Rapid identification of Staphylococcus aureus by using fluorescent staphylocoagulase assays

Area of Science:

• Microbiology
• Biochemistry
• Diagnostic Assays

Background:

• Staphylococcus aureus is a significant human pathogen.
• Accurate and rapid detection of S. aureus is crucial for timely clinical intervention.
• Staphylocoagulase is a key virulence factor produced by S. aureus.

Purpose of the Study:

• To develop and validate two rapid, fluorogenic assays for the detection of Staphylococcus aureus staphylocoagulase.
• To compare the performance of these novel assays against established methods like tube coagulase and latex agglutination.
• To assess the suitability of the fluorescent assays for high-throughput screening.

Main Methods:

• Development of two fluorogenic assays using N-t-boc-Val-Pro-Arg-7-amido-4-methylcoumarin (VPA) and N-t-boc-beta-benzyl-Asp-Pro-Arg-7-amido-4-methylocoumarin (BB) substrates.
• Testing of 406 clinical staphylococcal isolates.
• Comparison with traditional tube coagulase test and latex agglutination (LA).

Main Results:

• The VPA and BB assays demonstrated high positive and negative predictive values (98.9% and 99.1% for BB; 98.9% and 92.7% for VPA).
• The assays detected staphylocoagulase directly from colonies on solid media, eliminating the need for broth cultures.
• Assay performance was comparable to latex agglutination, with excellent accuracy.

Conclusions:

• The developed fluorogenic assays provide a rapid (1-h) and accurate method for detecting Staphylococcus aureus staphylocoagulase.
• These assays are highly sensitive and specific, with predictive values suitable for clinical diagnostics.
• The microtiter tray format makes these assays ideal for large-scale screening applications in clinical microbiology laboratories.