Contact with the surface may induce changes in the bacteria gene expression, for example the synthesis of extracellular polysaccharides is upregulated [2]. Also, within minutes microorganisms upregulate the secretion of intercellular signaling molecules; however, a strong attachment of microcolonies occurs only after a few hours [3].
The maturation of a biofilm is associated with the complex process of matrix formation and is dependent on nutrient availability, shear forces, and the influence of other microorganisms [4]. The maturation is complete only after 2–4 days following the initial attachment [3].
In the maintenance phase, there is relative stability within the community. However, several activities exist. For example, the biofilms continuously respond to desiccation by synthesis of extracellular polysaccharides molecules [4], and microorganisms (planktonic or microcolonies) are also shed into the environment [3]. The dispersion of biofilms might be mediated by ending the synthesis of matrix production, matrix degradation, and disruption of interactions between matric components [5].
Interactions between Microorganisms
When bacteria undergo the transition from a planktonic lifestyle to biofilm community, they must interact with microorganisms in close proximity [2]. Over a few years it has become clear that a communication among the microbial cells exists and that it plays an essential role in biofilm formation. Bacterial cells and also fungi produce certain molecules to which another microorganism can respond. The molecules produced by Gram-negative bacteria are acyl-homoserine lactones. Gram-positive bacteria may generate autoinducing peptide I and double-tryptophan signal peptide pheromone. Both Gram-positive and Gram-negative bacteria synthesize autoinducer 2, which is of importance in particular in interspecies communication [6]. Quorum sensing is controlled by the spatial distribution and the density of the bacterial cells [6]. In the process of biofilm formation, quorum sensing regulates the attachment of cells to surfaces [6]. Later, it regulates many cell processes, such as cell density in biofilms, exchange of genes, synthesis of bacteriocins, and biofilm dispersion. Synergistic and antagonistic behaviors occur among the microorganisms within a biofilm, for example microorganisms utilize the byproducts of others or compete on nutrients, and they coagregate or blanket the surface to compete for others [7, 8]. The close proximity of many different microorganisms is an important place for genetic exchange. It is of importance to note that it provides a reservoir for transferring antibiotic resistance genes [9].
Biofilm Matrix
Mature biofilms have a complex structure, consisting of a polysaccharide matrix and fluid-filled channels [2]. A fully hydrated biofilm is composed of about 15% microbial cells and 85% matrix material [3]. The matrix is of importance as it provides stability and protects against environmental insults; it consists of polysaccharides, water, lipids, proteins, and considerable amounts of extracellular DNA [4]. The exopolysaccharides mask bacterial ligands and decrease the ability of phagocytes to recognize and to phagocytose bacteria in a biofilm [10]. Microorganisms synthesize different exopolysaccharides. For example, for Pseudomonas aeruginosa three different polysaccharides have been described, an alginate being a mucoid negatively charged polymer, a glucose-rich polysaccharide being of importance at the liquid-air interface, and a pentasaccharide acting as scaffold for other bacterial cells [10]. The proteins of the matrix originate from bacterial outer membrane vesicles and from cytoplasmic proteins; after being recycled, they polymerize into amyloid-like structures like curli fibers and flagella [5]. Proteins may also form a hydrophobic layer around a mature biofilm [5] which protects the biofilm against environmental insults. The extracellular DNA in the biofilm matrix originates from lysed bacterial cells or is actively released from membrane vesicles at bacterial cell surfaces; interestingly, not all bacterial DNases are able to degrade it [11]. Its functions in a biofilm is discussed as a nutrient reservoir, the dissemination of genes between microorganisms, and a strengthening of the biofilm structure [11].
Fig. 2. Specimens of PMMA loaded with gentamicin and contaminated in vitro with an S. aureus strain for 2 days (photograph: Center for Electron Microscopy, University Hospital of Jena, Jena, Germany).
Biofilms in Medicine
Biofilms are omnipresent.In mountain lakes, they may serve as indicators of environmental influences [12]. Biofilms are found in water drinking distributors [13] and in water hydraulic systems [14]. Biofilms might be a reservoir for pathogens, water systems may distribute Chlamydiales, Legionella sp. [15], and drains are a source of Pseudomonas aeruginosa [16].
After being neglected for several decades, biofilms are gaining more and more interest in medicine. There are infections associated with biofilm formation on incorporated materials or medical devices used for treatment. Furthermore, other infections are clearly associated with biofilms, such as chronic wound infections and lung infections in cystic fibrosis. The diagnostic criteria for identifying a biofilm-associated infection are a localized chronic or foreign infection, or a medical history of predisposing factors for biofilm formation, such as cystic fibrosis, a recurrent infection, and antibiotic failure, among others [3].
Chronic wound infections, such as diabetic food ulcera, develop slowly and are difficult to eradicate. The majority of these infections are biofilm associated, the healing time is prolonged as microorganisms in biofilms are protected against host response, and furthermore, the biofilm may act as a barrier against re-epithelialization [17]. Cystic fibrosis is a congenital disorder and affects many organs, clinically characteristic is the production of mucous and repeated respiratory infections. Often the lower respiratory tract is infected with P. aeruginosa as a biofilm-growing species, which means the host response is unable to clear the chronic infection [18].