# Treatment of Biofilm Communities: An Update on New Tools from the Nanosized World
## 2 Biofilm Formation  
As previously mentioned, bacteria are traditionally considered unicellular organisms. Initial observations that led to the formulation of the hypothesis of the formation of sessile communities that are able to stick to surfaces date back to the 1970s thanks to the contributions of [PERSON] and [PERSON] [12; 13; 14]. Over the last forty years, progress has been made in the understanding of this form of bacterial association, understanding many different mechanisms related to biofilm biology, excellently reviewed recently [15; 16]. Concerning formation, schematized in Figure 1, the first step is adherence to a surface. It can be mediated both by non-specific factors, like physico-chemical factors, as well as by specific bacterial protein components. The former mainly involves on one side the nature of the bacterial cell-wall and superficial proteins and on the other the hydrophobicity of the surface, as analyzed in different bacterial species, like _Staphylococcus aureus_ (Gram-positive) [17] and _Pseudomonas aeruginosa_ (Gram-negative) [18; 19]. In these cases, interaction of cell wall components, surfaces proteins and cellular protrusions have been reported to play a role in the attachment to a surface. However, the contribution of single components of the bacterial surface is still debatable, especially due to the methods of analysis and their relevance in in vivo conditions [15]. Another relevant physical factor recognized to be important for biofilm formation is shear stress that allows the formation of a much stronger biofilm when it is applied during formation of the community and influences the dispersal process, increasing the chance to disseminate the infection. Many works established the influence of this physical factor on biofilm [20; 21; 22; 23], including also natural biofilm in rivers in which a particular form of biofilm microcolonies, termed streamers, are aligned to the flow with a sessile \"head\" and a flowing unattached \"tail\" [24]. Recently, the molecular basis of mechanosensing of shear was clarified in _P. aeruginosa_, further underlining the relevance of this physical factor [25]. Regarding in vivo conditions, upon interaction with body fluids, any surface becomes covered with ExtraCellular Matrix (ECM) components, thus coating the surface. The case of _S. aureus_ Cell Wall Anchored (CWA) proteins details the specific adhesion to defined ECM molecules. Fibrinogen [26; 27], fibronectin [28; 29] and collagen [30; 31] are the ligands of specific staphylococcal proteins that have been extensively studied and recently re-classified according to their structure [32]. Even specific biofilm-associated proteins have been reported [33]. These proteins have been substantially shown to bind to their respective ligand and thus promoting specific adherence to a coated surface. Similar cases are reported for Gram-negative bacteria [34]. These specific interactions provide a more realistic base for the first step of biofilm formation in medically relevant conditions. Figure 1: Biofilm formation stages. Phenol-Soluble Modulins (PMSs) (Modified with permission, from [15]). The second step is the maturation of the biofilm where increase in biomass and the production of the Extracellular Polymeric Substance (EPS) occur to achieve a complex 3D structure. The main component of EPS is water (up to 97%) [16] followed by different biological macromolecules as polysaccharides, proteins and extracellular DNA (eDNA). Specific types of polysaccharides are produced when bacteria are embedded within biofilm: poly-_N_-acetyl-glucosamine (PNAG or PIA--Polysaccharide Intercellular Adhesin) for staphylococcal species mainly [35; 36] and alginate together with Psl, a mannose-rich polysaccharide from gene cluster _psl_ (polysaccharide locus) for _P. aeruginosa_[37]. The expression of these polysaccharides is strictly regulated by operons and regulons that are specifically activated at precise moments of biofilm growth.