Ex Vivo Tracer Efficacy in Optical Imaging of Staphylococcus Aureus Nuclease Activity

Abstract

The key to effective treatment of bacterial infections is a swift and reliable diagnosis. Current clinical standards of bacterial diagnosis are slow and laborious. There are several anatomical imaging modalities that can detect inflammation, but none can distinguish between bacterial and sterile inflammation. Novel tracers such as smart activatable fluorescent probes represent a promising development that allow fast and specific testing without the use of ionizing radiation. Previously, a smart activatable probe was developed that is a substrate for the micrococcal nuclease as produced by Staphylococcus aureus. In the present study, the function of this probe was validated. Practical applicability in terms of sensitivity was assessed by incubation of the probe with 26 clinical S. aureus isolates, and probe specificity was verified by incubation with 30 clinical isolates and laboratory strains of various bacterial pathogens. The results show that the nuclease-specific probe was activated by all tested S. aureus isolates and laboratory strains with a threshold of ~106–107 cells/mL. The probe was also activated by certain opportunistic staphylococci. We therefore propose that the studied nuclease probe represents a significant step forward to address the need for a rapid, practical, and precise method to detect infections caused by S. aureus.

The smart activatable P2&3TT probe allows accurate, fast, and highly sensitive detection of Staphylococcus aureus in clinical blood culture samples

“…In worst case, empirical antimicrobial therapy does not cover the causative micro-organism, potentially leading to death of the patient. These concerns are particularly relevant for SAB, because of the high S. aureus virulence 18,20 .…”

“…A highly promising molecular detection approach for rapid and culture-independent detection of S. aureus infections is based on enzymatic activity of micrococcal nuclease (MN), a protein also referred to as staphylococcal nuclease (SNase) or thermonuclease (TNase), which is specifically and invariably secreted by S. aureus [10][11][12][13][14][15] . The signal-amplifying ability of MN and its relative abundance provide a very sensitive means of detecting S. aureus, possibly enabling diagnosis of SAB in hours instead of days 4 .…”

“…The present study was aimed at validating the potential for clinical implementation of a nuclease-activatable fluorescent probe named P2&3TT for diagnosis of SAB. Of note, in addition to MN, S. aureus produces a second nuclease (Nuc2), a surface-bound nuclease that exhibits substantially less activity than MN 13,16,17 . Upon specific cleavage of this probe by the secreted MN, it emits a readily detectable fluorescence signal 12,18 .…”

“…Of note, in addition to MN, S. aureus produces a second nuclease (Nuc2), a surface-bound nuclease that exhibits substantially less activity than MN 13,16,17 . Upon specific cleavage of this probe by the secreted MN, it emits a readily detectable fluorescence signal 12,18 . Here we report that the P2&3TT probe can distinguish clinical S. aureus-positive blood cultures from clinical non-S. aureuspositive blood cultures and culture-negative blood, rapidly and with high sensitivity.…”

Fluorescent imaging of bacterial infections and recent advances made with multimodal radiopharmaceuticals

“…In a mouse model bearing S. aureus-induced muscle infection, both FAM and Cy5.5 substrates were injected intravenously and yielded activation by bacteria and allowed imaging of the infection (Fig. 8b) [47].…”