Staphylococcus epidermidis adhesion to He, He/O2 plasma treated PET films and aged materials: Contributions of surface free energy and shear rate

https://doi.org/10.1016/j.colsurfb.2008.04.017Get rights and content

Abstract

Adhesion studies of bacteria (Staphylococcus epidermidis) to plasma modified PET films were conducted in order to determine the role of the surface free energy under static and dynamic conditions. In particular, we investigated the effect of the ageing time on the physicochemical surface properties of helium (He) and 20% of oxygen in helium (He/O2) plasma treated polyethylene terephthalate (PET) as well as on the bacterial adhesion. Treatment conditions especially known to result in ageing sensitive hydrophilicity (hydrophobic recovery) were intentionally chosen in an effort to obtain the widest possible range of surface energy specimens and also to avoid strong changes in the morphological properties of the surface. Both plasma treatments are shown to significantly reduce bacterial adhesion in comparison to the untreated PET. However, the ageing effect and the subsequent decrease in the surface free energy of the substratum surfaces with time – especially in the case of He treated samples – seem to favor bacterial adhesion and aggregation. The dispersion-polar and the Lifshitz–van der Waals (LW) acid–base (AB) thermodynamic approaches were applied to calculate the Gibbs free energy changes of adhesion (ΔGadh) of S. epidermidis interacting with the substrates. There was a strong correlation between the thermodynamic predictions and the measured values of bacterial adhesion, when adhesion was performed under static conditions. By decoupling the (ΔGadh) values into their components, we observed that polar/acid–base interactions dominated the interactions of bacteria with the substrates in aqueous media. However, under flow conditions, the increase in the shear rate restricted the predictability of the thermodynamic models.

Introduction

Implantation of artificial organs and medical devices, and therefore the use of synthetic materials, has become an indispensable part in almost all fields in medicine. In spite of non-septic conditions during the surgical process and systematic administration of antibiotics, infection impedes the materials’ long-term use [1], [2]. While a variety of microorganisms may be involved as pathogens, coagulase-negative staphylococci, most notably, Staphylococcus epidermidis, have been identified as a predominant cause of infection in the immunocompromised host or in the presence of a medical device [3]. There are two main characteristics of S. epidermidis that allow persistence of infection. These are the ability of the bacteria to adhere to surfaces, followed by the production of a mucoid substance, more commonly known as slime, and the formation of multilayered cell clusters. The adherent bacteria and slime are collectively known as biofilm [4]. Slime may protect the bacteria from antibiotic therapy, physiologic shear and possibly host cell-mediated defenses [5], [6].

The critical step in the development of infections related to implanted or intravascular devices is bacterial adhesion to the biomaterial substrate, which is mediated by interactions between the material and the bacterial surfaces [7]. Both specific (i.e., receptor–ligand) and non-specific (i.e., colloidal-type) interactions as well as flow conditions contribute to the ability of the bacterial cell to attach to the biomaterial surface. However, their relative contribution is not completely understood [8], [9], [10]. In the absence of specific ligand–receptor binding, bacteria may bind directly to the biomaterial surface via non-specific–physicochemical interactions. In this case, the initial adhesion phase is largely governed by a complicated interplay of forces between the bacteria and the substrate such as electrostatic, dispersion/Lifshitz–van der Waals (LW) and polar/acid–base (AB) [7]. As soon as microorganisms reach the surface under static conditions, they will be attracted or repelled by it, depending on the sum of these interactions and, as we reported previously, adhesion may be analyzed by a thermodynamic approach [11], as the electrostatic charges and therefore interactions can be neglected due to the induced charge balance caused by overlapping double layers in high ionic strength solutions [12]. However, since the process of bacterial adhesion to indwelling medical devices is associated in most cases with flow of body fluids [10], physical forces such as shear generated by local hemodynamics may modulate the adhesion process and restrict the predictability of the thermodynamic models.

In this study, we expand our investigation of non-specific adhesion of S. epidermidis to various He and He/O2 plasma treated Polyethylene Terephthalate (PET) films to consider the thermodynamics of the adhesion. Moreover, we examine adhesion not only under static conditions, as is the case with most studies based on thermodynamics so far [13], [14], [15], [16], [17], [18], but also under well-defined shear rates correlating to the normal range of hemodynamics.

In this direction, PET was chosen because it is used in certain medical implants such as artificial heart valve sewing rings and artificial blood vessels due to its excellent mechanical properties and relatively high biocompatibility [19]. However, as is the case with most biomaterials, its long-term use is impeded by infections. Helium is the most efficient of the inert gases for the crosslinking of the uppermost few monolayers of the polymer. Reactive oxygen, which originates within the polymer during the plasma process, can induce surface oxidation [20]. Both crosslinking and oxidation may be attributed to the large amount of energy which He is able to transfer to the polymer surface and to the surface reorganization which increases the material free energy. Oxygen is one of the so-called reactive gases which have surface functionalization as a main effect. Oxygenated functional groups such as ether, hydroxyl, carbonyl or carboxyl may be grafted onto the surface, increasing the polar character of the material and the surface free energy [21]. When a gaseous mixture of O2 and He is used for the material treatment, oxygenated functional groups are grafted simultaneously with a crosslinking reaction. Therefore, a more cohesive and dense layer is obtained between the uppermost surface and the bulk material, and this surface presents good stability with time [22], [23], [24], [25]. This time dependent stability is not accomplished with the He treated PET films. In this case the surface free energy decreases with ageing time [22], [23], [24], and this gives us the opportunity to investigate, in a direct manner, the effect of a broad range of surface free energy values on bacterial adhesion. Moreover, it permits the quantitative evaluation of thermodynamic approaches in predicting adhesion. Flow conditions permit the examination of the modifying roles of physiological shear forces.

Section snippets

Materials

100 μm thick PET films, supplied by Goodfellow, were cut in 43 mm diameter discs and ultrasonically cleaned in methanol for 1 min.

Plasma treatment

The plasma treatments were performed in a cylindrical, 160 mm in diameter, stainless steel chamber that has two parallel round stainless steel electrodes with a diameter of 55 mm and an interelectrode distance of 25 mm [26]. PET films were mounted on the grounded electrode surface using a stainless steel ring. The edge between PET and the stainless steel holder induces

Surface morphology

A change of PET surface morphology is expected as a consequence of the observed etching by He and He/O2 plasmas. AFM topographic images of the untreated PET, the He treated PET and the He/O2 treated PET (see Figure S-1 (a), (b) and (c) respectively, in Supplementary Information) revealed that the untreated PET surfaces are relatively smooth with granular structures of conical shape and moderate roughness: 3.1 ± 0.9 nm, while the He treated PET shows an enhancement of the already existing granular

Discussion

In this study we investigated the relation between the bacterial adhesion to plasma modified PET surfaces and the thermodynamic properties of these surfaces. The variation of the thermodynamic properties, in terms of the surface free energy, was achieved through the ageing of He and He/O2 plasma treated PET films. We observed that the untreated PET yielded the highest number of adherent bacteria, in comparison to both He and He/O2 plasma treated samples. However, the ageing effect results in an

Concluding remarks

We demonstrated that the increase in the free energy of PET surfaces by He and He/O2 plasma treatments significantly reduced the adhesion of a specific strain of S. epidermidis. The results are consistent with the thermodynamic analysis of the adhesion process, for both approaches: “dispersion-polar” and “Lifshitz–van der Waals acid–base”, and reveal that the polar/acid–base interactions dominate the interactions of bacteria with the substrates in aqueous media. However, simulated hemodynamic

Acknowledgments

The authors wish to thank the Associate Professor I. Spiliopoulou, from the Department of Microbiology, School of Medicine, University of Patras, for providing us with the bacteria and for the use of specific equipment, as well as the Ceramic and Composite Materials Laboratory of University of Patras and specifically Prof. P. Nikolopoulos and Mr. V. Ioannidis for performing the contact angle measurements on the samples.

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