Antibiotic resistance represents the main challenge of Helicobacter pylori infection worldwide. This study investigates the potential bactericidal effects of fosfomycin combinations with clarithromycin, metronidazole, ciprofloxacin, amoxicillin, rifampicin, and doxycycline against thirty-six H. pylori strains using the checkerboard and time-kill assay methods. The results showed that ≥ 50% of the strains were resistant to the six antibiotics. Remarkably, only six strains exerted resistance to these antibiotics, with the minimum inhibitory concentrations (MICs) ranges of (3.2–12.8 mg/l), (32–256 mg/l), (3.2–51.2 mg/l), (3.2–25.6 mg/l), (1.6–3.2 mg/l), and (25.6 > 51.2 mg/l), respectively. The seven antibiotics were evaluated through in silico studies for their permeability and ability to bind UDP-N-acetylglucosamine1-carboxyvinyltransferase (MurA) of H. pylori. The results indicated that fosfomycin exhibited the highest predicted membrane permeability (membrane ∆G insert = − 37.54 kcal/mol) and binding affinity (docking score = − 5.310 kcal/mol) for H. pylori MurA, compared to other tested antibiotics. The combinations of fosfomycin with these antibiotics exerted synergistic interactions (Fractional inhibitory concentration, FIC index < 1) against the six strains. Importantly, the combinations of fosfomycin with clarithromycin, doxycycline and rifampicin achieved bactericidal effects (reduction ≥ 3.0 Log₁₀ cfu/ml) against the most resistant H. pylori strain. Notably, these effects increased with presence of metronidazole, which enhanced the activity of the fosfomycin combination with amoxicillin from a weak inhibition to bactericidal effect. This study provides evidence that the combination of fosfomycin with either clarithromycin, amoxicillin, doxycycline, or rifampicin (especially with the presence of metronidazole) could be a promising option for treating MDR H. pylori infection.

• Clarithromycin
• Docking
• Fosfomycin
• Helicobacter pylori
• Metronidazole
• Resistant
• Synergism