## 2 Biofilm Concepts and Formation  
Biofilms can be monomicrobial or polymicrobial. A monomicrobial biofilm encases a community of a single species of microorganisms, while polymicrobial biofilms contain a diverse community of multiple species of microorganisms, conceding different features and properties [1; 10]. Polymicrobial biofilms tend to be more robust than monomicrobial biofilms. Moreover, due to symbiotic interactions between distinct species of microorganisms, polymicrobial biofilms have different architectures, and also altered tolerance to antimicrobial agents and other adverse environmental conditions (e.g., limited nutrition supply, the host immune system) [11]. Polymicrobial biofilms are major causes of oral, wound, respiratory, skin, and bone infections [12]. Regarding microorganism behavior within the biofilm, microorganisms can interact with each other in a synergetic/cooperative or inhibitory/antagonistic way, having a significant impact on the pathogenesis of infection [13]. Microbial synergism produces an effect or effects not achieved by individual species alone. These effects include (i) passive resistance--protecting from antibiotic killing activity; (ii) metabolic cooperation--producing adenosine  
Figure 1: Schematic illustration of the review structure. triphosphate (ATP), carbon, and nitrogen to synthesize metabolites and maintain redox balance; (iii) generation of metabolic by-products--a cooperative effect denoting metabolic cross-feeding (or syntrophy) where one species generates a metabolic by-product capable of increasing the growth of the surrounding species; (iv) quorum sensing (QS) systems--signal exchange, allowing cell-to-cell communication and triggering a wide range of biological processes within the biofilm (e.g., detachment) [14; 15; 16; 17; 18; 19; 20; 21]. Conversely, microbial antagonism (or antibiosis) is defined as the suppression and inhibition of one microbial species by another. In those scenarios, antagonistic mechanisms can be developed, such as (i) the production of factors that kill or inhibit the growth of the surrounding species; (ii) the production of chemical signals, negatively affecting the behavior and physiology of the surrounding species; and (iii) limitation of nutrients, leading to starvation [18]. Biofilm formation can be divided into four distinguished stages: (i) attachment--organic/inorganic molecules and microorganisms are adsorbed on a surface substrate (e.g., bone) through electrostatics, electric double layer forces, and hydrophobic and van der Waals interactions, leading to the irreversible attachment of microorganisms to the surface mediated by production of extracellular polymeric substance (EPS) matrix; (ii) formation of microcolonies--microorganisms grow within multicellular surface-attached communities; (iii) the biofilm's maturation--cell-to-cell interactions allow cells within the biofilm to take on specialized functions, secreting mature biofilm components, namely, proteins, polysaccharides, and extracellular DNA; (iv) detachment--microorganisms/microcolonies from the biofilm are dispersed and released into surrounding media as free-floating microorganisms, which can further spread to and colonize another site, causing other surface contamination, systemic infections, or septicemia. Biofilms disperse because of several factors, such as lack of nutrients, intense competition, and an outgrowth population [1; 2; 3; 22]. Recent advances in research have significantly deepened our understanding of the genetic and molecular mechanisms that underpin the resilience of biofilms. Biofilms, complex communities of microorganisms embedded in a self-produced matrix, exhibit remarkable resistance to antibiotics and the host immune system. This resilience is largely driven by a variety of genetic and molecular processes [23]. One key discovery is the role of specific gene regulatory networks that enable bacteria within biofilms to enter a dormant state, making them less susceptible to antimicrobial agents [24].