Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.

Bacterial infections are a frequently occurring and major complication in human healthcare, in particular due to the rapid increase of antimicrobial resistance and the emergence of pan-drug-resistant microbes. Current anatomical and functional imaging modalities are insufficiently capable of distinguishing sites of bacterial infection from sterile inflammation. Therefore, definitive diagnosis of an infection can often only be obtained by tissue biopsy and subsequent culture and, occasionally, a definite diagnosis even appears to be impossible. To accurately diagnose bacterial infections early, novel imaging modalities are urgently needed. In this regard, bacteria-targeted imaging is an attractive option due to its specificity. Here, different bacteria-targeted imaging approaches are reviewed, and their promising future perspectives are discussed.

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.

Conflict of interest: KPF reports that he is an employee of PerkinElmer Inc., the manufacturer of optical and PET imaging equipment. GMVD is CEO, founder, and shareholder of AxelaRx/ TRACER group and CSO AxelaRx Biosciences Inc. JMVD has filed a patent application on the use of 1D9 (US 9,944,694 B2), which is owned by his employer University Medical Center Groningen.

Positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (18F-FDG) can be applied to detect infection and inflammation. However, it was so far not known to what extent bacterial pathogens may contribute to the PET signal. Therefore, we investigated whether clinical isolates of frequently encountered bacterial pathogens take up 18F-FDG in vitro, and whether FDG inhibits bacterial growth as previously shown for 2-deoxy-glucose. 22 isolates of Gram-positive and Gram-negative bacterial pathogens implicated in fever and inflammation were incubated with 18F-FDG and uptake of 18F-FDG was assessed by gamma-counting and µPET imaging. Possible growth inhibition by FDG was assayed with Staphylococcus aureus and the Gram-positive model bacterium Bacillus subtilis. The results show that all tested isolates accumulated 18F-FDG actively. Further, 18F-FDG uptake was hampered in B. subtilis pts mutants impaired in glucose uptake. FDG inhibited growth of S. aureus and B. subtilis only to minor extents, and this effect was abrogated by pts mutations in B. subtilis. These observations imply that bacteria may contribute to the signals observed in FDG-PET infection imaging in vivo. Active bacterial FDG uptake is corroborated by the fact that the B. subtilis phosphotransferase system is needed for 18F-FDG uptake, while pts mutations protect against growth inhibition by FDG.

Purpose Due to an increased human life expectancy, the need to replace arthritic or dysfunctional joints by prosthetics is higher than ever before. Prosthetic joints are unfortunately inherently susceptible to bacterial infection accompanied by biofilm formation. Accurate and rapid diagnosis is vital to increase therapeutic success. Yet, established diagnostic modalities cannot directly detect bacterial biofilms on prostheses. Therefore, the present study was aimed at investigating whether arthroscopic optical imaging can accurately detect bacterial biofilms on prosthetic joints. Methods Here, we applied a conjugate of the antibiotic vancomycin and the near-infrared fluorophore IRDye800CW, in short vanco-800CW, in combination with arthroscopic optical imaging to target and visualize biofilms on infected prostheses. Results We show in a human post-mortem prosthetic knee infection model that a staphylococcal biofilm is accurately detected in real time and distinguished from sterile sections in high resolution. In addition, we demonstrate that biofilms associated with the clinically most relevant bacterial species can be detected using vanco-800CW. Conclusion The presented image-guided arthroscopic approach provides direct visual diagnostic information and facilitates immediate appropriate treatment selection.

Staphylococcus aureus bacteraemia (SAB) is associated with high mortality and morbidity rates. Yet, there is currently no adequate diagnostic test for early and rapid diagnosis of SAB. Therefore, this study was aimed at exploring the potential for clinical implementation of a nuclease-activatable fluorescent probe for early diagnosis of SAB. To this end, clinical blood culture samples from patients with bloodstream infections were incubated for 1 h with the “smart” activatable P2&3TT probe, the total assay time being less than 2 h. Cleavage of this probe by the secreted S. aureus enzyme micrococcal nuclease results in emission of a readily detectable fluorescence signal. Incubation of S. aureus-positive blood culture samples with the P2&3TT probe resulted in 50-fold higher fluorescence intensity levels than incubation with culture-negative samples. Moreover, incubation of the probe with non-S. aureus-positive blood cultures yielded essentially background fluorescence intensity levels for cultures with Gram-negative bacteria, and only ~ 3.5-fold increased fluorescence intensity levels over background for cultures with non-S. aureus Gram-positive bacteria. Importantly, the measured fluorescence intensities were dose-dependent, and a positive signal was clearly detectable for S. aureus-positive blood cultures with bacterial loads as low as ~ 7,000 colony-forming units/mL. Thus, the nuclease-activatable P2&3TT probe distinguishes clinical S. aureus-positive blood cultures from non-S. aureus-positive blood cultures and culture-negative blood, accurately, rapidly and with high sensitivity. We conclude that this probe may enhance the diagnosis of SAB.

Purpose Fracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery. Methods Extracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e., vanco-800CW). Subsequently, the devices were imaged, and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar. Results Importantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging can provide surgeons with real-time visual information on the presence and extent of infection. Conclusion Here, we present the first clinical application of fluorescence imaging for the detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate, and real-time visual diagnostic information on FRI in the clinical setting, even in the case of low-grade infections. Graphical abstract