## 5 Biofilm and Heterogeneity of _A. baumannii_ Populations
### Formation of A. baumannii Biofilms
baumannii_[130]. Although the Lon-deficient mutant produced thicker capsule compared to wild-type (WT) cells, it displayed lowered adherence to polystyrene surfaces, decreased motility, and formed a weak pellicle biofilm, but  
Figure 2: _A. baumannii_ forms biofilms on solid surfaces and pellicles at the air-liquid interface. (**A**) Extracellular appendages involved in biofilm/pellicle formation include exopolysaccharideacrylates (capsular polysaccharides, poly-\(\beta\)-(1-6)-\(N\)-acetylglucosamine (PNAG), alginate), and pili [108]. Csu pili are assembled via the chaperone-usher pathway. The Csu e adhesin, which is located at the pilus tip, exposes three hydrophobic finger-like loops that may insert into cavities in abiotic surfaces [109]. Type IV pili (T4P) are composed of PiIA subunits with variable amino-acid sequences in different _A. baumannii_ strains. Depending on the PiIA structure, the pili promote twitching motility or biofilm formation [110]. The chaperone-usher P pili are overproduced in _A. baumannii_ pellicles [105]. The homotrimic Ata autotransporter binds to extracellular matrix components and abiotic surfaces. The transmembrane anchor domain (TM) facilitates the export of the passenger domain (PSD) to the cell surface through a pore formed by the TM. Flexible PSDs allow interactions with different surfaces [111]. Bap and Bap-like proteins (BLP1, BLP2) stabilize the three-dimensional biofilm structure on abiotic surfaces and play a role in the adhesion of _A. baumannii_ to the host cell [112; 113]. Three resistance-modulation-division (RND) efflux pumps (AdeABC, AdeFGH, and AdelJK) affect _A. baumannii_ biofilm development [114; 115; 116]. The AdeFGH efflux pump participates in the transport of quorum-sensing (QS) molecules [116]. OmpA is responsible for the attachment of _A. baumannii_ to plastic surfaces and epithelial cells [67]. The CarO, OprC, and OprD porins may be involved in the uptake of metabolites required for the synthesis of siderophores in pellicles [105]. The iron uptake systems, including acietobactin and enterobactin receptors, are upregulated during pellicle formation [117; 105]. (**B**) The formation of the _A. baumannii_ biofilm and pellicle is regulated by the nucleotide second messengers, two-component signal transduction systems, and QS. cAMP inhibits pellicle formation [104]. The synthesis of Csu pili depends on cyclic di-GMP (c-di-GMP) [118] and the BfmRS and GacSA systems [119; 120; 48]. The hybrid two-component regulator CheA/Y controls the expression of Csu pili and acietin-505 via QS [121; 106]. The QS system of _A. baumannii_ consists of an Abal inducer and its cognate receptor AbaR. Abal is an autoinducer synthase producing \(N\)-acyl homoserine lactone (AFHL) molecules bound by AbaR. The AbaR-AHL complexes activate the synthesis of Abal and the expression of QS-dependent genes, which in turn triggers the production of acietin-505 and Csu pili [122; 122]. Biofilm formation may be inhibited by quorum-quenching (QQ) enzymes, which degrade AHLs [123; 124], as well as high concentrations of Fe\({}^{\text{III}}\) ions that bind AHLs [125]. On the other hand, Fe\({}^{\text{III}}\) ions are required for pellicle development [117; 105]. The AdeABC (controlled by the two-component signal transduction AdeRS system) and AdeFGH efflux pumps participate in biofilm formation [114; 116]. However, other studies revealed that the overproduction of efflux pumps may result in decreased biofilm/pellicle growth [115]. ppGpp regulates the expression of genes encoding the efflux pump’s components [126] and inhibits the production of AbaR and acietin-505 [127]. strongly upregulated a surface antigen, encoded by \(surA1\). The exact mechanism of biofilm regulation by Lon remains to be elucidated. _A. baumannii_ produces an additional surface exopolysaccharideacharide, poly-\(\beta\)-(1-6)-\(N\)-acetylglucosamine (PNAG). Proteins involved in the polymerization and secretion of PNAG are encoded by the _pgaABCD_ operon widely distributed among _A. baumannii_ clinical isolates [131].