# 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
### Efflux Pump Activity  
In the context of the protective function of _H. pylori_ biofilm components, one cannot fail to mention efflux pumps and the influence of these structures on the antibiotic resistance of this pathogen. Efflux pumps are proteinaceous transporters anchored in the bacterial cell membrane, the amount of which is highly expressed, e.g., under exposure to antimicrobial substances [106]. Their activity is related to the removal of substances harmful to the microbial physiology into the extracellular environment, and therefore, they are currently considered as one of the most important factors determining the antibiotic resistance of microbes. Moreover, the efflux pumps can also participate in the export of factors related to the microbial communication (autoinducers) or substances constituting the building units of matrix, thus playing an important role in the process of biofilm development [107; 108].  
In many studies on _H. pylori_ biofilms, it was found that cells in the biofilm phase are less susceptible to antibiotics [23; 36; 37; 38; 53; 76; 39; 35; 73; 36]. It was determined that minimal biofilm eradication concentrations (MBECs) were several times higher than minimal bactericidal concentrations (MBCs) or minimal inhibitory concentrations (MICs) recorded for planktonic cells, although in some cases the values were dramatically different. In a study conducted in 2020, on a large pool of clinical _H. pylori_ strains [23], MBECs for amoxicillin were shown to be 1000-fold higher than MICs for planktonic forms (16 ug/mL vs. 0.016 ug/mL). Additionally, MBEC values for clarithromycin and tetracycline were 31-fold higher, while for levofloxacin and metronidazolezo, they were increased by 16 and 8 times, receptively. On the basis of the obtained results, the correlation between MBECs and the biofilm production capacity of _H. pylori_ strains (expressed as optical density of crystal violet stained-biofilms) was determined. There was a significant correlation between the biofilm amount and MBECs for amoxicillin, levofloxacin, and clarithromycin. However, no correlation was noticed when determining the MICs of planktonic forms, i.e., values that are classically checked in the laboratory diagnosis of _H. pylori_ resistance. On this basis, the authors of the article postulated the need to verify MBECs in the routine _H. pylori_ antibiotic resistance determination [23].  
The efflux pumps play an important role in generating _H. pylori_ biofilm resistance. Independent results of various research teams show that biofilm _H. pylori_ cells possess a several-fold increase in the expression of genes encoding the efflux pumps compared to planktonic cells [37; 38; 39; 53; 54; 55; 76]. Such an observation was proven for 22 genes of the efflux pumps, including HP0471 (_leftB_) [38; 76], HP0497 [38; 76], HP0605 (_leftA_) [37; 53; 54; 55], HP0939 (_vckI_) [38; 76], HP1165 (_tetA_) [39; 54; 55], HP1174 (_glnP_) [39; 55], HP1327 (_crdB/hefG_) [37; 53], HP1486 (_ybhS_) [39; 55], and HP1489 [37; 53]. In addition, it was found that the expression level of the efflux pump genes was higher in clinical, multidrug-resistant _H. pylori_ strains than in susceptible isolates of this bacterium [38; 39; 76], showing a direct correlation between the production of transporter proteins and the resistance phenotype. It has also been shown that _spoT_ is expressed at a higher level in coccoid _H. pylori_ forms [75], and this gene plays an important role in controlling the biofilm structure and efflux pump activity [38; 39; 76]. Thus, in line with previous reports pointing to the important role of SpoT in the transition to the stationary phase of microbes [109], it is postulated that this protein is involved in the global monitoring of adaptability and response to environmental stress of _H. pylori_, including morphological variability, efflux pumps activity, and biofilm production.