Novel luminescent affiprobes for molecular detection of Staphylococcus aureus using flow cytometry

Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus.

To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA₁ ) and fluorescent dimeric affibody (fA₂ ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA₁ and fA₂ had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA₂ ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA₁ ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results.

The results show that 'fA₁ ' affiprobe and 'fA₂ ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus.

We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.