Assessing Vancomycin‐Formyl Peptide Conjugate Binding to the Surface of Resistant Staphylococcus aureus via Flow Cytometry

Staphylococcus aureus strains have emerged with resistance mechanisms that reduce the efficacy of last resort antibiotics and evade the immune system. One strategy to combat antimicrobial resistance is to modulate host immunity to eliminate infections more effectively. This has led to the development of immunotherapeutics consisting of vancomycin conjugated to formyl peptides (fPeps), with vancomycin targeting the cell wall and the fPeps engaging host innate immunity. Here, we used flow cytometry to quantify the binding of vancomycin=fPep conjugates to S. aureus clinical isolates. This revealed reduced binding of vancomycin=fPeps compared to vancomycin alone and quantified the interaction between the conjugates and the bacterial cell surface, which is important to quantify to then control the chemotactic gradient established by the fPep cargo. The direct antimicrobial activity of these conjugates was also reduced when compared to vancomycin, reflecting the reduced binding of these conjugates to S. aureus . This flow cytometry method allows quantification of vancomycin=fPep binding to bacteria and will assist in future studies to understand how attached fPeps and other immune signalling cargoes can stimulate innate immune cell activation leading to bacterial phagocytosis.