# Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of _Helicobacter pylori_
## 5 Holistic Model Describing the Transition of _H. pylori_ into the Biofilm Phase  
The process of _H. pylori_ adhesion to the surface is stimulated by the negative charge of the substrate [24]; hence, the secretion of OMVs with an anchored eDNA (a molecule generating their negative charge) may facilitate the first stages of adhesion [25; 97; 98]. However, it should be remembered that this mechanism is not obligatory, but rather may help to facilitate adhesion. The interaction of OMVs with bacterial cells is conditioned by both the interaction of eDNA with many positively charged _H. pylori_ proteins [113] and also directly by the presence of numerous adhesins incorporated into OMVs [101; 102]. The processes described above take place in the early stages of biofilm formation (the microcolony formation), which seems to be confirmed by studies suggesting the importance of eDNA in the early but not late stages of _H. pylori_ biofilm formation [24].  
Further steps in biofilm development appear to be largely controlled by global regulatory proteins [21; 22; 39; 43; 52; 112]. Within them, some are induced during the transition into biofilm phase, including HspR, HrcA, and CrdR, all of which increase the expression of adhesins and flagella [49; 66; 67]; aconitase stimulates the production of flagella and the activity of urease and hydrogenase [62]; RsfS inhibits metabolic intensity and proteins translation [68]; SpoT supervises the biofilm matrix production and the efflux pumps activity [39; 76]. Other global regulators, such as ArS and LuxS, both controlling the mobility and maintaining bacteria in planktonic forms, are inhibited [26; 48; 52; 69].  
During the maturation of the microcolony into biofilm, a high number of morphological transformations occur, which ultimately result in the presence of a large population of coccoid forms of this bacterium [70; 71]. This morphotype is associated with an increase in the expression of adhesins, urease, and hydrogenase as well as transporter proteins/efflux pumps [75; 77; 78]. In addition, spherical forms intensively secrete OMVs, which additionally stabilize the biofilm structure, but also participate in the process of genetic recombination and tolerance to environmental stress (e.g., by the presence of numerous degradative enzymes) [25; 104; 105]. Efflux pumps, intensively expressed by the coccoid forms of _H. pylori_, take part in matrix production in the process of active ejection of building components into the extracellular environment and protect microbial cells against the bactericidal effect of antimicrobials [38; 39; 76]. Flagella, which are also highly expressed during biofilm formation, may be an independent, additional stabilizer of this structure [21; 22]. Numerous protein componentsof \(H\). _pylori_ biofilms, including adhesins, flagella, or efflux pumps, are important at all stages of biofilm formation [21; 22; 24; 36]. This is confirmed by the results showing the ability of proteinases to significantly degrade the biofilm of this bacterium both in the early and late stages of this structure development [24]. Sugar components are also part of the biofilm matrix and are mainly derived from LPS and peptidoglycan, indicating that the active OMVs secretion, efflux pump activity, and/or cell lysis contribute to the deposition of sugars in this polymer [20; 90].  
In summary, biofilm formation is a stepwise and highly complex process (Figure 6). A number of changes in the expression and translation of \(H\). _pylori_ genes appear to be involved in the transition from planktonic to biofilm phase. Moreover, the process of biofilm structure development is influenced by various environmental factors and these of microbial origin, i.e., virulence and adaptive changes (morphological variability, OMVs secretion, metabolism, and efflux pumps activity).