incubation. Harvested cell pellet was re-suspended in 10 mM potassium chloride (KCl) followed by further washing and centrifugation. This step was repeated four times and finally the cells were re-suspended in 10 mM KCl solution to OD (600 nm) = 1. The resultant solution of bacterial cells was further diluted 1,000 times in 5 mL of 10 mM KCl. Measurements were conducted in electric field 2.5 V cm⁻¹ and 2 Hz. All measurements were done in triplicate and for each sample the final EPM quoted represented the average of five successive ZetaPALS readings, each of which consisted of 14 cycles per run.

High resolution AFM studies showed that application of a polyterpenol coating significantly altered the surface topography of the substrates, decreasing the substrate average surface roughness (R_a) from approximately 0.4 nm to 0.25 nm and 0.13 nm for 10 W and 25 W polyterpenol coatings, respectively (scan size 1 μm × 1 μm). A statistical analysis of the roughness parameters showed that there was a statistically significant difference in the surfaces roughness parameters for each of the coated and unmodified substrates used in this study. The extent of bacterial attachment and EPS production was then analyzed using SEM and CSLM. The CSLM and SEM analyses clearly demonstrated that s aureus and P. aeruginosa presented notably different patterns of attachment onto surfaces ofpolyterpenol thin films fabricated under the two conditions. The smoothon the scale of nanometers 25 Wpolyterpenol coating resulted in enhanced attachment and adherence of two taxonomically differentbacterial strains, the behavior previously described in our recent works. However, bacterial attachmentcan be further controlled by changing the surface chemical functionality, by, for example, coating thesubstrate with terpinen-4-ol, a compound with known bacterial inhibitory activity. Despite beingcharacterized as smoother on a nanometer scale than glass surfaces, the degree of attachment, EPSproduction, and cell proliferation on the polyterpenol thin films fabricated under 10 W was found to begreatly reduced compared to that obtained on the unmodified surface or on the polyterpenol thin filmsfabricated under 25 W deposition power conditions. The added advantages of this coating includeuniform coverage, good adhesion to the substrate, and film stability. Furthermore, the antibacterialefficacy is based on the presence of a natural antimicrobial compound with no use of antibiotics. Thus, this work suggests that such an antimicrobial and antifouling coating could be effectively integrated into medical and other clinically relevant devices to prevent bacterial growth, thus minimizing bacteria-associated adverse host responses.