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Staphylococcus xylosus, a coagulase-negative, non-pathogenic bacterium, responsible for opportunistic infections in humans and bovine mastitis, has the ability to form biofilms, which are responsible for persistent infections and antibiotic resistance. In our study, azithromycin significantly inhibited biofilm formation by altering protein expression. Of the 1764 proteins measured by the isobaric Tag for Relative and Absolute Quantification (iTRAQ) technique, only 148 proteins showed significantly different expression between the azithromycin-treated and untreated cells. Most ribosomal proteins were markedly up-regulated, and the expression of the proteins involved in histidine biosynthesis, which, in turn, influence biofilm formation, was down-regulated, particularly imidazole glycerophosphate dehydratase (IGPD). Previously, we had observed that IGPD plays an important role in biofilm formation by S. xylosus. Therefore, hisB expression was studied by real-time PCR, and the interactions between azithromycin and IGPD were predicted by molecular docking analysis. hisB was found to be significantly down-regulated, and six bond interactions were observed between azithromycin and IGPD. Many active atoms of azithromycin did not interact with the biologically active site of IGPD. Surface plasmon resonance analysis used to further study the relationship between IGPD and azithromycin showed minimum interaction between them. Histidine content in the azithromycin-treated and untreated groups was determined. We noted a slight difference, which was not consistent with the expression of the proteins involved in histidine biosynthesis. Therefore …