[Tolerance analysis of Helicobacter pylori internalization to commonly used antibiotics in clinical practice]

Objective: To explore the intracellular tolerance of Helicobacter pylori (Hp) to common clinical antibiotics. Methods: One strain of Hp26695, HpATCC43504, and HpHXW02 each were used to infect human gastric mucosal epithelial cells. Divide human gastric mucosal epithelial cells into intracellular and extracellular groups. In the intracellular group, infect cells with the selected bacterial strain that causes the most severe vacuolization effect, and establish an intracellular infection model; Extracellular group: cultivate an equal amount of bacterial strains in conventional liquid culture without infecting cells. Using immunofluorescence staining to locate Hp and human gastric mucosal epithelial cells, analyzing fluorescence signals using confocal microscopy to verify the phenomenon of Hp entering cells. The minimum inhibitory concentrations (MIC) of four commonly used clinical antibiotics, clarithromycin, metronidazole, tetracycline, and amoxicillin, against the Hp26695 strain were determined using the microbroth dilution method. Then, four antibiotics with concentrations ranging from 0 to 4 MIC were used to treat the intracellular and extracellular groups for 12 h, respectively. The bacterial strains were cultured using the plate coating method, and the colony forming units (CFU), survival rate and relative survival rate of the two groups were compared.Three parallel experiments were set up for each group of antibiotics with different concentrations, and the experiment was repeated three times. Results: Among the three strains of Hp26695, HpATCC43504, and HpHXW02, the Hp26695 strain caused the most severe vacuolization lesions in human gastric mucosal epithelial cells. Therefore, the Hp26695 strain was used for subsequent experiments. Immunofluorescence and confocal microscopy observations confirmed that the strain can invade host cells. The MIC values of Hp26695 strain for clarithromycin, metronidazole, tetracycline, and amoxicillin are 0.031, 0.500, 0.125, and 0.250 mg/L. After antibiotic treatment, the CFU of Hp in the intracellular group were higher than those in the extracellular group: clarithromycin [at 1/2 MIC (162±9) vs (79±5), at MIC (115±6) vs (25±5), at 2 MIC (93±6) vs (15±5), at 4 MIC (81±6) vs (12±1)]; metronidazole [at 1/2 MIC (166±12) vs (110±12), at MIC (161±14) vs (55±7), at 2 MIC (93±8) vs (18±5), at 4 MIC (60±6) vs (7±3)]; tetracycline [at 1/2 MIC (153±13) vs (113±10), at MIC (131±12) vs (45±7), at 2 MIC (113±11) vs (28±6), at 4 MIC (95±8) vs (16±7)]; amoxicillin [at 1/2 MIC (168±14) vs (109±11), at MIC (136±10) vs (47±7), at 2 MIC (120±8) vs (32±7), at 4 MIC (109±12) vs (19±6)] (all P<0.001). After the concentration of antibiotics increased, the survival rates of Hp in both the intracellular and extracellular groups decreased to varying degrees, and the survival rate of Hp26695 strain in the intracellular group was higher than that in the extracellular group (all P<0.05). The relative survival rate of bacteria in the intracellular/extracellular groups was 0.99-7.24. Conclusion: After internalization, Hp exhibit enhanced tolerance to clarithromycin, metronidazole, tetracycline, and amoxicillin.