Antibacterial and Synergistic Effects of Enzybiotics in Combination with Antimicrobial Peptides and Outer Membrane Permeabilizers Against Helicobacter pylori

Abstract This study systematically evaluated the in vitro antibacterial activity, synergistic effects, and outer membrane permeabilizer-mediated enhancement of enzybiotics (AB469, Lysozyme, Ply187) and antimicrobial peptides (Nisin, S16) against Helicobacter pylori. To our knowledge, this is the first report demonstrating in vitro synergistic anti-Helicobacter pylori activity between engineered enzybiotics and antimicrobial peptides. Results demonstrated that AB469 exhibited the strongest antibacterial activity with a minimum inhibitory concentration (MIC) of 0.1 μM (3.9 μg/mL), significantly outperforming Lysozyme (1.3 μM/19.5 μg/mL), Ply187 (1.4 μM/25 μg/mL), S16 (3.3 μM/8 μg/mL), and Nisin (3.6 μM/12.5 μg/mL). Synergistic assays revealed that the combination of AB469 and Lysozyme achieved the most potent synergy (FIC index = 0.249), reducing their MICs by 8-fold each. Similarly, S16 and Lysozyme showed synergistic effects (FIC = 0.249), with MICs reduced by 8-fold. In contrast, Nisin combined with AB469 or Lysozyme displayed indifferent effects (FIC = 2.0). Additionally, outer membrane permeabilizers (EDTA-2Na, lactoferrin, and citric acid) significantly enhanced the antibacterial activity of AB469 and Lysozyme. For example, EDTA-2Na, lactoferrin, and citric acid reduced AB469’s MIC by 16-fold, 8-fold, and 16-fold, respectively. This study highlights a dual-targeting strategy combining peptidoglycan degradation and outer membrane disruption, providing critical insights for combating drug-resistant Helicobacter pylori.