Supplementary Figures

Transcription

1 Supplementary Figures Supplementary Figure S1. In vitro determination of the detection limit of vanco-800cw (a) Specificity of vanco-800cw for different clinical bacterial isolates. The in vitro imaging analyses were performed using an IVIS Spectrum (Caliper LS, Hopkinton MA, USA; excitation 710 nm, emission 800 nm, acquisition time 1-60 s, binning 4, F-stop 1 or 2, field of view 12 8). The cutoff for a positive result was 10 5 net counts. (b) Determination of the lower limit of detection of S. aureus per mm 3 by fluorescence imaging. The cutoff for a positive reaction was set at 1 2x10 4 net counts. Open dots ( ) represent six replicate measurements, with the mean depicted by filled squares ( ).

2 a b c d Supplementary Figure S2. In vivo imaging of bacterial myositis Bioluminescent S. aureus Xen29 was used to induce a hind limb myositis in NMRI mice by injection of 5x10 7 CFU per mouse. Two days post-infection, 1 mg/kg vanco-800cw was intravenously administered to infected mice and non-infected mice. After 24 h, infected and non-infected mice were euthanized and imaged with an IVIS Lumina II to detect fluorescence (excitation 710 nm; filter: ICG; acquisition time 1-10 s, binning 2, F-stop 2, FOV12 5) and bacterial bioluminescence (excitation block, filter open, acquisition time 300 s, binning 2, F- stop 1, FOV12 5). (a) Upper panel, left side: Bioluminescence imaging of a mouse with S. aureus-induced myositis after injection with vanco-800cw (+/+); upper panel, right side: uninfected mouse after injection with vanco-800cw (-/+). Lower panel, left: NIR detection of S. aureus (Xen29)-induced myositis after injection with vanco-800cw; lower panel, right side: uninfected mouse after injection with vanco-800cw. (b) Excised muscle tissue of infected (left) and non-infected mice (right) as in panel a. (c) Light microscopy of infected muscle tissue (Giemsa staining). An individual S. aureus cell is indicated by the black arrow. (d) Fluorescence microscopy of infected muscle tissue after fluorescent in situ hybridization (FISH) with an EUB-fluorescein isothiocyanate (FITC) probe. An individual S. aureus cell is indicated by the red arrow.

3 a Counts b 40,000 30,000 20,000 Vanco-800CW + Vanco-800CW - 10,000 Counts Supplementary Figure S3. Bladder and urine signals (a) Ventral images of a mouse 24 h after administration of 1.8 mg/kg vanco-800cw were recorded with the IVIS SpectrumCT Imaging System as in Figure 2a (excitation 745 nm, emission 840 nm, FOV 12.8 cm, f-stop 2, 0.5 s acquisition time). A clear fluorescence signal was observed at the position of the bladder (left image), which disappeared upon bladder draining (right image). (b) Upon euthanasia, urine was collected from mice treated with or without vanco-800cw and imaged with the IVIS Spectrum (excitation 710 nm, emission 800 nm; acquisition time 60 s; binning 4; F-stop 1; FOV 12 8). Left: urine from a mouse intravenously injected with vanco-800cw (+). Right: urine from a mouse without vanco- 800CW (-).

4 Bioluminescence-µCT Fluorescence-µCT BLI-FLI co-registration E. coli (Xen 16) S. aureus (Xen 36) E. coli (Xen 16) S. aureus (Xen 36) E. coli (Xen 16) S. aureus (Xen 36) Supplementary Figure S4. Co-registration of bioluminescence and fluorescence Micro-Computed Tomography (μ-ct) imaging of a mouse with E. coli (Xen16)-induced myositis in the left hind limb and S. aureus (Xen36)-induced myositis in the right hind limb 24 h after intravenous administration of 1.8 mg/kg vanco-800cw. Imaging was performed as described for Figure 2c. Left panel, bioluminescence (rainbow scale); middle panel, fluorescence (red-yellow scale; excitation 745 nm, emission 800 nm); right panel, bioluminescence (BLI) and fluorescence (FLI) co-registration. A fluorescent signal from the bladder is detectable behind the spine (middle panel).

5 Supplementary Figure S5. Bioluminescence of S. aureus Xen29 during growth in vitro Overnight cultures of S. aureus Xen29 were diluted into fresh TSB medium to an OD 600 of 0.05 and grown for 24 h at 37 C under vigorous shaking (220 rpm). Samples were collected at hourly intervals for the first 8 h of growth and after 24 h to measure the bioluminescence (solid line) and the optical densitiy at 600 nm (OD 600, dashed line). After 7-8 h, when the cells entered the stationary growth phase, bioluminescence was no longer observed. Data represent the mean of three independent experiments ± the standard deviation (SD). Bioluminescence was measured with a luminometer LB 9501 (Berthold, Bad Wildbad, Germany).

6 Supplementary Figure S6. Microscopic analysis of bacterial myositis. A mouse with S. aureus (Xen36)-induced myositis in the right hind limb and E. coli (Xen16)-induced myositis in the left hind limb was intravenously administered 1.8 mg/kg vanco-800cw as shown in Figure 2 of the main manuscript. 24 h after the vanco-800cw administration, the mouse was imaged (Figures 2 and S4) and sacrificed. Fluorescence microscopy of infected muscle tissue was performed as in Figure 2d. Slides with fixed muscle tissue were prepared for NIR fluorescence microscopy, as described in the Methods section. The left panels show slides with 5µm-thick tissue sections as imaged with the IVIS SpectrumCT (excitation 745 nm, emission 800 nm, FOV 6.6 cm, f2, 50 s acquisition time). Note the strong fluorescent signal of tissue infected with S. aureus (upper left panel) in contrast to the background noise observed for the tissue infected with E. coli (lower left panel). Clusters of vanco-800cwlabeled Gram-positive cocci are detectable upon fluorescence microscopy of the S. aureusinfected tissue (upper right panel; red stain), while no vanco-800cw-labled bacteria are detectable in the tissue infected with E. coli (lower right panel). DAPI-stained cell nuclei are labeled green. Settings for fluorescence microscopy: vanco-800cw - excitation 710 nm, emission >785 nm; DAPI - excitation 360 nm and emission >458 nm.

7 Infected muscle Non-infected muscle Heart Kidney Liver Spleen Intensity (average counts) Supplementary Figure S7. Tissue distribution of vanco-800cw The diagram shows the intensities of the fluorescence signals of ex vivo analyzed tissues 24 h after administration of vanco-800cw (1 mg/kg) to mice with S. aureus (Xen29)-induced myositis. Fluorescence intensities were determined with the IVIS Lumina II as described for Figure S2 (n=5; 5x10 7 CFU per mouse). Mice were sacrificed, and muscle tissue and peripheral organs were harvested aseptically. Note that only muscle tissue was infected, while all other examined tissues remained non-infected. Mean signal intensity (-) ± 2SD ( ) is given.

8 Counts Supplementary Figure S8. IVIS Spectrum analysis of the human post-mortem implant model Two osteosynthesis plates (1 and 3) bearing S. epidermidis biofilms and one plate without biofilm (2) were surgically applied onto the fibula of a post-mortem ankle. Plates 1 and 2 were treated with vanco-800cw prior attachment to the fibula. Before imaging with a clinical camera system, the fluorescence signal from plate 1 was imaged with the IVIS Spectrum (excitation: 710 nm, emission: 800 nm, acquisition time 5 s, binning 4, F-stop 2, FOV 21 2). The plates without biofilm or vanco-800cw treatment (2 and 3, respectively) showed no fluorescence signal.

9 Supplementary Figure S9. UV-Vis spectral overlay of diluted aliquots from preparative HPLC purification of de novo synthesized vanco-800cw LLC and LLC B denote consecutively collected HPLC fractions. Net absorbances were obtained by background subtraction over the range of 900 to 910 nm. Average net absorbances were used to determine the product concentration in respective HPLC fractions.

10 Supplementary Figure S10. Low-Resolution Mass Spectrum (electrospray ionization) of purified de novo synthesized vanco-800cw The purified product was more readily detected in the negative rather than positive ion channel. The strongest m/z signal was deconvoluted by the instrument software and found to correspond to the triple-anion of the parent compound.

11 Supplementary Figure S11. Analytical HPLC data for purified vanco-800cw The compound shows absorbance signals at 780, 680, 280, and 230 nm, indicating the presence of IRDye 800CW. The peak height ratios of the absorbance intensities are consistent with the full UV-Vis spectrum in Supplementary Fig. S9.

12 Supplementary Figure S12. Analytical HPLC of unlabeled vancomycin Absorbance signals were only detectable at 280 and 230 nm, indicating the absence of IRDye 800CW. The unlabeled ligand eluted at an earlier retention time (tr = 4.10 minutes) than the dye-labeled compound (see Supplementary Fig. S11; tr = 5.58 minutes). This was expected as the dye confers additional hydrophobic surface area to interact with the reverse-phase column.

13 Radiance Supplementary Figure S13. Dose-response analysis for S. aureus limb myositis in mice. Bioluminescent S. aureus Xen29 was used to induce a hind limb myositis in immunocompetent NMRI mice by injection with 1x10 6, 1x10 7 or 1x10 8 CFU per mouse. Bioluminescent images were taken at 2 h, and 1 to 6 days (d) post infection, using the IVIS Lumina II with exposure time of 60 sec and medium binning. Radiance is indicated in p/sec/cm 2 /sr. The depicted mice are representative for the particular cohorts (n =3). The minimal dose to reproducibly induce a hind limb myositis was determined at 5x10 7 CFU.