Avaliação da motilidade e formação de biofilme por Helicobacter pylori

Abstract

Helicobacter pylori is a Gram-negative, spiral, microaerophilic bacterium containing four to six unipolar flagella that aid in locomotion in the gastric mucosa. It presents several virulence factors that are important for the process of colonization and pathogenesis in the gastric mucosa, ensuring its survival in a hostile environment such as the stomach. Numerous studies have demonstrated that sub-inhibitory concentrations (sub-MICs) of antimicrobials can alter bacterial virulence factors. The demonstration that amoxicillin can act on the different aspects of H. pylori and its virulence factors, as well as on the host response, when in concentrations below the inhibitory concentration, may be one of the justifications for the therapeutic failure of the proposed regimens, aggravating the process. inflammatory and compromising the cure of the disease. Our study aimed to evaluate bacterial motility and biofilm formation by H. pylori 43504 when treated with subinhibitory concentrations of tetracycline, clarithromycin, amoxicillin and clarithromycin + amoxicillin association. The determination of the minimum inhibitory concentration was performed using the broth microdilution technique and, thus, the subinhibitory concentrations of amoxicillin, clarithromycin, amoxicillin + clathrythomycin and tetracycline were determined. For the swimming and swarming motility assays, bacterial suspensions of H. pylori 43504 were prepared with and without the sub-MICs of amoxicillin, clarithromycin, association amoxicillin + clarithromycin, and tetracillin and for swimming type motility, a culture medium with 0 .35% agar supplemented with 5% FBS and swarming type 0.5% agar supplemented with 5% FBS. To determine biofilm formation, bacterial suspensions (109 CFU/mL) of Helicobacter pylori 43504 were prepared in BHI broth supplemented with different 1-10% SBF and incubated for 3, 6, and 10 days at 37 °C in microaerophilia. To evaluate the formation of sub-MICs, bacterial suspensions (109 CFU/mL) were prepared in 2.5% FBS plus sub-MICs (0.063 μg/mL amoxicillin, 0.031 μg/mL clarithromycin, 0.0625 μg/mL amoxicillin + 0.031 μg/mL clarithromycin, 0.5 μg/mL tetracycline), incubated under the same conditions described above. Biofilm biomass was evaluated using the crystal violet method. The untreated strain was used as control at different incubation times. H. pylori showed higher swimmingtype motility, compared to the untreated strain. H. pylori, when incubated at subinhibitory concentrations of amoxicillin, clarithromycin, and tetracycline, showed strong biofilm production, a result similar to the untreated control, after 3 days of incubation. In view of this, further studies should be carried out using the treatment regimen recommended according to the IV Maastricht Consensus and IV Brazilian Consensus on Helicobacter pylori to assess the capacity of biofilm formation by H. pylori when treated with subinhibitory concentrations of antimicrobials, increasing knowledge of their effects on virulence factors related to biofilm formation and motility, investigating new strategies for H. pylori eradication.