Boosting the Anti‐Helicobacter Efficacy of Azithromycin through Natural Compounds: Insights From In Vitro, In Vivo, Histopathological, and Molecular Docking Investigations

ABSTRACT Background Antimicrobial‐resistant Helicobacter pylori ( H. pylori ) poses a significant public health concern, especially given the limited therapeutic options for azithromycin‐resistant strains. Hence, there is a necessity for new studies to reconsider the use of azithromycin, which has diminished in effectiveness against numerous strains. Thus, we aimed to augment azithromycin's anti‐ Helicobacter properties by combining it with curcumin in different formulations, including curcumin in clove oil, curcumin nano‐gold emulsion, and curcumin nanoemulsion. Methods The antimicrobial activities of the investigated compounds, both individually and in combination with other anti‐ Helicobacter drugs, were evaluated. Their antibiofilm and anti‐virulence properties were assessed using both phenotypic and genotypic methods, alongside molecular docking studies. Our findings were further validated through mouse protection assays and histopathological analysis. Results We observed high anti‐ Helicobacter activities of curcumin, especially curcumin nanoemulsion. A synergistic effect was detected between curcumin nanoemulsion and azithromycin with fraction inhibitory concentration index (FICI) values <0.5. The curcumin nanoemulsion was the most active anti‐biofilm and anti‐virulence compound among the examined substances. The biofilm‐correlated virulence genes ( babA and hopQ ) and ureA genes were downregulated (fold change <1) post‐treatment with curcumin nanoemulsion. On the protein level, the anti‐virulence activities of curcumin nanoemulsion were documented based on molecular docking studies. These findings aligned with histopathological scoring of challenge mice, affirming the superior efficacy of curcumin nanoemulsion/azithromycin combination. Conclusion The anti‐ Helicobacter activities of all curcumin physical forms pose significant challenges due to their higher minimum inhibitory concentration (MIC) values exceeding the maximum permissible level. However, using curcumin nanoemulsion at sub‐MIC levels could enhance the anti‐ Helicobacter activity of azithromycin and exhibit anti‐virulence properties, thereby improving patient outcomes and addressing resistant pathogens. Therefore, more extensive studies are necessary to assess the safety of incorporating curcumin nanoemulsion into H. pylori treatment.