Identification of metabolic pathways modulated by GAM and NGAM in the inhibition of Staphylococcus aureus biofilm formation

Amirmohammed Afsharnia¹Arjen Nauta²Andre Groeneveld²Blanca Fernandez-Ciruelos³Mostafa Asadpoor¹Gert Folkerts¹Saskia Braber¹Marc M.s.m. Wosten^3*

• ¹Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht, Netherlands
• ²Koninklijke FrieslandCampina NV, Amersfoort, Netherlands
• ³Universiteit Utrecht Departement Biomolecular Health Sciences, Utrecht, Netherlands

The prevalence of antibiotic-resistant bacterial strains, particularly Staphylococcus aureus, poses an alarm for global health. The ability of S. aureus to form biofilms reduces the efficacy of antibiotics. Therefore, the need for innovative anti-biofilm strategies to improve the efficacy of antibiotic therapy is crucial, particularly when biofilms cause treatment failure. In this study, we investigated the effects of glucosamine (GAM) and its acetylated derivative, N-acetylglucosamine (NGAM) on the biofilm formation of the multidrug-resistant S. aureus strain Wood 46. The minimum biofilm inhibitory concentration (MBIC) assay was used to evaluate the inhibition of biofilm formation, revealing that 2-8% of GAM significantly inhibited S. aureus biofilm formation. However, only high concentration of NGAM (8%) showed partial inhibition against biofilm. RNA-seq analysis of the treated biofilms indicated that, compared to NGAM, GAM causes a more pronounced downregulation of S. aureus adhesion genes (eno, ebps, and sraP), as well as genes in the arginine biosynthesis and tricarboxylic acid (TCA) pathways, essential for biofilm proteinaceous structure. The decreased pH in the biofilm environment treated with higher GAM concentrations supports its observed anti-biofilm activity and is likely linked to impaired pH homeostasis resulting from ureABC downregulation and disruption of urea metabolism, a process interconnected with the arginine biosynthesis. In conclusion, unlike its acetylated form (NGAM), GAM is a potent anti-biofilm agent that effectively inhibits S. aureus Wood 46 biofilm formation and significantly alters the gene expression profile associated with biofilm formation.