# Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of _Helicobacter pylori_
## 2 Characteristics of _H. pylori_ biofilms Produced in Laboratory Conditions  
The first study showing the ability of _H. pylori_ to form biofilm took place in 1999 [20]. Since then, the knowledge on this subject has expanded significantly, and the presence of _H. pylori_ biofilms has been demonstrated in numerous in vitro studies using plastic or glass surfaces [21, 22, 23, 24, 25], cell cultures [26, 27], gastric biopsies from animals [28] or humans [29, 30], and even vegetable surfaces [31] or water distribution tanks [32, 33, 34]. It is worth noting that initially _H. pylori_ biofilms were strongly associated by scientists with water distribution systems and were considered as a survival mechanism outside the host and a source of fecal-oral infections [35]. In the course of the last 20 years, however, this trend has changed and now the most attention is focused on the biomedical aspect of this structure and its potential contribution to the therapeutic difficulties of _H. pylori_[22, 23, 36, 37, 38, 39]. Under laboratory conditions, _H. pylori_ biofilm may be formed during both liquid or solid cultures. In the first case, there are several types of biofilms that can develop under such conditions, i.e., a biofilmformed on the liquid-air surface (most often as a macroscopically visible ring, possibly accompanied by a pellicle development) [22; 40; 41; 42], a biofilm formed as sediment of cells adhered to a solid surface (cells are completely immersed in the medium) [21; 22; 41], and a biofilm present as multicellular aggregates freely drifting in the liquid phase (cells are immersed in the medium but do not sink to the bottom) [22; 41; 43]. In the case of culture on a solid substratum, the so called \"colony biofilm\" [38; 39] or \"lawn biofilm\" [44] are most often indicated. Since the vast majority of research on _H. pylori_ biofilms focuses only on these formed during liquid culture, the data discussed in this review will mainly spotlight such types of bacterial communities. Environmental and cultivation conditions constitute parameters that may affect the process of _H. pylori_ biofilm formation in the laboratory [21; 22; 24; 45; 46]. Adhesion of _H. pylori_ to the surface, a first stage conditioning the biofilm formation, largely depends on the flow rate (inversely associated with the adhesion) and the surface charge (the negative surface charge is preferred), while the ambient temperature does not significantly affect the adhesion [24; 45]. The pH of the environment is a factor that modulates the subsequent processes of biofilm formation. It was found that with decreasing pH, the ability of _H. pylori_ to produce this structure declined, especially at very low values [22]. Exposure of the preformed biofilm to a slightly acidified environment (pH = 5.5) had no effect on the _H. pylori_ biofilm, but still, a highly acidified medium (pH = 3) eliminated this structure. These results indicate the existence of a regulator of _H. pylori_ that senses changes in the pH of the local environment and modulates the ability of this bacterium to form biofilm, especially in its early stages. An example of this type of regulator is ArsRS, which conditions the maintenance of _H. pylori_ in a planktonic form in the strongly acidic environment of gastric juice [41]. Its more detailed characteristics and influence on the formation of _H. pylori_ biofilm can be found in later parts of this article (\"Transcriptomic and proteomic analysis of _H. pylori_ biofilm forms\"). Apart from environmental conditions, the chemical composition of the culture medium and the incubation period are other important parameters influencing the biofilm of _H. pylori_[21; 46]. Laboratory experiments showed that the low serum content (2%) and the three-day culture were optimal for the maximal growth of _H. pylori_ biofilms [21].