Fluorescence imaging of extracted osteosynthesis devices
Osteosynthesis devices extracted from 13 trauma patients were included in this study. During surgery, the removed plates were divided in two by using nippers. One half of the plate was used for fluorescence imaging, the other half was used for regular diagnostics (described in the following section). A number of extracted screws, all retrieved from the same surgical site, were used for regular microbial diagnostics, and some for fluorescence imaging. Upon extraction, the osteosynthesis devices were washed with phosphate-buffered saline (PBS), and incubated with vancomycin-IRDye800CW (vanco-800CW, 0.14 nmol mL-1, Li-COR Biosciences, Nebraska, USA) for 15 min at 37oC. Subsequently, the incubated devices were washed twice with PBS to remove unbound vanco-800CW and imaged in the near-infrared range with an IVIS Lumina II imaging system (Excitation 710 nm, Emission filter ICG, Exposure times 1-10 s, PerkinElmer Inc., USA) and an intra-operative Explorer Air camera coupled to a closed-field imaging box (Vault, Excitation 760 nm, Emission filter Semrock FF01-819/44-25, Exposure times 100-200 ms, SurgVision B.V. Groningen, NL). To assess autofluorescence of the extracted device, it was imaged prior and post tracer incubation (Amersham Typhoon Biomolecular Imager, Filter IRLong 825BP30). To verify and visualize Gram-positive bacterial biofilms on extracted devices, microscopic imaging was performed with a two-photon confocal laser scanning microscope (objective 5x/0.16, filters 500-550/575-610, wavelength 850 nm, Zeiss LSM 7MP) using vancomycin-BODIPY FL as a tracer (0.14 nmol mL-1, Thermo Fisher Scientific, USA). Of note, the use of a two-photon microscope was necessary due to the shape and size of the materials tested (plates and screws) in order to have enough distance between the sample and objective. In turn, this required the application of vancomycin-BODIPY FL instead of vanco-800CW, because our two-photon microscope cannot image fluorescence in the near-infrared range. The fluorescence imaging workflow of extracted osteosynthesis devices is schematically represented in Figure 1.
Sonication and culturing of extracted osteosynthesis devices
The presence of microorganisms on the halved extracted plates and screws selected for regular diagnostics (see the afore-going section) and on collected tissues was independently investigated at the diagnostic microbiology laboratory of the University Medical Center Groningen (UMCG). Sonication of extracted osteosynthesis devices was performed as described by Trampuz et al. [11], with subsequent culturing of the sonicate on aerobic blood agar (BA), chocolate agar, anaerobic BA, and in blood culture bottles (BD BACTEC) as liquid culture. The decision whether osteosynthesis devices and/or tissues were infected was taken by the medical team, including a trauma surgeon, infectious disease specialist and a clinical microbiologist according to the standard protocol [4]. In addition, extracted osteosynthesis devices were replica plated on BA plates to correlate fluorescent signals to the presence of bacteria.
Vancomycin-BODIPY FL binding to vancomycin resistant enterococci
Vancomycin resistant Enterococcus faecium (VRE) strains carrying the vanA or vanB genes, vancomycin sensitive Enterococcus faecalis, and the Gram-negative control bacteria Escherichia coli and Klebsiella oxytoca were grown overnight in 3 mL Tryptic Soy Broth (TSB) at 37°C under constant agitation (250 RPM). Overnight cultures were diluted up to an optical density at 600 nm (OD600) of 2 in a final volume of 1 mL. The bacteria were harvested by centrifugation for 3 min at 14.000 RPM. Subsequently, the bacteria were resuspended in 300 µL of 0.14 nmol mL-1 vancomycin-BODIPY FL (Thermo Fisher Scientific, USA) and incubated for 15 min in the dark at 37°C. The bacteria were then washed once with PBS to remove any unbound tracer and 100 µL aliquots of each sample were transferred in triplicate to a flat-bottomed transparent 96-well plate for fluorescence measurements in a Biotek Synergy 2.0 plate reader (BioTek Instruments, Inc., USA). Fluorescence was measured with filters optimal for FITC, the excitation was set at 480 nm and emission at 520 nm, with the optics position at ‘bottom’. For control, triplicate samples of the bacteria incubated and washed only in PBS were also included in the plate. All measurements were carried out in triplicate at 37°C without shaking. In addition, fluorescence of the same 96-well plate was imaged with an Amersham Typhoon Biomolecular Imager (filter Cy2 525BP20).
Data analysis and statistics
Macroscopic fluorescence images and mean fluorescence intensity values were analyzed using ImageJ (National Institutes of Health, Maryland, USA) and Living Image 4.7.3 (PerkinElmer Inc, USA) software. Microscopic images were analyzed using Imaris 9.5.0 software. Regions of interest (ROIs) were drawn around the stained osteosynthesis devices after which the fluorescence signal was quantified. The background signal was quantified by drawing ROIs in uncontaminated biomaterials. Mann-Whitney U tests were used to determine the statistical significance of differences in fluorescence between infected or non-infected extracted devices and devices that had not been incubated with vanco-800CW. A p-value of <0.05 was considered statistically significant.
Ethical approval
Only surplus osteosynthesis devices that were considered unnecessary for clinical diagnosis were used for the present bacteria-targeted imaging with vanco-800CW or vancomycin-BODIPY FL in accordance with the permission obtained from the Medical Ethical Review Board of the UMCG (permission number METc 2016/481). All experiments were performed with adherence to the guidelines of the Declaration of Helsinki and local regulations. Patient data were used pseudo-anonymously based on informed consent.