## 5 Biofilm and Heterogeneity of _A. baumannii_ Populations
### Formation of A. baumannii Biofilms
The ability of _A. baumannii_ to form biofilms on abiotic surfaces depends on the production of pili assembled via the CsuA/BABCDE chaperone-usher secretion system which is controlled by the BfmR global regulator [109; 119]. OmpA also participates in the development of biofilms on plastic surfaces. In contrast to the CsuA/BABCDE pili system, OmpA is required during the attachment to _Candida albicans_ filaments and human alveolar epithelial cells. After the attachment, OmpA triggers apoptosis of the eukaryotic cells [67]. The giant Bap protein, consisting of 8621 amino acids, is involved in the formation and stabilization of the complex three-dimensional biofilm architecture on abiotic surfaces, and it plays a role in adhesion of _A. baumannii_ to the host cell [112; 113]. Bap possess immunoglobulin-like (Ig-like) repeats that seem to be typical for proteins involved in biofilm development, for example, Bap-like proteins, BLP1 and BLP2, produced by some _A. baumannii_ strains [112; 132]. Another surface adhesion in _A. baumannii_, the trimeric Ata autotransporter, is involved in biofilm production by binding various extracellular matrix/basal membrane (ECM/BM) components, including the basement protein laminin and collagen types I, III, IV, and V. During tissue damage, ECM/BM proteins become exposed serving as docking sites for _A. baumannii_ and a niche supporting biofilm growth. Ata is also responsible for self-adhesion of _A. baumannii_ cells and biofilm formation on various abiotic materials [111; 133; 134]. Other proteins and structures contributing to _A. baumannii_ biofilm development comprise CarO, Omp33, the resistance-modulation-division (RND) efflux pumps, Pap pilus, and alginate [102; 103; 108; 114; 116; 135; 136]. Depending on the experimental conditions, various structures and mechanisms responsible for pellicle formation were identified [104; 105; 106; 117; 137]. Two independent studies demonstrated that the iron uptake systems, as well as CarO, OprD, and OprC porins, are required to develop pellicles [105; 117] In addition, the overexpression of multiple pili systems, including Fil and Csu pili, was also observed [105; 117; 137]. At least three two-component systems regulate surface motility and biofilm/pellicle formation: the aforementioned BmfRS pathway, GacSA, and CheA/Y [48; 106; 120]. CheA/Y is a hybrid sensor histidine kinase/response regulator that controls the _csuA/ABCDE_ operon and the Abal-dependent quorum-sensing (QS) pathway [106]. The QS system of _A. baumannii_ consists of Abal autoinducer synthase and the AbaR receptor protein for the autoinducer, \(N\)-acyl homoserine lactone (AHL) (for more details, see Figure 2B) [138]. Different types of AHLs were detected in _A. baumannii_[139; 140; 141]. Interestingly, C8-AHL and 3-oxo-C8-AHL were produced by both soil and nosocomial _A. baumannii_ strains, whereas long-chain AHLs with C10, C12, C14, and C16 acyl chains were detected only in the nosocomial isolates [140]. During biofilm formation, the AdeFGH efflux pump participates in the transport of AHLs [116]. It was suggested that QS signals may initiate twitching motility and the attachment of _A. baumannii_ to abiotic surfaces via the CsuA/BABCDE secretion system [122]. Other studies reported that AbaI is required for the later stages of biofilm development [139; 141]. Since QS signaling molecules in some bacteria are strong iron chelators, ferric iron (Fe\({}^{3+}\)) limitation increases the AHL level in _A. baumannii_ in a dose-dependent manner, leading to a stress response and biofilm formation [125]. Nucleotide second messengers such as cAMP, cyclic di-GMP (c-di-GMP), and penta/tetra-guanosine phosphate ((p)ppGpp) are key regulators of numerous bacterial traits including adaptation to harsh environments, as well as transition from biofilm to motility, mutualism to commensalism, acute to chronic virulence characteristics, and cell division to cell differentiation [142; 143]. The exact functions of these messengers in _A. baumannii_ were only partially examined.