Ethics statement
All animal experiments were approved by the University of Nottingham Animal Welfare and Ethical Review Board and performed in accordance with the UK Home Office Licence rules, under Project Licence 40/3821. All injected volumes were in line with ASPA guidelines.
Statistical analysis
Statistical analysis was performed using Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). Where appropriate, analyses were performed by student's t-test (for in vitro analysis) and Mann-Whitney U test (for in vivo analysis). Unless otherwise stated, for in vitro data, error bars show standard error of the mean (SEM); for in vivo data, bars on graphs show data median.
Synthesis and analysis of [18F]FDG-6-P
[18F]FDG-6-P was prepared from commercially available [18F]FDG (PETNET Solutions, Nottingham, UK) following a method previously described [31]. The purity of the [18F]FDG-6-P product was determined by reverse-phase high-performance liquid chromatography (RP-HPLC) on an Agilent 1260 Series LC (Agilent Technologies, Sta. Clara, CA, USA) connected to Flow-RAM™ sodium iodide detector (LabLogic Systems Limited, Sheffield, UK). A SphereClone SAX 5 μm 250 × 4.6 mm column (Phenomenex, Torrance, CA, USA) was used for RP-HPLC at a flow rate of 0.8 ml min−1 with UV detection at 295 nm, using an isocratic method with 20 mmol l−1 potassium phosphate (pH 7.2) as the mobile phase. Instant thin layer chromatography (iTLC) was carried out using reverse-phase plates (Merck F254 aluminium sheet silica plates, 1.0 × 7.0 cm; Merck & Co., Whitehouse Station, NJ, USA). Radiolabelling efficiency on iTLC strips was measured using a Bioscan radio-TLC Scanner (LabLogic, Sheffield, UK). To ensure the stability of [18F]FDG-6-P over time, 2 MBq of [18F]FDG-6-P was incubated at 37°C for up to 3 h in 1 ml water or for 1 h in 1 ml blood (extracted from mice and heparinised). The products were analysed by HPLC (water) or iTLC (blood).
[18F]FDG-6-P uptake assays
S. aureus RN6390 and a UHPT-deficient strain S. aureus RN6390 ΔuhpT were grown overnight in tryptic soy broth (TSB, Oxoid Microbiology Products; Thermo Fisher Scientific, Basingstoke, Hampshire, UK) at 37°C, with shaking at 250 rpm. The overnight cultures were diluted to OD600 0.1 and incubated until mid-exponential phase was reached. One millilitre of cells was harvested (1 × 108 CFU), washed and resuspended in 1 ml RPMI 1640 (Life Technologies, Carlsbad, CA, USA).
Jurkat, AGS, THP1 and HL60 cell lines were grown in RPMI 1640 tissue culture medium supplemented with L-glutamine, 10% v/v foetal calf serum (FCS) and 1% w/v penicillin/streptomycin (P/S). The cells were grown in 75-cm2 flasks at 37°C and 5% CO2. The cells were maintained at 0.5 to 1 × 106 cells ml−1. HL60 cells were supplemented with 1.3% v/v DMSO for 3 to 4 days followed by 24 h without DMSO prior to experimentation in order to increase expression of CD11b. AGS cells were seeded into 6-well plates (Corning Inc., Corning, NY, USA) prior to experimentation.
HIB-1B and 3 T3-L1 cell lines were grown in DMEM (Gibco; Life Technologies) tissue culture medium, supplemented with 10% v/v FCS, 1% w/v P/S and 1% w/v sodium pyruvate. The cells were grown in 75 cm2 flasks at 37°C and 5% CO2. The cells did not exceed 80% confluence. HIB-1B cell lines were seeded into 6-well plates and underwent differentiation by incubation with DMEM supplemented with 10% v/v FCS, 1% P/S, 1% sodium pyruvate, 3-isobutyl-L-methylxanthine (IBMX, 500 μM), dexamethasone (DEX, 250 nM), insulin (170 nM) and triiodo-L-thyronine (T3, 10 nM) for 48 h. The cells were then incubated with fresh DMEM supplemented with 2% v/v FCS, T3 (10 nM) and insulin (170 nM) every 48 h for 8 days. The 3 T3-L1 cells were seeded into 6-well plates and underwent differentiation by incubation with DMEM supplemented with 10% v/v FCS, 1% w/v P/S, 1% w/v sodium pyruvate, IBMX (500 μM), DEX (250 nM) and insulin (170 nM) for 48 h. The cells were then incubated in fresh DMEM supplemented with 10% v/v FCS, 1% P/S, 1% w/v sodium pyruvate and insulin (170 nM) every 48 h for 8 days.
For experimentation, non-adherent cell lines (HL60, THP1, Jurkat; 1 × 106 cells ml−1) were harvested, washed and resuspended in 1 ml tissue culture medium. The adherent cell lines (AGS, HIB-1B and 3 T3-L1) were maintained in 6-well plates and were 80% confluent. Each cell type (bacterial or mammalian) and controls without cells were incubated with 2 MBq [18F]FDG or 2 MBq [18F]FDG-6-P for 1 h at 37°C. Bacteria and non-adherent mammalian cells were harvested by centrifugation (600 × g, 5 min) and washed three times by centrifugation. All supernatants were collected for each sample in scintillation vials. After washing, the cells were transferred into scintillation vials. Adherent cell lines were washed three times by replacing the well medium. Supernatants were collected into scintillation vials. The cells were removed from the wells by trypsin treatment and placed into scintillation vials. The scintillation vials for cells and supernatants were counted by gamma counter (1480 Automatic Gamma Counter, PerkinElmer Inc., Waltham, MA, USA), and the results were obtained as counts per min (cpm).
Results were normalised for controls (no cells) and by calculating the percentage of activity in the cell containing scintillation vials compared with the total counts (cells and supernatant combined). The percentage of activity associated with the cells was normalised so that [18F]FDG cell uptake was equivalent to 1. The samples were incubated in triplicate, and the data were presented from at least two independent repeats.
Animal models and infection
Female BALB/c mice, 19 to 22 g (Charles River Laboratories, Tranent, UK) were used for all experimentation. All injections were performed using a 25-G insulin needle and syringe (Becton Dickinson and Co., Franklin Lakes, NJ, USA). Silicone 5-mm-long catheter sections (scale Fr 8, RUSCH Brilliant Pediatric, Teleflex Inc., Wayne, PA, USA) were implanted subcutaneously (sc) into the right flank of the mice as previously described [32,33], with the following modifications. Analgesia (carprofen (Rimadyl), 5 mg kg−1, Pfizer Inc., New York, NY, USA) was administered by sc injection 60 min prior to anaesthesia (inhalation of 2% isoflurane in oxygen). The incision site was shaved and cleansed with chlorhexidine gluconate (Hydrex Surgical Scrub) clear solution (Ecolab, Northwich, Cheshire, UK). After insertion of the catheter, the incision site was sealed with tissue adhesive (GLUture, Abbott Laboratories, Maidenhead, UK). The incision sites were healed for at least 7 days prior to inoculation with bacteria.
The bioluminescent (BL) S. aureus strain Xen29 (1 × 107 CFU) in 50 μl saline (n = 6) or saline only (n = 5) was administered directly into the catheter lumen. Mice were imaged optically using an IVIS Spectrum (PerkinElmer) to confirm the presence of BL bacteria. The images were captured for 30 s with small (4 × 4) binning. Optical data was processed with Living Image 3.2 software (Caliper Life Sciences, Hopkinton, MA, USA). All mice were maintained under anaesthesia by inhalation of 2% v/v isoflurane in oxygen for the duration of imaging (optical or nuclear).
NanoScan PET-CT imaging
At 24 h post bacterial inoculation, mice were injected with 10 MBq [18F]FDG (n = 3 infected, n = 3 uninfected) or [18F]FDG-6-P (n = 3 infected, n = 2 uninfected) by intraperitoneal (ip) injection. All scans were carried out 1 h later using a nanoScan PET-CT (Mediso Medical Imaging Systems, Budapest, Hungary) small animal scanner with the following parameters: CT: 1 field of view (FOV), maximum FOV, full scan, 720 projections; tube 35 kVP, 170 ms exposure, 1:1 binning. PET: coincidence 1:5, scan time 20 min, packet timestamp. Scanning and subsequent reconstruction were carried out using Nucline software (Mediso). Reconstruction parameters are as follows: 3D, whole body, non-dynamic with 1:3 binning.
NanoScan PET-CT data analysis
All image analysis was performed with VivoQuant software (inviCRO LLC, Boston, MA, USA). All data were normalised for min/max counts based on exact radiopharmaceutical injection dose; radiopharmaceutical signal from bladders was masked and all images were scaled equally. 3D regions of interest (ROIs) were drawn manually or using integrated thresholding tools (global) where appropriate. Standardised uptake values (SUVs) were calculated for each ROI using the following formula: ROI concentration (MBq mm−3)/(Injected dose (MBq)/Mouse weight (kg)). To determine the infected to uninfected (I/UI) ratio of infected vs. uninfected catheters, the mean SUV for the infected mouse catheter was divided by the mean catheter SUV of the uninfected mouse.