Biofilms: Microbial Life on Surfaces

Rodney M. Donlan

Disclosures

Emerging Infectious Diseases. 2002;8(9) 

In This Article

Attachment

The solid-liquid interface between a surface and an aqueous medium (e.g., water, blood) provides an ideal environment for the attachment and growth of microorganisms. A clear picture of attachment cannot be obtained without considering the effects of the substratum, conditioning films forming on the substratum, hydrodynamics of the aqueous medium, characteristics of the medium, and various properties of the cell surface. Each of these factors will be considered in detail.

The solid surface may have several characteristics that are important in the attachment process. Characklis et al.[6] noted that the extent of microbial colonization appears to increase as the surface roughness increases. This is because shear forces are diminished, and surface area is higher on rougher surfaces. The physicochemical properties of the surface may also exert a strong influence on the rate and extent of attachment. Most investigators have found that microorganisms attach more rapidly to hydrophobic, nonpolar surfaces such as Teflon and other plastics than to hydrophilic materials such as glass or metals.[7,8,9] Even though results of these studies have at times been contradictory because no standardized methods exist for determining surface hydrophobicity, some kind of hydrophobic interaction apparently occurs between the cell surface and the substratum that would enable the cell to overcome the repulsive forces active within a certain distance from the substratum surface and irreversibly attach.

A material surface exposed in an aqueous medium will inevitably and almost immediately become conditioned or coated by polymers from that medium, and the resulting chemical modification will affect the rate and extent of microbial attachment. Loeb and Neihof[10] were the first to report the formation of these conditioning films on surfaces exposed in seawater. These researchers found that films were organic in nature, formed within minutes of exposure, and continued to grow for several hours. The nature of conditioning films may be quite different for surfaces exposed in the human host. A prime example may be the proteinaceous conditioning film called "acquired pellicle," which develops on tooth enamel surfaces in the oral cavity. Pellicle comprises albumin, lysozyme, glycoproteins, phosphoproteins, lipids, and gingival crevice fluid;[11] bacteria from the oral cavity colonize pellicle-conditioned surfaces within hours of exposure to these surfaces. Mittelman noted that a number of host-produced conditioning films such as blood, tears, urine, saliva, intervascular fluid, and respiratory secretions influence the attachment of bacteria to biomaterials.[12] Ofek and Doyle[13] also noted that the surface energy of the suspending medium may affect hydrodynamic interactions of microbial cells with surfaces by altering the substratum characteristics.

In theory, the flow velocity immediately adjacent to the substratum/liquid interface is negligible. This zone of negligible flow is termed the hydrodynamic boundary layer. Its thickness is dependent on linear velocity; the higher the velocity, the thinner the boundary layer. The region outside the boundary layer is characterized by substantial mixing or turbulence. For flow regimes characterized as laminar or minimally turbulent, the hydrodynamic boundary layer may substantially affect cell-substratum interactions. Cells behave as particles in a liquid, and the rate of settling and association with a submerged surface will depend largely on the velocity characteristics of the liquid. Under very low linear velocities, the cells must traverse the sizeable hydrodynamic boundary layer, and association with the surface will depend in large part on cell size and cell motility. As the velocity increases, the boundary layer decreases, and cells will be subjected to increasingly greater turbulence and mixing. Higher linear velocities would therefore be expected to equate to more rapid association with the surface, at least until velocities become high enough to exert substantial shear forces on the attaching cells, resulting in detachment of these cells[14] This finding has been confirmed in studies by Rijnaarts et al.[15] and Zheng et al..[16]

Other characteristics of the aqueous medium, such as pH, nutrient levels, ionic strength, and temperature, may play a role in the rate of microbial attachment to a substratum. Several studies have shown a seasonal effect on bacterial attachment and biofilm formation in different aqueous systems.[17,18] This effect may be due to water temperature or to other unmeasured, seasonally affected parameters. Fletcher[19,20] found that an increase in the concentration of several cations (sodium, calcium, lanthanum, ferric iron) affected the attachment of Pseudomonas fluorescens to glass surfaces, presumably by reducing the repulsive forces between the negatively charged bacterial cells and the glass surfaces. Cowan et al.[21] showed in a laboratory study that an increase in nutrient concentration correlated with an increase in the number of attached bacterial cells.

Cell surface hydrophobicity, presence of fimbriae and flagella, and production of EPS all influence the rate and extent of attachment of microbial cells. The hydrophobicity of the cell surface is important in adhesion because hydrophobic interactions tend to increase with an increasing nonpolar nature of one or both surfaces involved (i.e., the microbial cell surface and the substratum surface). Most bacteria are negatively charged but still contain hydrophobic surface components, as noted by Rosenberg and Kjelleberg.[22] Fimbriae, i.e., nonflagellar appendages other than those involved in transfer of viral or bacterial nucleic acids (called pili), contribute to cell surface hydrophobicity. Most fimbriae that have been examined contain a high proportion of hydrophobic amino acid residues.[22] Fimbriae play a role in cell surface hydrophobicity and attachment, probably by overcoming the initial electrostatic repulsion barrier that exists between the cell and substratum.[23] A number of aquatic bacteria possess fimbriae, which have also been shown to be involved in bacterial attachment to animal cells.[23] Rosenburg et al.[24] and Bullitt and Makowski[25] provided evidence for the role of fimbriae in bacterial attachment to surfaces.

Other cell surface properties may also facilitate attachment. Several studies have shown that treatment of adsorbed cells with proteolytic enzymes caused a marked release of attached bacteria,[26,27] providing evidence for the role of proteins in attachment. Bendinger et al.[9] found that mycolic acid-containing organisms (Corynebacterium, Nocardia, and Mycobacterium) were more hydrophobic than were nonmycolic acid-containing bacteria, and increase in mycolic acid chain length generally coincided with increase in hydrophobicity. For most strains tested, adhesion was greater on hydrophobic materials. The O antigen component of lipopolysaccharide (LPS) has also been shown to confer hydrophilic properties to gram-negative bacteria. Williams and Fletcher[28] showed that mutants of P. fluorescens lacking the O antigen adhered in greater numbers to hydrophobic materials.

As early as 1971, Marshall et al.[29] provided evidence based on SEM that attached bacteria were associated with the surface via fine extracellular polymeric fibrils. Fletcher et al.[30] found that treatment of attached freshwater bacteria with cations resulted in contraction of the initial adhesives (decrease in the cell distance from the substratum), supporting the idea that this material was an anionic polymer. Cations have been shown to cross-link the anionic groups of polymers (such as polysaccharides), resulting in contraction. Beech and Gaylarde[31] found that lectins inhibited but did not prevent attachment. Glucosidase and N-acetylglucosaminidase (NAG) reduced attachment for P. fluorescens, while NAG reduced attachment for Desulfovibrio desulfuricans. Lectins preferentially bind to polysaccharides on the cell surface or to the EPS. Binding of lectins by the cells would minimize the attachment sites and affect cell attachment if polysaccharides were involved in attachment. Zottola[32] confirmed the role of polysaccharides in attachment in studies with Pseudomonas fragi.

Korber et al.[33] used motile and nonmotile strains of P. fluorescens to show that motile cells attach in greater numbers and attach against the flow (backgrowth) more rapidly than do nonmotile strains. Nonmotile strains also do not recolonize or seed vacant areas on a substratum as evenly as motile strains, resulting in slower biofilm formation by the nonmotile organisms. Flagella apparently play an important role in attachment in the early stages of bacterial attachment by overcoming the repulsive forces associated with the substratum.

In light of these findings, cell surface structures such as fimbriae, other proteins, LPS, EPS, and flagella all clearly play an important role in the attachment process. Cell surface polymers with nonpolar sites such as fimbriae, other proteins, and components of certain gram-positive bacteria (mycolic acids) appear to dominate attachment to hydrophobic substrata, while EPS and lipopolysaccharides are more important in attachment to hydrophilic materials. Flagella are important in attachment also, although their role may be to overcome repulsive forces rather than to act as adsorbents or adhesives.

The attachment of microorganisms to surfaces is a very complex process, with many variables affecting the outcome. In general, attachment will occur most readily on surfaces that are rougher, more hydrophobic, and coated by surface "conditioning" films. An increase in flow velocity, water temperature, or nutrient concentration may also equate to increased attachment, if these factors do not exceed critical levels. Properties of the cell surface, specifically the presence of fimbriae, flagella, and surface-associated polysaccharides or proteins, also are important and may possibly provide a competitive advantage for one organism where a mixed community is involved. Table 1 summarizes the variables important in cell attachment and biofilm formation.

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