# Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of _Helicobacter pylori_
## 4 Phenotypic Variability as a Modulator of _H. pylori_ Biofilm Structure
### Cocooid Forms  
In the initial stages of biofilm formation, _H. pylori_ is in a spiral form [45; 56; 58]. This morphological form is highly mobile and associated with the colonization of new niches [11; 70]. After effective adhesion to the surface and multiplication, a morphological transformation occurs, which is accompanied by the creation of high shapes heterogeneity (spiral, rod-shaped, curved, coccoid, and filamentous forms) [70]. However, in the case of prolonged cultivation, all cells in the biofilm finally transform to a coccoid form, for which participation in survival and higher tolerance to adverse environmental factors is indicated [71; 72]. It is worth mentioning that the morphological transition of _H. pylori_ to coccoid forms is accompanied by numerous physiological changes, including reduction of cell size,  
Figure 4: Representative photos of cellular and extracellular structures involved in the formation of _H. pylori_ biofilm (based on multidrug resistant, clinical _H. pylori_ 8064). From above: outer membrane vesicles (OMVs) visible as tiny, oval structures of 50–300 nm diameters; flagella noticeable as long structures entwining bacterial cells; and pili seen as very fine, thin filaments connecting bacteria cells. All the above-mentioned structures are marked with yellow arrows. The scale bar shows 1 μm. limitation of intracellular ATP, changes in cell membrane potential, and generation of endogenous oxidative stress-dependent modifications in the structure of DNA and proteins [71]. These processes are associated with the acquisition of a viable but non-culturable (VBNC) phenotype by spherical _H. pylori_ forms and loss of culturability [73], although there are also reports showing the possibility of the existence of spherical _H. pylori_ forms in a culturable form [74]. A recently published report by [PERSON] et al. [74] has described an _H. pylori_ strain with changed phenotype (overproduction of mucus on agar plates occurring only in a spherical morphotype; hence, it was defined as a mucoid-cocoid strain). In contrast to the parental strain with a typical phenotype and high antibiotic sensitivity, from which the _H. pylori_ mucoid-cocoid strain was derived, the latter was resistant to all tested antibiotics. This shows the link between the overproduction of the extracellular matrix and the presence of spherical _H. pylori_ forms with tolerance to antimicrobial substances. Transcriptomic analysis by [PERSON] et al., determining the effect of the morphology change of _H. pylori_ on the _spoT_ status, showed 30 times higher expression of this gene in coccoid forms than in their spiral counterparts [75]. SpoT is a bifunctional enzyme responsible for both the synthesis and degradation of (p)ppGpp (referred to as alarmone). For SpoT, it has been shown that this enzyme contributes to the biofilm formation and supervision of efflux pumps, i.e., proteins associated with antimicrobial removal and resistance generation [39; 76]. A detailed description of the involvement of efflux pumps in the formation of _H. pylori_ biofilm and the induction of antibiotic tolerance will be presented in the \"Efflux pump activity\" section. Apart from the increased level of SpoT, proteomic studies by [PERSON] et al. [77] and [PERSON] et al. [78] showed an increase in the production of the following components in spherical _H. pylori_ forms: outer membrane proteins (Omp7 (HopF), Omp8 (HopG), Omp11 (HorE), Omp15 (HopE), and Omp25 (HopI)), transporter proteins/efflux pumps, flagellin A, urease and hydrogenase subunits, proteins regulating the metal ions concentration (e.g., NikR being crucial for urease and hydrogenase activity [79]), CagE and CagV (stabilizing the T4 SS pilus biogenesis [80]), VacA (related to cytotoxicity and immunosuppression induction [81]), lipoprotein LPP20 and TNF-\(\alpha\)-inducing protein (both contributing to the carcinogenesis promotion [82]), as well as MreB (influencing the action of urease, hydrogenase, and VacA [83]). Research on the transcriptome/proteome of coccoid _H.