## 2 Characterization of Bacterial Biofilms
### Antibiotic Resistance in Bacterial Biofilm
Undoubtedly, multicellular behaviors such as migration and cell-cell signaling seem to play an important role in bacterial biofilm formation, life cycle, and resistance to antibiotics and other antimicrobial agents. Interestingly, the high density of the EPS matrix and its binding properties to antimicrobial agents build an effective barrier that can restrict diffusion of antibiotics to various layers of the biofilm [29]. Additionally, toxic compounds can interact with this polymeric matrix and decrease its activity through enzymatic reactions or chelation of the antibiotics [42; 43]. It is commonly known that alignates are able to block the diffusion of gentamicin or tobramycin. Moreover, the exopolysaccharide can protect biofilms of _P. aeruginosa_ from aminoglycos by directly binding these cationic antibiotics [44]. The persister cells form a small subpopulation of slow-growing or growth-arrested bacteria. Their occurrence in the biofilm structure is related to poor diffusion of nutrients and oxygen into periphery region of the biofilm. Interestingly, the persister cells become highly tolerant to antibiotics. This state of resistance is not achieved by genetic changes [45]. It is proposed that this phenomenon relates to the lack of active targets that antibiotics can  
Figure 2: Bacterial biofilm layers and factors that can inhibit activities of antibiotics. Mature biofilm may be divided into three layers: low-density layer, high-density layer, and microenvironmental layer with slow growth and persister cells. All of the layers contain anti-antibiotic factors and can resist antibiotic activity in different ways. corrupt (i.e., no replication of persister cells and slow death) [27]. Importantly, persister cells represent a small fraction (0.1-10%) of the entire population of biofilms formed by _P. aeruginosa, E. coli, S. aureus, Acintobacter_ spp., _Salmonella_ spp., and other bacteria [45]; however, they can survive the presence of 1000-fold the minimum inhibitory concentration of various antibiotics [32]. Therefore, persister cells appear to be responsible for the inability to eliminate chronic infection by using antibiotic treatment [46]. The antibiotic resistance of biofilms may also be conditioned by the presence of bacterial cells carrying resistance genes encoding enzymes, such as, e.g., \(\beta\)-lactamases or aminoglycosoide adenylyltransferases, which can inactivate or modify antimicrobial agents. Such enzymes are secreted into the biofilm matrix and prevent these agents from reaching their cellular nonresistant targets [29; 47]. For instance, _K. pneumoniae_ biofilms produce and secret \(\beta\)-lactamase that was found to effectively degrade ampicillin and prevent it from reaching other cells within the biofilm [47]. In turn, genes encoding aminoglycosoide-modifying enzymes have been found in _E. coli, Salmonella enterica_, and _P. aeruginosa._ They occur in a high number of variants, and their products can utilize aminoglycosoide antibiotics as substrates [48]. In effect, products of antibiotic-resistant genes expressed in some bacterial cells in biofilm can protect other nonresistant bacteria against the action of antibiotics. Importantly, cells carrying such genes are resistant to antibiotics, irrespective of whether they are present in a biofilm or not. It is worth mentioning that antibiotic tolerance in the bacterial biofilms may also be related to oxygen limitation in the lower layers of its structure. Antibiotic resistance in this system is likely due to the fact that anaerobic conditions restrict bacterial metabolic activity to a narrow zone adjacent to the air interface. Interestingly, outside this zone, bacteria included in the biofilm are not easily killed by antibiotics. Importantly, an oxygen gradient is a common feature of life in biofilm, and such conditions may be correlated with aminoglycosoide and fluoroquinoline tolerance of the _P. aeruginosa_ biofilm system [49].