Determination of antimicrobial activity, mechanism of action and synergistic effect of bicarinalin antimicrobial peptide on Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach which results in various gastrointestinal-related infections, diseases, and cancer. Eradication has become difficult until today due to the increasing antibiotic resistance of the bacteria. The objective of our study is to investigate the activity and synergistic activity with treatment antibiotics of the ant venom derived from Tetramorium bicarinatum known as Bicarinalin antimicrobial peptide against H. pylori. For this purpose, disk/well diffusion and microdilution experiments were conducted to find the antibacterial activity of determined concentrations of Bicarinalin AMP against H. pylori reference and clinical isolates in our stock. Positive and Levofloxacin were used as control. MIC99 values were determined by measuring the OD at 600nm. Then, Bicarinalin effect on the intracellular proteins and DNA was analyzed using colorimetric assay. Bradford method was used for the estimation of protein leakage. Subsequently, the cellular damage effects were verified by Scanning Electron Microscopy (SEM). Finally, through the disk diffusion method, the synergistic effect of Bicarinalin was tested in combination with Levofloxacin, Clarithromycin and Tobramycin. As a result, the MIC99 value of Bicarinalin was determined as 4.8μg/mL and 4.9μg/mL for the reference strain and clinical isolate, respectively. For the mechanism of action, the protein and DNA leakage that are secreted to the external environment were determined to be 33.25% and 55.10%, respectively. In SEM analysis, the cell membrane structure is disrupted, cells penetrate each other, morphological structure is deteriorated. Finally, the synergistic activity of Bicarinalin was evaluated and it was determined that the AMP increases the inhibition zones of the currently used antibiotics, Levofloxacin, Clarithromycin and Tobramycin.