Bacterial adhesion and the subsequent formation of biofilm are major concerns in biotechnology and medicine. The initial step in bacterial adhesion is the interaction of cells with a surface, a process governed by long-range forces, primarily van der Waals and electrostatic interactions. The precise manner in which the force of interaction is affected by cell surface components and by the physiochemical properties of materials is not well understood. Here, we show that atomic force microscopy can be used to analyze the initial events in bacterial adhesion with unprecedented resolution. Interactions between the cantilever tip and conf luent monolayers of isogenic strains of Escherichia coli mutants exhibiting subtle differences in cell surface composition were measured. It was shown that the adhesion force is affected by the length of core lipopolysaccharide molecules on the E. coli cell surface and by the production of the capsular polysaccharide, colanic acid. Furthermore, by modifying the atomic force microscope tip we developed a method for determining whether bacteria are attracted or repelled by virtually any biomaterial of interest. This information will be critical for the design of materials that are resistant to bacterial adhesion.Bacterial adhesion onto inanimate surfaces is a critical issue in processes ranging from the biofouling of industrial equipment to dental decay to infections of biomaterials for medical use. Bacterial infections associated with the formation of biofilms refractile to antibiotic therapy is one of the main reasons for the failure of devices such as catheters, vascular grafts, joint prostheses, and heart valves (1-3). The first step in bacterial adhesion is the immediate attachment of bacteria onto a substratum which is a reversible, nonspecific process (3-5). This initial interaction between bacteria and artificial surfaces is a key determinant in biofilm formation. If the approach of bacteria to a surface is unfavorable, cells must overcome an energy barrier to establish direct contact with the surface. Only when bacteria are in close proximity to the surface do shortrange interactions become significant. Thereafter, proteinligand-binding events mediated by a plethora of microbial adhesins and in some cases the production of extracellular polymers render the binding process practically irreversible (6).Initial bacterial attraction or repulsion to a particular surface can be described in terms of colloidal interactions. Consequently, the force of interaction depends on physiochemical parameters such as surface-free energy and charge density (7-11). The propensity of bacteria to adhere onto surfaces has been estimated by counting the number of bacteria that remain attached to surfaces following incubation for a specified length of time (5, 12). This approach is qualitative, time consuming, and has low sensitivity. Moreover, the resulting number of adherent bacteria is determined by multiple factors in addition to long-range attractive/repulsive interactions. A direct and ...

The atomic force microscope (AFM) was used to directly measure the forces of interaction between E. coli D21 bacteria and hydrophilic glass or hydrophobic N-octadecyltrichlorosilane (OTS)-treated glass substrates coated with the block copolymers, poly(ethylene glycol) (PEG)-lysine dendron or Pluronic F127 surfactant, respectively. Short-range repulsive interactions between bacterial cells and substrates coated with the block copolymers were detected by the AFM over distances of separation comparable to the extended length of the PEG polymer chains. In contrast, glass and OTS-treated glass devoid of PEG-lysine dendron or Pluronic F127 exerted long-range attractive forces on E. coli D21 bacteria. Thus, polymeric brush layers appear to not only block the long-range attractive forces of interaction between bacteria and substrates but also introduce repulsive steric effects.

Bacterial infection of biomaterials represents one of the most important reasons for the failure of transdermal or implanted medical devices. The first and least understood step in biomaterial-associated infections is the initial interaction between bacteria and a surface. This initial interaction can be either attractive or repulsive depending on the physiochemical nature of the biological and synthetic surfaces, as well as the properties of the interstitial fluid. We have shown that atomic force microscopy (AFM) can be employed as an exquisitely sensitive and versatile tool for quantifying the interaction between bacteria and surfaces in physiological solutions. The forces of interaction between an AFM cantilever tip and a uniform lawn of bacteria immobilized on glass were determined. By comparing the interactions of cantilever tips with lawns of isogenic E. coli strains carrying genetic lesions that alter their cell surface composition, it was possible to evaluate the effect of macromolecules such as lipopolysaccharide and capsular polysaccharide on the adhesion process. Mutations that result in the synthesis of truncated lipopolysaccharide or in the overproduction of the negatively charged capsular polysaccharide colanic acid render the interaction of the bacteria with the AFM tip unfavorable due to increased electrostatic repulsion. Furthermore, AFM could be used to evaluate the adhesion of bacteria onto commercially relevant biomaterials. In one approach, micron-size polystyrene beads were attached to AFM tips which were then used to measure forces. Unfortunately, this approach is limited by the meager number of materials manufactured as beads of a size suitable for AFM measurements. As an alternative approach, AFM cantilever tips were coated with a confluent layer of bacteria and used to probe planar surfaces. In this configuration, AFM could be employed to measure the force of interaction between virtually any bacterium and surface of interest.