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Gadolinium-Labelled Aminoglycoside and its Potential Application as a Bacteria-Targeting Magnetic Resonance Imaging Contrast Agent Lei Lei Zhang, Yun Liu, Qingyang Zhang, Tiegang Li, Min Yang, Qingqiang Yao, Xilei Xie, and Hai-Yu Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04029 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018
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Analytical Chemistry
GadoliniumGadolinium-Labell Labelled Aminoglycoside and its Potential Application as a BacteriaBacteria-Targeting Magnetic Resonance Imaging Contrast Agent Leilei Zhang†, ‡, ∇, Yun Liu§, ⊥, ∇, Qingyang Zhang†, ‡, Tiegang Li†, Min Yang†, Qingqiang Yao⊥, Xilei Xieǁ and Hai-Yu Hu*, †, ‡ †
‡
State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China. §School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, Shandong, 250200, China. ⊥Institute of Materia Medica, Shandong Academy of Medical Sciences, Key Laboratory for Biotech-Drugs Ministry of Health, Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan, Shandong, 250062, China. ǁCollege of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Shandong Normal University, Jinan 250014, China. ABSTRACT: Magnetic resonance imaging (MRI) is a powerful diagnostic technique that can penetrate deep into tissue providing excellent spatial resolution without the need for ionizing radiation or harmful radionuclides. However, diagnosing bacterial infections in vivo with clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capabilities and bacterial penetration and specificity. Here, we report the development of the first gadolinium (Gd)-based bacteria-specific targeting MRI contrast agent, probe 1, by conjugating neomycin, an aminoglycoside antibiotic, with Dotarem (Gd-DOTA, an FDA approved T1-weighted MRI contrast agent). The T1 relaxivity of probe 1 was found to be comparable to that of Gd-DOTA; additionally, probe 1-treated bacteria generated a significantly brighter T1-weighted MR signal than Gd-DOTA-treated bacteria. More importantly, in vitro cellular studies and preliminary in vivo MRI demonstrated probe 1 exhibits the ability to efficiently target bacteria over macrophage-like cells, indicating its great potential for high-resolution imaging of bacterial infections in vivo.
Although much fundamental knowledge of antimicrobial therapy has been obtained, bacterial infections presently remain responsible for substantial patient morbidity and mortality globally.1 In addition, multidrug resistant (MDR) bacteria are increasingly challenging to treat, leading to life-threatening diseases, and the rise of MDR bacteria has been considerably by misdiagnosis and the inappropriate use of antibiotics. In situ imaging of bacteria offers the prospect of accurately diagnosing diseases and monitoring patient treatment response in real-time. However, by themselves, current clinical imaging modalities, including computed tomography (CT),2,3 magnetic resonance imaging (MRI),4,5 ultrasound (US),6 positron emission tomography (PET)7 and single-photon emission computed tomography (SPECT),8 are unable to differentiate bacterial infections from each other or from sterile inflammation.9 With the increasing prevalence of MDR bacteria, rapid and accurate diagnosing and monitoring of persistent bacterial infection are urgently needed.10 In this regard, bacteria-targeted imaging represents an attractive option because of its specificity.11-13
Although optical imaging techniques, including fluorescence and bioluminescence imaging, have significantly helped provide an understanding of disease pathogenesis for bacterial infections,14-20 they have not been extensively evaluated in the clinical setting for bacterial infections because they are limited by tissue penetration.21,22 MRI offers a non-radioactive alternative for the non-invasive detection of inflammatory diseases.23 In particular, MRI is essential for examination of underlying central nervous system and musculoskeletal infections.24 The contrast agents most frequently employed in MRI are gadolinium (Gd)-based, as these compounds do not elicit an immune response in cells.25 Conventional Gd chelates currently in clinical use, such as Multihance,26 Magnevist27 or Dotarem (GdDOTA),28 lack specificity and thus provide nonspecific enhancement of pathologies along with endogenous tissue contrast.29,30 For bacterial infections imaging applications, differentiating bacterial infection from each other or from sterile inflammation with these clinical, non-targeted MRI contrast agents is problematic.31 Therefore, bacteria-targeted MRI contrast agents will hold promise for the quick and accurate detection of infections.32
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Antibiotics are a type of antimicrobial drug used in the treatment of bacterial infections with high affinity to certain target sites within bacterial cells. Because antibiotics offer a targeted imaging approach to diagnose infections, there have been numerous attempts to develop bacterial imaging agents based on antibiotics. The most widely explored method was radiolabelled ciprofloxacin ([99mTc]-ciprofloxacin) which was anticipated to be a general reporter of Gram-positive and Gramnegative bacterial infections; however, in vivo investigations indicated that it may not enable a clear distinction between bacterial infection and sterile inflammation.33-36 Recently, several vancomycin-conjugated optical probes have been successfully explored as diagnostic agents of Gram-positive bacterial infection that function by accumulating at the cell wall of Gram-positive bacteria.21, 37-39 These probes could be detected selectively in the S. aureus infected sites, whereas no signal was detected for bacteria free controls. Whether this approach would be suitable for imaging deeper infections within the body, however, has not yet been reported. Aminoglycoside antibiotics kill bacteria by inhibiting protein synthesis as they bind to the A-site on the 16S ribosomal RNA of the 30S ribosome.40 Since aminoglycoside antibiotics act on processes that are unique to bacteria, we proposed that aminoglycoside antibiotics conjugated with an imaging contrast agent should also target bacteria. More recently, we constructed optical probes for a broad spectrum pathogenic bacteria by conjugating an aminoglycoside antibiotic, neomycin, with the Cy 5 fluorophore.16 These probes are selectively taken up by a broad spectrum of pathogenic bacteria over macrophage-like cells. Based on our previous results, we herein report the development of the first Gd-based bacteria-specific targeting MRI contrast agent, probe 1, by conjugating an aminoglycoside antibiotic, neomycin, with an FDA-approved T1-weighted MRI contrast agent, Gd-DOTA. The ability of probe 1 to target bacteria was investigated through LC-MS and MRI. The T1 relaxivity of probe 1 was found to be comparable to that of Gd-DOTA; moreover, for both Gram-positive and Gramnegative bacteria, probe 1-treated bacteria generated a significantly brighter T1-weighted MR signal than Gd-DOTA treated bacteria. More importantly, in vitro cellular studies and preliminary in vivo MRI demonstrated that probe 1 exhibits the ability to efficiently target bacteria over macrophage-like cells, indicating its great potential for high-resolution imaging of bacterial infections in vivo.
EXPERIMENTAL SECTION Materials and reagents. All chemicals and solvents were purchased from J&K (Beijing, China). Commercially available reagents were used without further purification. Solvents were purified by conventional methods before use. Cell culture reagents including Dulbecco’s modified Eagle’s medium (DMEM), tryptic soy broth (TSB), Luria-Bertani medium (LB) and foetal bovine serum (FBS) were obtained from BioDee Biotechnology Co. Ltd (Beijing, China). The bacterial strains (Escherichia coli (E. coli) (ATCC 25922), Staphylococcus aureus (S. aureus) (ATCC 29213)) used were pur-
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chased from American Type Culture Collection (ATCC), USA. Instrumentation and characterization. All 1H NMR and 13 C NMR spectra were taken on a Bruker Advance 400 or 600 MHz spectrometer (Bruker Co., Ltd., Germany). LC-MS analysis was performed on a ThermoFisher Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) equipped with a ThermoFisher Accela HPLC system (ThermoFisher Scientific, Bremen, Germany). The purity of compounds was measured with an Alltech HPLC 426 equipped with an evaporative light scattering detector (ESLD). OD values were recorded in a 10 mm path quartz cell on a Metash UV-5100B spectrometer (Metash instrument Co. Ltd, Shanghai, China). MRI experiments were conducted on a Bruker 7 T (Pharmscan 70/16 US, Switzerland). Synthesis of intermediate compounds of probe 1. A partially protected 5”-NH2 neomycin analogue (compound 9) was synthesized according to a previously reported procedure,41 and the protected lipid linker (compound 5) was prepared in four steps starting from 12-amino dodecanoic acid. Compound 15 was synthesized in six steps from cyclen according to modified procedures. The purity of compound 15 was assessed using analytical RP-HPLC with a Kromasil C18 column (250 mm × 4.6 mm, 5 µm particle size). Details of synthesis and characterization of intermediate compounds are described in Supporting Information. Gadolinium complex formation. Either compound 15 or DOTA was dissolved in H2O and the pH was adjusted to 6.57.0 by adding NaOH (0.1 M). GdCl3.6H2O aqueous solution was slowly added in stoichiometric amounts (1:1) and stirred at room temperature. The pH of the solution was periodically checked and maintained at 6.5-7.0 with the addition of NaOH (0.1 M). The reaction was stirred until the pH remained constant for 1 h (4 h reaction time). Upon completion, the solution was then adjusted to pH 9-10 by adding NaOH (0.1 M aq) and the reaction was stirred for an additional 20 min, and then filtered through a 0.45 µm syringe filter. The solution was concentrated to give the crude product, which was purified by RP-HPLC via reversed phase column chromatography (C18 silica, 2-90% MeCN: H2O). The product was lyophilized to afford probe 1 (72 mg, 58%), Gd-DOTA (18 mg, 13%) as white fluffy solids. NMR spectra of these Gd complexes were not recorded because of the paramagnetic properties. The molecular weights of probe 1 and control probe Gd-DOTA were confirmed by ESI-Orbitrap. Probe 1 calcd. for C51H94N12O20Gd: 1352.5943; found: 1352.5944 ([M+H]+). GdDOTA calcd. for C16H26N4O8Gd: 560.0986; found: 560.0986 ([M+H]+). Relaxivity measurements of probe 1 and Gd-DOTA. A Pharmscan 70/16 US (7 T, Bruker, Switzerland) MRI scanner fitted with an RF RES 300 1H 089/072 QSN TR AD volume coil was used at 25 °C. Samples of probe 1 and Gd-DOTA were prepared by dilution of 10 mM stock solutions. For acquisition of T1 relaxation times, a rapid acquisition with refocused echoes (RARE) T1 map pulse sequence was used. The following parameter values were utilized: static echo time = 8 ms; variable repetition time = 200, 400, 800, 1500, 3000, and 5500 ms; field of view = 55 × 55 mm2; matrix size = 256 ×
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Analytical Chemistry 192; number of axial slices = 1; slice thickness = 1.0 mm; and averages = 1. Paravision 6.0 software (Bruker) was used for T1 analysis by monoexponential curve-fitting of image intensities of selected regions of interest (ROIs) from each axial slice. MRI of bacteria labelled with probe 1. S. aureus cells were cultured for 12 h in TSB culture media at 37 °C. Bacterial strains cultured overnight were harvested and washed three times with phosphate-buffered saline (PBS, pH 7.4). The washed cells were resuspended in PBS to an OD600 of 6. Then, 500 µL aliquots in 600 µL Eppendorf tubes were (a) left untreated, (b) treated with 250 µM Gd-DOTA, or (c) treated with 250 µM probe 1. After incubation at 37 °C for 24 h, the cells were washed with PBS by centrifugation (6500 rpm, 3 min) to remove free contrast agents. MR images of the bacterial cell precipitates and suspensions were acquired at 7.0 T. For the acquisition of T1 relaxation times, the parameters and data analysis method used were the same as those mentioned above. Data analysis and determination of the cellular relaxation rate R1, cell were performed from the measured T1 values according to the literature.42 Cytotoxicity test. The cytotoxicity of probe 1 was evaluated according to an approach reported previously. RAW 264.7 cells, 293A cells, rat fibroblast L6 cells and HepG2 cells were seeded on a 96-well plate containing 7500 cells per well in 100 µL of DMEM and incubated overnight before adding the probe. After incubation with different concentration of probe 1 at 37 °C for 48 h, the cells were then incubated with cell culture medium containing 20% MTS (3-(4,5-di-methylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2Htetrazolium, inner salt). After 3 h incubation at 37 °C, the absorbance was measured at 490 nm using a TECAN Spark 10M microplate reader (Männedorf, Switzerland). Cell viabilities at various probe concentrations were given as the percentage of the control sample without probe. Each experiment was repeated three times. The cell survival rate from the control group was considered 100%. Identification of probe 1 in bacteria by LC-MS. Chromatographic separation was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5 µm particle size) connected to an Agilent guard column. A linear gradient from 98% A (0.1% formic acid in water) to 90% B (0.1% formic acid in acetonitrile) in 18 min at a flow rate of 1 mL/min was applied. The mass spectrometric data were collected from m/z 100 to 1500 in positive ion mode. To prevent PBS from entering the mass spectrometer ion source and having a negative effect on the electrospray performance, a switching valve was used to divert the solvent front to waste. Nitrogen was used as the sheath and auxiliary gas with flow rates of 40 psi and 10 L/min, respectively. The data were acquired in full scan MS mode at a resolving power of 35,000. An external calibration for mass accuracy was performed before the test. The other mass parameters were as follows: a capillary temperature at 320 °C, an ion spray voltage of 3.0 kV, a heater temp of 100 °C, an AGC target value of 1×106, and a maximum IT of 50 ms. Preliminary in vivo MRI. MRI was performed using a 7 T Bruker BioSpec whole body MR imager equipped with an RF RES 300 1H 075/040 QSN TR body volume coil. The imaging
parameters for a standard T1 FLASH sequence are as follows: repetition time (TR) = 300 ms, echo time (TE) = 2.5 ms, field of view (FOV) = 40 × 40 mm2, matrix size = 192 × 192, slice thickness = 0.8 mm, averages = 3. Male C57BL/6 mice (n = 6, acquired from Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) were anaesthetized (with isoflurane) and fixed in prostrate position. S. aureus cell suspensions (10 µL, OD600 = 12) prestained with probe 1 or Gd-DOTA were injected into their posterior thighs via intramuscular (IM) administration. Both legs were visualized simultaneously, allowing the comparison of one leg with the other, and the labelling of bacteria with the two contrast agents probe 1 and GdDOTA and their clearance were concurrently monitored over time. For quantitative measurements, signal intensities in specific ROIs were measured using Paravision 6.0 software (Bruker).
RESULTS AND DISCUSSION DISCUSSION Probe design and synthesis. Neomycin is a potent aminoglycoside antibiotic that shows highly specificity for targeting the 30S ribosomal subunit, leading to inhibition of bacterial protein synthesis. In our previous studies, we demonstrated that probes developed by conjugating the Cy 5 fluorophore and neomycin with or without a lipid chain as a linker showed high specificity for labelling bacteria -over mammalian cells. Moreover, the introduction of hydrophobic lipid chains increased bacterial membrane-targeting interactions. Therefore, we hypothesized that the conjugation of neomycin with an FDA-approved T1-weighted MRI contrast agent, Gd-DOTA, via a lipid linker could afford a Gd-based bacteria-specific targeting MRI contrast agent. However, to the best of our knowledge, a strategy to directly conjugate a Gd-based MRI contrast agent with an antibiotic to develop a bacteriatargeting MRI contrast agent has not yet been reported. Scheme 1. Synthesis of probe 1 by conjugating neomycin with MRI contrast agent Gd-DOTA. For a more detailed description of the chemistry, see the Supporting Information.
As shown in Scheme 1 and S1-S3, a partially protected 5”NH2 neomycin analogue (compound 9) was prepared in 4 steps from neomycin. Bn- protected 12-amino dodecanoic acid was synthesized and attached to DO3A-tris-tert-butyl ester (compound 10) affording compound 11. Compound 11 was
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conjugated with compound 9 after deprotecting the Bn- group. After deprotection of all the Boc and tBu groups with TFA, probe 1 was formed by incubation with GdCl3 at pH 6.5-7.0 for 24 h. MRI contrast agent Gd-DOTA was also synthesized as a control probe. (Detailed procedures for their syntheses are provided in the Supporting Information). Relaxivity measurements. The T1 signal enhancement of probe 1 in PBS was measured and compared with that of the FDA approved MRI contrast agent Gd-DOTA. A 10 mM stock solution of probe 1 or Gd-DOTA in PBS (pH = 7.4) was diluted to give six concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mM), and their abilities to decrease the T1 relaxation time were investigated (Figure 1). To obtain quantitative information about the efficiency of probe 1 and Gd-DOTA as MRI contrast agents, we determined their molar longitudinal relaxivity (r1) at 25 °C and 7.0 T using the inversion-recovery method.24 The longitudinal relaxivity value r1 found for probe 1 (4.1 mM–1 s−1) was comparable to that of Gd-DOTA (r1 = 3.9 mM–1 s−1, T1 = 828 ms, 0.2 mM). In vitro relaxivity studies indicated probe 1 as a potential MRI contrast agent that can effectively enhance the T1 signal (T1 = 804 ms, 0.2 mM) by reducing the bulk water T1 (T1 = 2303 ms) by 65%. In vitro, probe 1 and Gd-DOTA were stable at pH 6-8 with no detectable dissociation after 24 h of incubation in PBS.
Figure 1. Longitudinal relaxivity (r1) of probe 1 and Gd-DOTA in PBS (pH = 7.4) measured at 300 MHz, 25 °C and 7.0 T using the inversion-recovery method.
Bacteria-targeting LC-MS analysis of the uptake of probe 1 by bacteria. The permeability properties of the bacterial cell membrane are essential for developing bacteriatargeting MRI contrast agents. Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of compounds; however, the outer membrane of Gramnegative bacteria has a crucial role in providing an extra layer of protection to the organism without compromising the exchange of material required for sustaining life. Aminoglycosides can penetrate both aerobic Gram-positive and Gramnegative bacteria to exert their effects. Aerobic Gram-negative bacteria actively pump aminoglycosides into the cells, which is initiated by an oxygen-dependent interaction between the cationic antibiotic and anionic bacterial membrane lipopolysaccharides. Previous studies have shown that the modification
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of neomycin at 5”-OH will not cause a loss of activity;41 therefore, we inferred that neomycin conjugate probe 1 could be internalized by both aerobic Gram-positive and Gram-negative bacteria and accumulate inside these cells by binding with ribosomes and the membrane-bound respiratory chain. S. aureus, and E. coli. were used as examples of Gram-positive and Gram-negative bacterial strains, respectively. The bacteriatargeting properties of probe 1 were first investigated by LCMS analysis. S. aureus and E. coli. were each incubated with probe 1 (250 µM) for 24 h. The bacterial cells were washed 3 times to remove unbound probe and were subsequently broken up by ultrasonication. After centrifugation, the supernatant was analyzed by LC-MS. As shown in Figure 2, a peak at 5.47 min (m/z 451.5, [M + 3H]3+; m/z 338.9, [M + 4H]4+) was detected in the supernatant, which clearly demonstrates the presence of probe 1. The LC-MS analysis results revealed that probe 1 could bind to bacterial cells, which makes it a potential MRI contrast agent for bacterial imaging. We believe that the bacterial uptake mechanisms of probe 1 are driven by the active uptake of neomycin; however, the exact uptake mechanism of probe 1 remains to be elucidated further.
Figure 2. HPLC-MS analysis of S. aureus supernatant after incubation with probe 1. a) Extracted ion chromatograph (EIC) of the supernatant in the molecular weight range of 451.00-452.00; b) Mass spectrum of the peak at 5.47 min.
MRI bacteria incubated with probe 1. To evaluate the ability of the probe 1 to image bacteria, we investigated bacterial cells by using MRI and employing Gd-DOTA as a reference. In these cellular experiments, S. aureus cells were treated for 24 h with 250 µM probe 1 or Gd-DOTA, then were washed to remove unbound complex and finally were centrifuged at high speed to obtain the precipitates. Then, MR images of the bacterial precipitates were acquired. As shown in Figure 3a, the images obtained from the bacterial precipitates (n = 5) showed an unambiguous enhancement in signal when using probe 1 compared to that when using Gd-DOTA (T1: 92 ± 2 ms vs 851 ± 20 ms). The relaxation rates R1, cell of the S. aureus cells incubated with probe 1 were 10-fold greater than those of untreated bacterial cells, whereas the R1, cell of control Gd-DOTA treated S. aureus cells showed almost no difference from that of untreated cells (Figure 3b). The level of signal
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Analytical Chemistry enhancement demonstrates the high effectiveness of the bacteria-targeting ability of probe 1 for the visualization by MRI. Additionally, bacterial suspensions were also measured through rinse and re-suspension in fresh buffer after incubation (Supporting Information Figure S2, S5). In T1-weighted MR images, S. aureus cells labelled with probe 1 exhibited greater contrast enhancement, and were significantly brighter than S. aureus cells treated with Gd-DOTA, Gd-DOTA + Neo (the mixture of neomycin and Gd-DOTA, 1:1) or untreated bacterial cells (Supporting Information Figure S2A, S5A). As shown in Supporting Information Figure S2B and S5B, the relaxation rate R1, cell of the suspension of probe 1-treated S. aureus cells was 144% greater than that of the unlabelled cell suspension, while Gd-DOTA- or Gd-DOTA + Neo- treated cell suspension was indistinguishable from unlabelled cell suspension, which was consistent with the images in Figure 3. Similar studies were also conducted with E. coli. as a representative Gram-negative bacteria. In these studies, E. coli cells labelled with probe 1 (Supporting Information Figure S1-S2) showed a significant signal enhancement compared to GdDOTA-treated or untreated bacterial cells. The level of signal enhancement demonstrates the high effectiveness of the bacteria-targeting ability of probe 1 for visualization by MRI.
Figure 3. (a) T1-weighted MR images of untreated (control), GdDOTA-treated or probe 1-treated bacterial cells (red circle: bacterial cells). Images were recorded using a RARE pulse sequence with a matrix of 256 × 256 voxels over an FOV of 55 × 55 mm2, slice thickness of 0.5 mm, TR = 400 ms, TE = 8 ms, flip angle = 90°, and NEX = 4. (b) Cellular relaxation rates R1, cell (mean ± standard deviation, n = 5) of untreated (control), Gd-DOTAtreated or probe 1-treated bacterial cells. (***P < 0.001, ns: not significant vs. Control; ***P < 0.001 vs. [Gd-DOTA].)
Specific detection of pathogens has long been recognized as a vital strategy in the control of infectious diseases. A crucial challenge in imaging bacteria is to develop MRI contrast agents with high specificity for bacteria. To determine the
bacterial specificity of probe 1 , a mouse leukemic monocyte macrophage cell line (RAW 264.7) was chosen to mimic the macrophages in infected tissue. The cell morphology or viability did not change significantly during the imaging process. In addition, no signal enhancement could be observed from probe 1-treated macrophage-like cells after removing unbound complex (Supporting Information Figure S7-S8). Cellular MRI studies indicated that probe 1 may enable a clear distinction between bacterial infection and sterile inflammation. Cytotoxicity studies of probe 1. The cytotoxicities of probe 1, neomycin and Gd-DOTA were evaluated using standard MTS assays with RAW 264.7 cells, rat fibroblast L6 cells, 293A cells and HepG2 cells. The viability of the cells showed no significant change, and approximately 80% of the cells survived even in the presence of 500 µM probe 1. The results suggested that probe 1 has low cytotoxicity and good biocompatibility. Preliminary in vivo MR imaging. MRI is the non-invasive tool most commonly used in vivo to detect inflammatory diseases. However, it is unable to differentiate bacterial infections from each other or from sterile inflammation with current clinical non-targeted MRI contrast agents. Enhancement of the MRI contrast with bacteria-targeting contrast agent probe 1 would improve the diagnosis of bacterial infections. The bacteria-targeting abilities of the contrast agents were visualized and estimated by injecting probe 1-and Gd-DOTA-prestained S. aureus cells into each of leg muscles of the same mouse, allowing direct comparison of the labelling and clearance behavior of the two different contrast agents over time without background due to interanimal differences. Figure 4a shows representative images obtained before, immediately after, and 2 h and 4 h after IM injection of S. aureus cells prestained with one of the contrast agents. A significant MRI signal enhancement was detected in the probe 1-prestained bacteria. Compared with Gd-DOTA-prestained bacteria, the signal intensities (SIs) increased 53% (***P
