Lysostaphin-mediated fragmentation of microbial peptidoglycans for label-free electrochemical impedance immunoanalysis of Staphylococcus aureus

Lysostaphin is a Staphylococcus aureusspecific bacteriolytic enzyme that recognizes and cleaves the pentaglycine linkage of peptidoglycan layers in the cell wall of S. aureus. Its target-specific lytic property to the staphylococcal cell wall has attracted interest for the application of lysostaphin as natural antibiotics against S. aureus. However, studies that consider its applicability in the detection of S. aureus have not been reported yet. Based on this, lysostaphin-mediated peptidoglycan fragmentation principles were implemented in the label-free immunoassay of S. aureus. In this study, peptidoglycan layers of S. aureus were fragmented by lysostaphin and the obtained fragments were quantitatively analyzed by electrochemical impedance spectroscopy (EIS). A linear relationship between the logarithmic value of the S. aureus concentration and the increase in the EIS signal was acquired in a range between 102 and 107 colony-forming units (CFU)/mL. Compared to the whole cell-based analysis, the developed lysostaphin-mediated assay for S. aureus showed a 90% increase in sensitivity because the fragments can interact with the immunoanalytical surface more efficiently than the whole cell. In contrast to previous EIS-based bacterial immunoassays that utilize complex electrodes and/or additional signaling probes such as labeled antibodies for signal amplification, we could detect signals with high sensitivity on conventional electrodes without any signaling probes. In addition, by using the developed method, S. aureus was selectively determined in bacterial samples that also contained 106 CFU/mL of Escherichia coli and Corynebacterium glutamicum.