## 2 Characterization of Bacterial Biofilms
### Antibiotic Resistance in Bacterial Biofilm  
Biofilm formation imposes a serious threat for human health worldwide. Antibiotic resistance of bacteria in biofilm communities contributes to serious infections. Bacteria in biofilms reveal about a 1000-fold decrease in susceptibility to antibiotics compared with planktonic cells [24; 25; 26]. Currently, it is believed that over 80% of chronic infectious diseases are caused by biofilms, and it is known that conventional antibiotic medications are inadequate at eradicating these biofilm-mediated infections [5; 27]. The spreads of biofilm-related infections cause an intractable problem in modern medicine [28]. Regardless of their location, bacteria in biofilms are tolerant or resistant to the response of the host immune system, antibiotic therapy, antiseptic agents, or disinfectants and germicides [29; 30]. It is worth mentioning that the tolerance of bacterial biofilms to antimicrobials is multifactorial and is based on different molecular strategies of protection of bacterial cells from hostile conditions. Difficulties in controlling biofilm formation arise from intrinsic and acquired resistance mechanisms of microorganisms. As reported, the resistance of microorganisms in a biofilm may be related to (i) interaction of the biofilm matrix with antibiotics that can retard and lower their activities, (ii) slow growth rates of bacteria in which drugs are not effective, (iii) genetic changes of target cells or hiding the target sites, (iv) action of the modifying enzymes, (v) generation of persister cells which are tolerant to different antibiotics, (vi) alteration of the chemical microenvironment, (vii) multiple microbial species, and (viii) the age of the biofilm (Figure 2). Thus, this multifactorial nature of bacterial biofilms with respect to antimicrobial tolerance is a great challenge for the use of conventional antib biofilm therapeutic strategies [5; 31]. The bacterial resistance is related to the biofilm matrix structure which protects bacteria from a variety of physical, chemical, and biological factors, and which inactivates or impairs antimicrobial molecule spread through the polymer matrix [31]. Due to the impermeability of the biofilm matrix and the diversity of bacterial cells within this structure, antibiotics usually fail when treating biofilm-related infections [32; 33]. Therefore, these diseases often tend to be recurring, even when formed by opportunistic bacterial microorganisms [34]. In many cases, the use of antibiotics, such as colistin, imipenem, fluoroquinoles, beta-lactams, aminoglycos, and others, can only reduce the biofilm layers but cannot eliminate them completely [31; 35; 36]. EPSs hold bacterial cells together and lead to the development of multicellular consortia, allowing the biofilm to function as a multicellular system. The formation of these biofilm complex structures is often regulated by the communication between bacterial cells in the quorum sensing (QS) process that enables the bacterial community to survive environmental stresses and actions of antimicrobial agents [12; 37; 38]. The presence of antibiotic-tolerant subpopulations has been confirmed in biofilms formed by various bacterial species. [PERSON] et al. observed development of colistin-tolerant subpopulations in biofilms formed by _P. aeruginosa_. Notably, colistin-tolerant cells were able to migrate toward the dead microcolonies of the antibiotic-treated biofilm using type IV pili, where they initiated formation of new biofilm via QS [39]. Importantly, the components of the QS system have been identified as genetic determinants responsible for formation of biofilm in the presence of antibiotics by _E. faecalis_ and other bacterial species [40]. On the other hand, inhibition of QS system was shown to increase the susceptibility of _S. aureus_ biofilms toward different classes of antibiotics [41].