Biocidal action of ozone-treated polystyrene surfaces on vegetative and sporulated bacteria

Abstract

Surfaces of materials can be modified to ensure specific interaction features with microorganisms. The current work discloses biocidal properties of polystyrene (PS) Petri-dish surfaces that have been exposed to a dry gaseous-ozone flow. Such treated PS surfaces are able to inactivate various species of vegetative and sporulated bacteria on a relatively short contact time. Denaturation of proteins seems likely based on a significant loss of enzymatic activity of the lysozyme protein. Characterization of these surfaces by atomic-force microscopy (AFM), Fourier-transform infra-red (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) reveals specific structural and chemical modifications as compared to untreated PS. Persistence of the biocidal properties of these treated surfaces is observed. This ozone-induced process is technically simple to achieve and does not require active precursors as in grafting.

Introduction

The various possible interactions between microorganisms and surfaces of different materials have been receiving considerable attention in biomedical areas over the last decades. Two main research directions are emerging from these studies: (i) the adhesion of cells on surfaces (including their growth and proliferation) and (ii) the biocidal action of surfaces.

Various surface treatments/modifications have been extensively examined to control the adhesion and growth characteristics of cells on surfaces [1], [2], [3], [4], a matter of prime importance, for instance, for tissue engineering [5]. Understanding the relationship between cells and the physico-chemical properties of surfaces with which they interact (such as wettability, functional groups, topography) is, in fact, essential for the optimisation of cell adhesion, their spreading, and growth thereon [1]. Improved cellular attachment [3] is promoted by certain functional groups, which at the same time increase hydrophilicity, favouring the adsorption or adhesion of proteins [6]. For example, surface modifications with UV/O₃ [1] or with plasmas such as corona discharges [2] can increase proliferation and protein expression of cells or enhance their culture process. Similar action can be obtained with coatings with well-defined surface chemistries as they can also amplify, or prevent, bioadhesion of molecules, cells and, in some cases, bacteria [7], [8], [9], [10].

Antimicrobials properties resulting from surface modifications and specific coatings have been the object of a large number of studies. As a matter of fact, it is known since antiquity that some heavy metals such as silver and copper possess anti-infective activity. These metals can be impregnated on surfaces such as venous, vascular or urinary catheters [11], [12] or they can be immobilized in textiles and ceramics, providing antimicrobial properties [13]. Such treated materials are routinely used even nowadays in healthcare, for instance in burned skin treatment, to prevent infections [11], [13], [14].

More recently, surface coatings incorporating antimicrobial, antibiotic or antiadhesive molecules for preventing surface contamination have been proposed [12]. Conventional surface modification techniques for achieving coatings are usually based on the incorporation into a polymeric surface of a leachable antiseptic. Plasmas can be used in such processes for surface modification, deposition, or as a preliminary step before grafting reactions, as needed in the fabrication of specific medical devices and biomaterials [7], [15]. To illustrate the use of plasma in such applications, consider the work of Zhang et al. [16] reporting the covalent immobilization of antimicrobial bronopol and triclosan molecules by means of plasma-immersion ion implantation (PIII). This method gives excellent antimicrobial properties to polyethylene and PVC (medical grade) surfaces against Gram-positive and Gram-negative bacteria [16], [17]. Initially, the surface is activated by an O₂ plasma to provide more hydrophilic groups in order for triclosan and bronopol molecules to be integrated more efficiently on the surface. Other authors showed that surfaces could also be modified chemically (e.g. thiocyanation) by turning to various gas plasmas (Ar, O₂) [17], [18], with O₃ [19], with vacuum ultra-violet (VUV) irradiation in the presence of O₂ and with UV/O₃ exposure [2], [20] before grafting the antiseptic or other active species [21]. These modified surfaces exhibit antimicrobial effectiveness on a broad spectrum of Gram-positive and Gram-negative bacteria and in some case viruses and fungi [21], [13], [17].

The novel method that we present allows polystyrene (PS) Petri dishes to acquire biocidal features ensuring microorganism inactivation. These properties were demonstrated for both vegetative and sporulated bacteria. As a rule, surface modification studies conferring sporicidal properties are scarcer than those providing antibacterial properties [15], [22]. Special attention should nonetheless be paid to endospores because they can withstand severe treatments including heat, irradiation, chemicals and desiccation, and as such are generally used to validate sterilization processes. Our method requires no grafting reactions with chemical precursors, but simply exposure to an ozone flow. Recall that because of its optical, mechanical and chemical properties, PS has commonly been used for the cost-effective production and commercialization of culture vessels replacing glass, in the mid 1960s, for cell culture and biological assays [23].

The paper is organized as follows. Section 2 describes the materials used and the experimental methods. Section 3 characterizes the biocidal activity of treated PS surfaces and its effect on the structural integrity of various vegetative bacteria, bacterial spores and lysozyme (protein). In Section 4, surface diagnostic techniques such as Fourier-transform infra-red (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic-force microscopy (AFM) are used to characterize physical and chemical modifications of the PS surface after treatment. Summary and conclusion are presented in Section 5.