Bacterial attachment to surfaces or other cells can be seen as a physicochemical process determined by Van der Waals, electrostatic and steric forces acting between the cells and the attachment surface. A theory to quantitatively describe this interaction of charged surfaces through a liquid medium, designated the Derjaguin, Verwey, Landau and Overbeek (DLVO) theory, has been developed in the 1940s. Later, an extended DLVO theory was developed, which incorporated besides these long-range forces also hydrophilic/hydrophobic and osmotic interactions, resulting in more accurate predictions of bacterial adhesion [reviewed by Strevett and Chen (2003)]. These theories are not reviewed here, instead the wide variety of individual outer cell surface structures and molecules that are exposed on, or protrude from, the cell surface are described in detail. These structures shape the physicochemical surface properties of bacterial cells, and hence determine attachment and biofilm formation properties. However, the presence or absence of a certain structure on initial attachment or biofilm formation should be evaluated with care because multiple structures can be present, each with their own specific effects, and different structures could have diverse roles depending on the bacterium and the attachment surface.
Many bacteria are motile by virtue of peritrichous or polar flagella, and motility is generally regarded as a virulence factor facilitating the colonization of host organisms or target organs by pathogenic bacteria. Flagellar motility is critical for initial cell-to-surface contact and normal biofilm formation under stagnant culture conditions for Escherichia coli (Pratt and Kolter 1998), Listeria monocytogenes (Vatanyoopaisarn et al. 2000; Lemon et al. 2007; Todhanakasem and Young 2008) and Yersinia enterocolitica (Kim et al. 2008). Although lack of flagella also affected initial attachment under flow conditions for Y. enterocolitica and L. monocytogenes, further maturation was unaffected for Y. enterocolitica (Kim et al. 2008), and the formation of high density biofilms was not suppressed for L. monocytogenes (Todhanakasem and Young 2008). For Pseudomonas fluorescens, mutants lacking flagella showed a decreased attachment to a variety of plant seeds and inert surfaces such as sand (Deflaun et al. 1990, 1994) and a decreased colonization of potato roots (De Weger et al. 1987). Finally, initial attachment of L. monocytogenes to stainless steel can also be affected by flagella per se (Vatanyoopaisarn et al. 2000). These observations indicate that flagella can affect adherence and biofilm formation via different mechanisms depending on the type of bacterium. First, motility can be necessary to reach the surface by allowing the cell to overcome the repulsive forces between the cell and the surface. This mechanism is possibly more important under stagnant than under flow conditions. In addition, motility can be required to move along the surface, thereby, facilitating growth and spread of a developing biofilm. Finally, flagella themselves (as surface appendages) can directly mediate attachment to surfaces.
Fimbriae, thread-like structures that protrude from the cell surface, are classified on the basis of their adhesive, antigenic or physical properties, or on the basis of similarities in the primary amino acid sequence of their major protein subunits (Low et al. 1996). Type 1 fimbriae, which are rod-shaped and approximately 7-nm wide and 1-μm long, are the most common adhesins found in both commensal and pathogenic E. coli as well as in other Enterobacteriaceae (Klemm and Krogfelt 1994). Their role in biofilm formation has been studied exhaustively, demonstrating a critical role in initial stable cell-to-surface attachment for numerous E. coli strains (Pratt and Kolter 1998; Beloin et al. 2004; Ren et al. 2004) including Shiga toxin-producing strains (Cookson et al. 2002), in adherence to Teflon and stainless steel for Salmonella enterica serovar Enteritidis (Austin et al. 1998), and in promoting biofilm formation on abiotic surfaces (polystyrene) for Klebsiella pneumoniae (Schembri et al. 2005).
Besides Type 1 fimbriae, other types of fimbriae have been shown to affect biofilm formation. For example, Di Martino et al. (2003) showed that for a Kl. pneumoniae strain, which produced both Type 1 and Type 3 fimbriae, the latter constituted the main factor facilitating adherence to both glass and polypropylene, and the formation of a full-grown biofilm on polystyrene. Type 4 fimbriae promoted the rapid formation of strongly adherent biofilms for the opportunistic pathogen Aeromonas caviae (Bechet and Blondeau 2003), commonly found in water and foods (Neyts et al. 2000), and affected the binding of Pseudomonas aeruginosa to stainless steel, polystyrene and polyvinylchloride (Giltner et al. 2006). Genes involved in the biogenesis, regulation and secretion of Type 4 fimbriae were found to be up-regulated within 6 h of attachment to silicone tubing for Pseudomonas putida (Sauer and Camper 2001), often associated with spoilage of fresh milk and vegetables (Ternstrom et al. 1993; Garcia-Gimeno and Zurera-Cosano 1997). Type 4 fimbriae also played a role in the colonization and persistence of Vibrio vulnificus in oysters (Paranjpye et al. 2007). Vibrio vulnificus is a pathogen associated with human infections caused by raw oyster consumption (Blake et al. 1979) and an important cause of reported deaths from food-borne illness in Florida (Hlady et al. 1993). Furthermore, for enterohemorrhagic E. coli O157:H7, these structures not only affected attachment and biofilm formation but have also been implicated in virulence and transmission (Xicohtencatl-Cortes et al. 2009).
Curli fimbriae (called thin aggregative fimbriae in Salmonella) are proteinaceous, coiled filamentous surface structures, which are assembled by an extracellular nucleation/precipitation pathway (Olsen et al. 1989). The effect of curli on attachment and biofilm formation of E. coli O157:H7 appears to be variable. In one study, curli production enhanced the biofilm-forming capacity of a particular strain to stainless steel (Ryu et al. 2004b), although initial attachment was unaffected (Ryu and Beuchat 2005). In another study, different Shiga toxin-producing and enterohaemorrhagic E. coli strains showed an enhanced attachment to abiotic surfaces such as polystyrene and stainless steel when curli were produced (Cookson et al. 2002; Pawar et al. 2005). Probably, this increased attachment is strain dependent as shown in a study comparing the attachment of curli-producing and noncurli-producing E. coli O157:H7 strains to lettuce (Boyer et al. 2007). Interestingly, it cannot be excluded that the observed differences are not only strain dependent, but are also induced by other (nonevaluated) mechanisms or by the occurrence of dissimilar environmental triggers in the experiments.
In addition to curli, cellulose is also usually associated with biofilms of various salmonellae, including strains of the serovar Typhimurium (Solano et al. 2002; Jain and Chen 2007). The simultaneous production of cellulose and curli leads to the formation of a highly inert, hydrophobic extracellular matrix in which the cells are embedded (Zogaj et al. 2001). However, other capsular polysaccharides can be present in the extracellular biofilm matrix of Salmonella strains (de Rezende et al. 2005), and the exact composition depends upon the environmental conditions in which the biofilms are formed (Prouty and Gunn 2003). A variety of environmental cues such as nutrients, oxygen tension, temperature, pH, ethanol and osmolarity can influence the expression of the transcriptional regulator CsgD, which regulates the production of both cellulose and curli (Gerstel and Romling 2003). In addition, a study of 122 Salmonella strains indicated that all had the ability to adhere to plastic microwell plates and that, generally, more biofilm was produced in low nutrient conditions, as can be found in specific food-processing environments, compared to high nutrient conditions (Stepanovic et al. 2004).
Pili are structurally similar to fimbriae and are involved in a process of horizontal gene transfer called conjugation. Mostly, the transferred DNA is a conjugative plasmid encoding the formation of the conjugative pilus itself, and thereby mediates an intimate cell-to-cell contact. This conjugation process can stimulate biofilm development, because the conjugative pilus can act as an adhesion factor allowing nonspecific cell-solid surface or cell–cell contacts (Ghigo 2001; Reisner et al. 2003). Vice versa, the high density of bacterial populations in biofilms can stimulate conjugation and plasmid dispersal (Hausner and Wuertz 1999; Molin and Tolker-Nielsen 2003) and can therefore contribute to the spread of resistance genes, which are often also carried on the plasmid (Bower and Daeschel 1999). Luo et al. (2005) have demonstrated that conjugation enhanced the expression of CluA, a surface-bound clumping protein encoded by the chromosomally embedded sex factor, and subsequently facilitated biofilm formation in Lactococcus lactis. Furthermore, this enhanced biofilm-forming trait is transmissible by conjugation.
In addition to proteinaceous organelle-type surface appendages, some Gram-negative bacteria can produce autotransporter proteins. These are secretory proteins that contain in their primary structure all the information necessary to direct their own secretion across the cytoplasmic and outer membrane to the bacterial cell surface. Adhesive phenotypes such as aggregation and biofilm formation have been attributed to a subfamily of E. coli autotransporters, including antigen 43 (Ag43) (Danese et al. 2000a; Kjaergaard et al. 2000), the AIDA adhesin associated with some diarrheagenic E. coli (Sherlock et al. 2004), and the TibA adhesin/invasin from enterotoxigenic E. coli (Sherlock et al. 2005).
The lipopolysaccharide (LPS) outer layer of Gram-negative bacteria typically consists of a surface exposed O-antigen, a core structure and a lipid A moiety that is embedded in the outer membrane lipid bilayer. The LPS layer not only affects the bacterium’s susceptibility to disinfectants, antibiotics and other toxic molecules (Russell and Furr 1986), it also plays a role in biofilm formation. For example, O-antigen mutants of Salmonella enterica serovar Typhimurium showed reduced capacities to attach and colonize alfalfa sprouts (Barak et al. 2007). Alterations in the LPS of Salm. Typhimurium had also osmolyte-dependent effects on biofilm formation (Anriany et al. 2006). For E. coli, truncation of LPS (deep-rough phenotype) did not affect adhesion per se, but had a pleiotropic effect on the biosynthesis of Type 1 fimbriae and flagella, resulting in a reduced adherence (Genevaux et al. 1999). Alterations in the peptidoglycan structure exposed at the surface of Gram-positive bacteria can also have an effect on attachment, as shown by analysis of L. monocytogenes rough colony variants. The latter, characterized by an impaired cellular localization of several peptidoglycan-degrading enzymes such as the cell wall hydrolase A (CwhA), showed enhanced attachment to stainless steel (Monk et al. 2004).
Many bacteria produce and secrete extracellular polysaccharides (EPS). The polysaccharide-containing layers outside the cell are collectively defined as glycocalyx, but when the layers are rigid and organized in a tight matrix that excludes particles, the term capsule is used. If the layers do not exclude particles and are more easily deformed and detached, the term slime is used. These EPS are an important constituent of the extracellular matrix characteristically produced by many biofilms. The matrix often contains additional constituents, such as nucleic acids, proteins, glycoproteins and lipoproteins.
For Kl. pneumoniae, the capsule is considered to be a dominant virulence factor, and its synthesis blocked Type 1 fimbriae-promoted biofilm formation on abiotic surfaces (see above), thereby, actually reducing the bacterial adhesion to such surfaces (Schembri et al. 2005). For V. vulnificus, expression of capsular polysaccharides also inhibited attachment and biofilm formation on abiotic surfaces (plastic) (Joseph and Wright 2004). The EPS colanic acid (or M antigen) produced by most E. coli strains as well as by other species of the Enterobacteriaceae appears to be important for establishing the complex structure and depth of E. coli biofilms, but not for initial attachment to abiotic surfaces (Danese et al. 2000b; Prigent-Combaret et al. 2000). Overproduction of EPS can even inhibit initial attachment of E. coli O157:H7 to stainless steel (Ryu et al. 2004a). The unbranched polysaccharide, β-1,6-poly-N-acetyl-d-glucosamine (PGA), is involved not only in adhesion by staphylococci, but also in attachment to abiotic surfaces, intercellular adhesion and biofilm formation of E. coli (Wang et al. 2004). Furthermore, depolymerization of PGA led to dispersal of biofilms (Itoh et al. 2005). Colanic acid, PGA and cellulose production, but not LPS production, affected binding of E. coli O157:H7 to alfalfa sprouts as shown by mutational analysis (Matthysse et al. 2008).
These observations indicate contrasting roles for EPS (and LPS) in biofilm formation of different bacteria. The particular function of EPS in biofilm formation may depend on its structure, relative quantity and charge and on the properties of the abiotic surface and surrounding environment. Furthermore, EPS play a role not only in biofilm formation but also in the increased resistance of biofilm bacteria to biocides as described in section Implications of biofilm formation.