Microbial biofilm is defined as a structured consortium of cells immersed in a self-produced matrix. It is worth mentioning, however, that the biofilm may also include host components, e.g., fibrin, antibodies, platelets, or leukocytes. Biofilms can be attached to an abiotic or biotic surface, but they can also constitute a mobile, non-adhered structure floating in culture broth or body fluids.
H. pylori has the ability to reside in the stomach for many years, often for a lifetime . Although the presence of this bacterium may be associated with a number of gastrointestinal diseases, including gastric ulcers and cancers , more and more often the protective role against developing extra-gastric diseases, such as allergy, asthma, inflammatory bowel disease, or multiple sclerosis, is indicated . Survival of H. pylori in an unfavorable, gastric niche is enabled by an intensive urease secretion, a spiral shape, presence of numerous adhesins, and the production of cytotoxic proteins, e.g., vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA) . It is increasingly recognized, however, that the adaptive mechanisms of H. pylori in the stomach may also be linked to the ability of this pathogen to form biofilms .
The development of biofilm structure and the transition from planktonic to biofilm phase are influenced by changes in the expression and translation of H. pylori genes. These changes were determined experimentally using ‘omics’ techniques (transcriptomics, proteomics, and metabolomics), and despite some differences between the results obtained by independent research teams, it is still possible to identify the most important features of the biofilm forms of this bacterium. In these studies, an increased expression has been demonstrated for: global pleiotropic regulators , adhesins (especially outer membrane proteins from the Hop and Hom family) , lipopolysaccharide (LPS) , efflux pumps , flagella proteins , components of T4SS systems , enzymes regulating pH (e.g., urease or arginase)  and responsible for obtaining alternative energy sources (e.g., hydrogenase) , proteins related to the cell wall rearrangement , and proteins of the toxin-antitoxin system  (Figure 1). Covering the components for which a decrease in the expression in biofilm H. pylori forms has been shown, these were factors involved in metabolism , translation , and quorum sensing related to the autoinducer-2 (AI-2) activity  (Figure 1). The influence of the transition of H. pylori into the biofilm form on the amount of antioxidant proteins is difficult to determine, because Yang et al.  and Shao et al.  showed their increase, while Hathroubi et al.  showed an opposite situation. It is possible that this difference results from the type of analyzed biofilm. Yang et al.  and Shao et al.  studied biofilm cells that were exposed to oxidative stress (air-liquid biofilms or these formed as microaggregates in the medium), while the one studied by Hathroubi et al.  was a sedentary biofilm produced at the bottom of wells, where the oxygen concentration is significantly lower.
Figure 1. Diagram showing transcriptomic/proteomic changes in H. pylori cells during the transition from planktonic to biofilm phase. The scale bar shows 1 µm.
Among the proteins mentioned above, it is worth paying attention to the production of global regulators of H. pylori physiology, because they will influence the expression of other genes under their control. In general, negative regulators bind to the promoter, reducing the attachment of RNA polymerase and lowering the expression of specific genes. In the case of positive regulators, binding is observed in the promoter's upstream region, which determines the recruitment of polymerase and the increase in gene expression .
Shao et al.  showed that aconitase production increases in the biofilm phase. Aconitase acts as a pleiotropic regulator supervising antioxidant functions, motility and production of flagella, as well as the activity of some enzymes (urease and hydrogenase) . It was established that the high activity of H. pylori hydrogenase is a feature of strains with a high carcinogenic potential . Through hydrogenase, this bacterium can use molecular hydrogen (H2) to switch to the chemolithoautotrophic mode and use the energy from this process to bind CO2 . Hydrogenase is also crucial for the functioning of the T4SS systems, both Cag-T4SS (involved in cytotoxicity and oncogenicity) and ComB-T4SS (involved in DNA transformation) . It is worth noting that the ComB system is inactive at acidic pH and is stimulated in the environment with pH > 6.5, indicating that the transformation process takes place in close proximity to the gastric mucosa  and explaining the increase in urease production during biofilm formation (when microbial transformation is strongly intensified) .
The increased expression of H. pylori regulators during the transition to the biofilm phase, including HspR, HrcA, CrdR, and RsfS, was also demonstrated by the team of Hathroubi et al. . HspR and HrcA have a positive effect on the production of flagella and selected adhesins, while negative on the chaperone proteins (GroES, GroEL, and DnaK) . CrdR responds in H. pylori to the nitrosic stress (exposure to NO) . It was experimentally found that flagellar components, iron transporter (fecA, HP0807) and efflux pump (glnP, HP1169) were among the genes with the highest induction controlled by CrdR . For RsfS, participation in the inhibition of large and small subunits of ribosomes has been shown and, as a consequence, a reduction of protein synthesis (one of the most energy-consuming physiological processes) . This mechanism seems to explain the observations of Hathroubi et al., who noticed a decrease in the expression of translation genes during the transition of H. pylori to the biofilm phase .
Apart from global regulators having a positive effect on the production of biofilm, it is also worth paying attention to the existence of the ArsRS regulator, which is a two-component system negatively associated with the production of this structure . For ArsRS, the pH-dependent effect is indicated, during which exposure to the acidic pH of gastric juice inhibits the expression of genes related to biofilm formation, while in an environment with a neutral pH the effect of this system is inhibited . The ∆arsS mutants showed a higher degree of adhesion to the surface and more intensive biofilm production (twice after 1–2 days and four times after 3 days of incubation than the wild-type strain) . Moreover, in these mutants, an increase in the expression of genes encoding outer membrane proteins was noticed; these included alpB (omp21/homB), sabA (omp17/hopP), labA (omp2/hopD), and hopZ (omp1) .
The presented data show that global regulators can significantly influence the physiology of H. pylori and determine the transition from planktonic to biofilm form and vice versa.