## 5 Biofilm and Heterogeneity of _A. baumannii_ Populations  
### Formation of A. baumannii Biofilms  
Biofilms are multicellular consortia of single or multiple bacterial species enclosed in extracellular polymeric substances (EPS) which comprise polysaccharides, proteins, and nucleic acids secreted by bacteria. The structure of mature biofilms is often very complex with clusters of bacterial cellsseparated by fluid-filled channels. Diffusion of nutrients and oxygen is limited in biofilms; therefore, the environmental conditions are not homogeneous throughout a biofilm. This leads to the formation of heterogeneous cell subpopulations adapted to local microenvironments. Biofilm-dwelling bacteria are more resistant to antibiotics and other stressors than planktonic cells [99; 100]. A number of factors are known to lead to the enhanced antibiotic resistance of biofilms, e.g., impaired drug diffusion due to microbial aggregations and overexpression of the extracellular polymeric substance (EPS) matrix, biofilm-specific efflux pumps, alterations in microbial phenotypic and genotypic features due to stress responses, and specific microenvironment conditions that inactivate antibiotics and the presence of persister cells (see below) [101]. Antibiotics administered at concentrations below the minimum inhibitory concentration (MIC) often induce biofilm formation in a variety of bacterial species [100; 102]. _A. baumannii_ forms biofilms on both biotic and abiotic surfaces which contributes to its remarkable ability to survive in hospital environments. While extrinsic factors such as surface hydrophobicity, temperature, and oxygen concentration are reported to influence _A. baumannii_ biofilms, numerous physicochemical and microbial features (e.g., capsular polysaccharides, surface appendages, adhesins, and virulence and resistance determinants) facilitate the formation and maintenance of _A. baumannii_ biofilms (Figure 2A) [103]. In addition to biofilms on solid surfaces, _A. baumannii_ also forms \"pellicles\" at the air-liquid interface (Figure 2B) [104; 105]. The formation of these floating biofilms is a rare trait in clinical _A. baumannii_ isolates, and it is associated with surface-associated motility [104; 106]. The relationship between motility and pellicles or surface-attached biofilms is complex. Although a motile state seems to be the opposite to a sedentary lifestyle in biofilms, motility may be required for the formation of microcolonies at the early stages of biofilm development and during the reorganisation of mature three-dimensional biofilm structures [107]. A. baumannii does not produce flagella; however, it can move via surface-associated motility or twitching motility [104; 128]. Multiple genes required for surface-associated motility, including genes associated with purine and pyrimidine biosynthesis or natural competence, were recently identified [128], but its mechanism remains poorly understood. Twitching motility is mediated by the extension and retraction of type IV pili, which are composed of helically assembled PiA subunits. PiIA produced by various _A. baumannii_ isolates differ in amino-acid sequence and \(O\)-linked glycosylation. It was proposed that, when negatively charged residues dominate on the surface of the headgroup domain of PiIA, the pili retract from each other due to electrostatic repulsion and promote twitching motility. The opposite effect, i.e., pili bundling, cell-cell attachment, and biofilm formation, may occur in the case of PiIA variants without negatively charged headgroup domains [110]. Several studies showed that one of the main structures required for cell attachment and biofilm development is CPS [129]. For example, it was shown that, in the case of the _A. baumannii_\(\Delta\)_wza_, capsule-deficient strain biofilm growth and adhesion to epithelial cells were reduced [45]. However, under certain conditions, enhanced production of the capsule may be associated with biofilm reduction. It was recently reported that the Lon protease affects biofilm formation in _A.