# Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of _Helicobacter pylori_
## 6 Limitations and Challenges in _H. pylori_ Biofilm Research  
There is no doubt that research of _H. pylori_ biofilm is still in its infancy. Nevertheless, this subject, especially in recent years, has been gaining momentum. Therefore, it is worth paying attention to factors that may inhibit or slow down the speed of this development.  
Unquestionably, transcriptomic and proteomic studies have great cognitive value in the analysis of \(H\). _pylori_ transition to the biofilm phase, which has been strongly outlined in this review article. It should be emphasized here that their results largely depend on the knowledge about the function of specific genes and proteins. As can be seen quite easily, even in recent 'omics' research on \(H\). _pylori_, the function of about 1/3 rd of the genes or proteins that are defined as being significantly altered in expression is unknown (termed as \"hypothetical protein\") [21; 22; 77; 78; 102]. It seems, therefore, that the substantial amount of future research on \(H\). _pylori_ should focus on determining their role in the physiology of this pathogen.  
Differences in the medium composition and culture conditions significantly affect the obtained results. This is particularly important in the case of 'omics' techniques, which, because of their high sensitivity, bring out all such differences. At this point, it is worth recalling the previously discussed example of discrepancies in the expression of genes encoding antioxidant proteins [22; 43; 56], which were most likely the result of the analysis of distinct types of _H. pylori_ biofilms. Some groups studied biofilm cells formed as microcolonies or a pellicle at the air-liquid interface (both exposed to oxidative  
Figure 6: Stages of _H. pylori_ biofilm development. Transition of bacteria from planktonic to biofilm phase is associated with the transformation of spiral forms into coccoids, as well as increased expression of structural components (adhesins, flagella, and efflux pumps) and intensified secretion of outer membrane vesicles (OMVs). All of them determine the stabilization of the _H. pylori_ biofilm structure.  
stress) [43; 56], while others focused on biofilm created as a bacterial sediment (exposure to oxygen was minimal) [22]. In the opinion of the authors of this review article, the standardization of all research procedures is not necessarily needed, because their diversity is a factor resulting in various interesting observations. However, the research procedure and the type of the analyzed bacterial cells should be very precisely indicated each time, so that the subsequent analysis of the results by other research teams is as easy as possible.  
Another future challenge is to establish a specific procedure to verify the sensitivity of _H. pylori_ biofilms and incorporate it into the routine diagnosis of antibiotic resistance of this bacterium [23]. It is certainly a task that will neither be easy to perform nor achievable in the coming years. However, this challenge is worth to be taken, because more and more studies have indicated the protective role of _H. pylori_ biofilm in antibiotic recalcitrance, especially in the course of therapeutically difficult, recurrent cases of infections caused by this microorganism. In contrast to the previous paragraph, here, the authors of the review strongly encourage the establishment of a single, well-defined procedure allowing the comparison of minimum inhibitory or bactericidal values of _H. pylori_ biofilms between different research teams/diagnostic laboratories.