**19. Biofilms**

An appreciation for the fact that in nature bacteria adhere to many abiotic or biotic surfaces, embedded in an extracellular matrix, and form communities known as "biofilms" has emerged over the past few decades [90]. Biofilm formation conferred on individual bacteria the ability to collaborate and to adapt to a range of harsh environmental conditions and, perhaps most of all, to evade predation by phagocytic microbes. The formation of a biofilm provides a microbe with a small measure of control over the local environment, including fluctuations in temperature, pH, ultraviolet light, starvation, and exposure to toxic agents [91, 92].

Advances in medical biofilm research have led to the understanding that biofilms represent the prevalent form of bacterial life during tissue colonization and may occur in more than 80 % of microbial infections in the body [93].

Members of a biofilm community, which can be of the same or multiple species, show varying stages of differentiation and exchange information, metabolites, and genes with each other. As a result, members of the biofilm community are in a diversity of physiologies influenced by the unequal sharing of nutrients and metabolic by-products, which results in subpopula‐ tions with increased tolerance to antimicrobials and environmental stresses, the host immune system, and predatory microorganisms [19, 94, 95, 96, 97, 98].

Canonically, biofilm development has been grouped into five stages that are reflective of conditions in many, but not all, biofilms: (1) reversible aggregation of planktonic cells on a surface, (2) irreversible adhesion, (3) formation of microcolonies, (4) biofilm maturation, and (5) detachment and dispersion of cells [99]. The events that are of special significance for ocular infections and the treatment of biofilm infections will be discussed in greater detail, while the reader is referred to several excellent reviews for details on other biofilm-related subjects [19, 100,101].

The biofilms involve the production of an extracellular matrix (ECM) that embedded the cells and, in some cases, binds the cells together and that can be composed of polysaccharides, lipopolysaccharides, proteins, or extracellular DNA [10]. This process may be active or passive, in that cells on the surface of an adherent colony that are lysed by the ejection of neutrophil antimicrobial factors may encase and protect siblings below in a matrix consisting simply of cell lysate. Whatever the nature of the matrix, its chemical and physical properties contribute to the differentiation of cells within the encased population, a process that can protect the bacteria from the action of antimicrobial agents, host immune responses, bacteriophages, and phagocytic amoeba [19].

As the microcolony grows through cell division or the recruitment of more planktonic cells, the biofilm grows and takes on a three-dimensional structure that often includes open water channels [19, 103].

The three-dimensional organization of the biofilm causes gradients of oxygen, pH, and nutrients, resulting in the development of different microniches [104, 105, 106]. The cell's individual physiological adaptations to these microniches result in physiological heterogene‐ ity [98]. Cells near the surface of the biofilm will be exposed to more nutrients and oxygen and are therefore more metabolically active, while cells in the deep regions will be less active or even dormant. This heterogeneity results in a range of responses to antimicrobial agents, with metabolically active cells at the surface being rapidly killed, while more internal, dormant cells are comparatively unaffected [106]. This, together with potential effects on the diffusion of antimicrobial molecules within the biofilm, causes some cells in a biofilm to be recalcitrant to antimicrobial treatment, with antibiotic susceptibilities reduced by 10- to 1,000-fold compared to their planktonic counterparts [106].

The high local concentration of cells in a biofilm creates an ideal environment for information exchange through cell-to-cell communication and lateral gene transfer. Cell signaling medi‐ ated by secreted, accumulating messenger molecules, known as quorum sensing, allows bacteria to sense and respond to their environment and couple cell density and other envi‐ ronmental cues with gene expression in ways that allow adaptive phenotypic responses. Quorum sensing has been shown to be involved in the control of biofilm formation and the production of virulence and colonization factors in a variety of organisms of medical impor‐ tance [106]. Cell-to-cell signaling is also involved in biofilm dispersion, which is of general and medical interest [107].
