Rapid and Sensitive Detection of Staphylococcus aureus in Blood Cultures Using a Nuclease-Activatable Fluorescent Probe

Staphylococcus aureus bacteremia (SAB) is a severe infection causing significant morbidity and mortality. Current diagnostic methods rely on blood cultures, which can take several days to yield results, delaying appropriate treatment and potentially worsening patient outcomes. Early and accurate identification of S. aureus is crucial for timely intervention. Micrococcal nuclease (MN), a protein uniquely secreted by S. aureus, presents a promising target for rapid, culture-independent diagnostics. MN's signal-amplifying capabilities and abundance make it suitable for sensitive detection. This study aimed to evaluate the clinical feasibility of a nuclease-activatable fluorescent probe, P2&3TT, for detecting S. aureus directly in blood cultures, potentially reducing diagnosis time from days to hours. The probe's design allows for highly specific detection of MN activity, providing a sensitive means of identifying S. aureus infections.

Previous research has established the unique and consistent production of MN by S. aureus. Studies have demonstrated the potential of using MN activity as a diagnostic marker for S. aureus infections. Several probes and methods have been explored for detecting MN, including signal-amplifying techniques. The need for rapid and accurate diagnostic tools for SAB has driven the development of these methods, highlighting the clinical significance of faster detection in improving patient outcomes. While some progress has been made, a robust and clinically applicable method for rapid, culture-independent detection of S. aureus remains an unmet need.

This study evaluated the P2&3TT probe's performance using blood culture samples. Samples included those positive for S. aureus (n=17), those positive for other Gram-positive bacteria (n=17), those positive for Gram-negative bacteria (n=15), and culture-negative samples (n=7). S. aureus-positive blood cultures were processed by spiking with calcium chloride, heat treatment (to inactivate MN inhibitory antibodies), and centrifugation. Supernatants were then incubated with the P2&3TT probe (3.9 µM). Fluorescence intensity was measured and normalized to the probe signal in buffer. Statistical significance between groups was analyzed using the Kruskal-Wallis test.

The P2&3TT probe showed significantly higher fluorescence intensity in S. aureus-positive blood cultures compared to other groups (P<0.0001). A clear distinction was observed between S. aureus-positive cultures and those positive for other Gram-positive or Gram-negative bacteria, as well as culture-negative samples. The results suggest high sensitivity and specificity of the P2&3TT probe in detecting S. aureus in clinical blood cultures. One sample, co-infected with S. hominis, S. saprophyticus, and S. epidermidis, exhibited unexpectedly high fluorescence, suggesting potential interference or the presence of an undetected S. aureus fraction.

This study demonstrates the potential of the P2&3TT probe as a rapid and sensitive diagnostic tool for SAB. The probe's ability to distinguish S. aureus from other pathogens in blood cultures highlights its clinical utility. The rapid detection enabled by this method could significantly impact patient management by allowing for timely administration of appropriate antibiotics, potentially reducing complications and improving outcomes. Further studies are warranted to fully assess the probe's clinical performance in a larger, more diverse patient population.

The P2&3TT probe offers a promising approach for rapid and culture-independent detection of S. aureus in blood cultures. Its high sensitivity and specificity make it a potential game-changer in SAB diagnosis, enabling faster treatment and improved patient care. Future studies should focus on validating the probe's performance in a large-scale clinical trial to confirm its diagnostic accuracy and establish its clinical utility.

The study involved a relatively small sample size. The potential interference from other nucleases or co-infections needs further investigation. The impact of varying concentrations of MN in different infections requires exploration. Large-scale clinical validation is necessary before widespread clinical implementation.