TITLE:
Evaluation of Helicobacter pylori in Vitro Biofilm Formation by Scanning Electron Microscopy and Associated Genetic Determinants
AUTHORS:
Sibel Gulseroglu
KEYWORDS:
Helicobacter pylori, Biofilm Formation, Scanning Electron Microscopy, Biofilm Genes
JOURNAL NAME:
Journal of Biosciences and Medicines,
Vol.13 No.11,
November
17,
2025
ABSTRACT: Background: Helicobacter pylori is associated with gastroduodenal ulcer, chronic gastric ulcer, and gastric cancer and is a global public health problem. H. pylori develops biofilm on the surface of the human gastric mucosa. Biofilm reduces H. pylori eradication by increasing antimicrobial resistance. Detection of biofilm-associated genes by molecular methods may be a step in biofilm eradication strategy. Materials and Methods: E Test was applied for clarithromycin, metronidazole, levofloxacin, and tetracycline MIC studies of the strains. The clarithromycin MIC study was compared using the microbroth method. The crystal violet (CV) staining technique (measured at OD595) was used to evaluate the biofilm formation ability of H. pylori clinical strains. Biofilm formation images of the strains and antibiofilm images formed by exposing the biofilm to clarithromycin were evaluated using scanning electron microscopy (SEM). After detection of three biofilm-associated genes by PCR, the evolutionary similarities of the strains were evaluated by Sanger sequencing. Results: This study investigates the relationship between antibiotic resistance, in vitro biofilm formation, and the presence of specific genetic determinants in three clinical isolates and one standard strain of Helicobacter pylori. Using antimicrobial susceptibility testing, crystal violet assays, scanning electron microscopy, and PCR, it was found that clinical isolates were highly resistant to clarithromycin and that all strains formed biofilms. The study successfully identified three biofilm-related genes (luxS, rpoD, homD) across all strains and demonstrated that clarithromycin treatment reduced biofilm biomass in most but not all strains. Six of the nine target genes (hypothetical protein K747_10375, hypothetical protein K747_09130, flagellar protein, alpha-(1,3)-fucosyltransferase, hypothetical protein K747_06625, cag pathogenicity island protein) were not detected. In the results obtained by the Sanger sequencing method, it was determined that there was phylogenetic similarity between the H. pylori standard strain ATCC 26695 and the three clinical H. pylori strains. Conclusions: The formation of biofilms by H. pylori can be genetically defined based on the capability of the strains to form a biofilm and the number of genes related to this ability.