Appl Environ Microbiol 65(11): 4995-5002

Bacterial Adhesion at Synthetic Surfaces

Macromolecular Science Department, Institute of Food Research, Reading Laboratory, Reading RG6 6BZ, United Kingdom

^{Corresponding author. Mailing address: Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, United Kingdom. Phone: (44) (0) 1603 255000. Fax: (44) (0) 1603 507723. E-mail for C. Alexander:}ku.ca.crsbb@rednaxela.noremac. E-mail for E. N. Vulfson: ku.ca.crsbb@nosfluv.aynej.

Received 1999 May 11; Accepted 1999 Aug 17.

Abstract

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ · m^{. Protein adsorption experiments were performed with}^{H-labelled bovine serum albumin and cytochrome}c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.

Abstract

The prevention of contamination caused by pathogenic microorganisms during the manufacture, processing, and packaging of food is of considerable importance to public health and consequently is a major issue for industry (30). In particular, there is increasing concern associated with the contamination arising from bacterial biofilms which develop on materials used during food manufacture (6, 19, 40). These communities of bacteria, often embedded in a matrix of organic polymers exuded by the cells, can be extremely difficult to remove, and complete eradication of the pathogens is difficult, time-consuming, and expensive (27). In general, the formation of bacterial biofilms is believed to take place over at least three stages: a reversible adsorption step (26), primary adhesion of microorganisms to a surface, and colonization (9). The rates of these processes vary widely depending on the environmental conditions and the type of microorganisms, but the adhesion and colonization stages are considered to be relatively slow compared to the first step of cell adsorption (17). In principle, it should be possible to retard, if not prevent, the formation of biofilms on substrates by using materials to which bacteria cannot initially attach, and such a material or surface coating would be of considerable commercial interest (7). In practice, however, synthetic materials that are capable of preventing bacterial adsorption have proved rather elusive, despite a significant volume of research (8, 11). Properties of the substrate, such as hydrophobicity (32), hydrophilicity (23), steric hindrance (24), roughness (22), and the existence of a “conditioning layer” at the surface (1), are all thought to be important in the initial cell attachment process.

In addition, there are large discrepancies in the data available on the adhesion of microorganisms to various synthetic materials. These disparities are mainly due to the different experimental conditions and protocols used in different laboratories as well as to the fact that the substrates employed are sometimes incompletely characterized both in terms of their functionality, i.e., the actual chemistry of the groups present, and in terms of their density at the surface. Consequently, it is often difficult to draw a meaningful comparison between results reported by different authors, and no simple structure-function correlation has yet emerged (33). The objective of this investigation was to carry out a comprehensive comparative study of bacterial adsorption by using a broad range of surfaces with controllable and precisely defined chemistry, such that various chemical and physicochemical factors which may be important in defining the surface at the molecular level could be assessed for their effect on the adhesion of the representative food pathogens Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli.

ACKNOWLEDGMENTS

This work was funded by the Ministry of Agriculture Fisheries and Food.

We would like to thank Frans Llevat and Laura Magraner for help with some of the experimental work; John Tsibouklis, Adrian Thorpe, and Simon Young, University of Portsmouth, for contact angle measurements; and Terry Roberts (Ministry of Agriculture, Fisheries, and Food) for many helpful discussions.

ACKNOWLEDGMENTS

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