Summary Staphylococcus xylosus (S. xylosus)‐induced cow mastitis is an extremely serious clinical problem. However, antibiotic therapy does not successfully treat S. xylosus infection because these bacteria possess a strong biofilm formation ability, which significantly reduces the efficacy of antibiotic treatments. In this study, we developed ceftiofur‐loaded chitosan grafted with β‐cyclodextrins (CD‐g‐CS) nanoparticles (CT‐NPs) using host–guest interaction. These positively charged nanoparticles improved bacterial internalization, thereby significantly improving the effectiveness of antibacterial treatments for planktonic S. xylosus. Moreover, CT‐NPs effectively inhibited biofilm formation and eradicated mature biofilms. After mammary injection in a murine model of S. xylosus‐induced mastitis, CT‐NPs significantly reduced bacterial burden and alleviated inflammation, thereby achieving optimized therapeutic efficiency for S. xylosus infection. In conclusion, this treatment strategy could improve the efficiency of antibiotic therapeutics and shows great potential in the treatment of S. xylosus infections.
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