Proteomic assay for rapid characterization of Staphylococcus aureus antimicrobial resistance directly from blood cultures

An efficient management of bloodstream infections requires a fast identification of the pathogen and a determination of its antimicrobial resistance profile. Staphylococcus aureus is among the most common pathogen causing bloodstream infection. A prompt characterization of methicillin-resistant Staphylococcus aureus (MRSA) and their aminoglycoside resistance profile is therefore crucial to quickly adapt the treatment and improve clinical outcomes. Among analytical technologies, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising tool to detect resistance mechanisms in clinical samples. Herein we present a rapid proteomic workflow to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus: PBP2a, PBP2c, APH(3’)-III, ANT(4’)-I, and AAC(6’)-APH(2’’), directly from positive blood cultures and in less than 70 minutes. This approach provided 99% sensitivity for PBP2a (n=98/99 strains) detection. Sensitivity was 100% for PBP2c (n=5/5), APH(3’)-III (n=16/16) and ANT(4’)-I (n=20/20), and 94% for AAC(6')-APH(2') (n=16/17). Across the entire collection, 100% specificity was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4’)-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in strains all harboring the ant(4’)-Ia gene. The LC-MS/MS method presented herein demonstrates its ability to provide a reliable and in-depth profiling of S. aureus resistance, directly from positive blood culture and in a short turnaround time, as required in clinical laboratories.