Fluorescent imaging of bacterial infections and recent advances made with multimodal radiopharmaceuticals
Today, both radioactive SPECT and PET imaging radiopharmaceuticals are used for clinical diagnosis of bacterial infections. Due to the possible applications in image-guided surgery, fluorescent imaging of infections has gained interest. We here present the highlights and recent developments in the use of fluorescence imaging for bacterial infections. In this overview, we also include the latest developments in multimodal bacterial imaging strategies that combine radioactive and fluorescent imaging. Based on this literature, we present our future perspectives for the field including the translational potential.
In the current review, we complement earlier reports with the most recent fluorescent and multimodal radiopharmaceuticals for bacterial infection imaging. Where possible, in this review, the chemical structure of the compounds and clinical images were shown.
A total of 35 out of 77 original articles on pre-clinical and clinical imaging of bacterial infections with fluorescent tracers and multimodality radiopharmaceuticals were included for reviewing.
In our view, the highest translational potential lies with compounds that are based on targeting vectors that are specific for bacteria: e.g., fluorescently labelled UBI29–41, polymyxin B, vancomycin, ZnDPA and a M. tuberculosis-specific β-lactamase-cleavable linker CNIP800. Multimodal concepts using dually labelled UBI29–41, vancomycin, and ZnDPA help connect optical imaging to the more traditional use of radiopharmaceuticals in infectious diseases. Multimodal bacterial imaging is a promising strategy not only to diagnose bacterial infections but also to evaluate the effectivity of surgical treatment for infections.
KeywordsBacterial infection Fluorescence imaging Molecular imaging Radioactivity Infectious diseases
Class of tracers
Imaging achievements in animals
Clinical evaluation and testing in ex vivo patient material
Negatively charged lipopolysaccharide residues on the outer membrane
Imaging infections with both Gram-positive and Gram-negative bacteria, apoptosis and tumours
Antimicrobial peptides 
Negatively charged residues and receptors on the outer membrane, intracellular targets (mitochondria, DNA)
Imaging infections with Gram-positive and Gram-negative bacteria, discrimination between infection and sterile inflammation
Cell wall synthesis, DNA and/or protein synthesis
Bacterial labelling, imaging of infections selectively with Gram-positive bacteria (vancomycin)
Vanco-IRDye800CW in ex vivo patient material
Discrimination of bacterial infections from sterile inflammation in mice
Building blocks for replication, membrane synthesis and energy source
Discrimination of infections from sterile inflammation, selective imaging of Gram-positive or Gram-negative bacteria
β-Lactamase, protease, sortase
Detection of intracellular bacteria
Coagulase activity with prothrombin, lysostaphin lectin binding
Coagulase-positive S. aureus bacteria detected in endocarditis, S. aureus in bloodstream
In the current review, we complement earlier reports [6, 7, 12, 13] with the most recent fluorescent and multimodal radiopharmaceuticals for bacterial infection imaging. Where possible, the chemical structure of the compounds and clinical images were shown. In addition, we provide some future perspectives for the field.
Imaging endogenous bacterial fluorescence
The authors illustrated the potential benefits of using this technique in open wounds, where it may assist the clinicians in confirmation whether a wound is infected at the bedside, accurate sampling of the wounds, and in treatment monitoring . This detection method, despite having the advantage of being independent of the use of exogenous tracers, is limited to bacteria that produces fluorescent molecules on the surface and subsurface of the skin. The detection limit of this method mainly depends on the number of bacteria present in the wounds. A potentially valuable secondary side effect of this technology could be that the UV light used to excite the endogenous fluorophores, potentially applies photodynamic therapy to the bacteria .
Imaging bacteria with ZnDPA
Imaging infections with fluorescent antimicrobial peptides
Although the autofluorescence of background tissue disturbed the sensitivity of imaging and increased the detection limit, initial results were promising for imaging bacteria in vivo. Another issue is the chance of proteolysis and oxidation of peptide-based tracers in vivo, and to reduce this complication, a cyclic analogue of the UBI peptide was included in the study as well (Fig. 4c). The imaging results were comparable with the linear variant of the tracer (Fig. 4b, d).
Imaging infections with antibiotics
The use of fluorescein-labelled vancomycin is limited by the inability to detect deep tissue infections because of scattering and absorption of photons by tissue. To overcome this limitation, a hybrid vancomycin-based radiopharmaceutical was introduced that contained the fluorescent dye rhodamine B as well as the radioisotope 125I (creating 125I-Rho-vancomycin) (Fig. 7d) . Mice were infected in the right thigh muscle with methicillin-resistant S. aureus (MRSA), whereas the contralateral thigh muscle was infected with E. coli reflecting the bias in labelling Gram-positive bacteria. Within 2 h after injection, 125I-Rho-vancomycin showed an 8.7-fold higher accumulation in MRSA-infected thigh muscles than in muscles infected with E. coli. (Fig. 7e). Fluorescence imaging revealed a 3.9-fold increase in uptake in MRSA-infected thigh muscles with 125I-Rho-vancomycin compared to control tissue (Fig. 7f). Imaging of pulmonary infections by MRSA with 125I-Rho-vancomycin yielded about 8.9- to 13.3-fold higher lung-to-background ratios than a control radiopharmaceutical (non-cell binding variant of 125I-Rho-vancomycin). This study underlines that 125I-Rho-vancomycin allows accumulation on bacterial membranes of Gram-positive bacteria.
Imaging bacteria with enzyme-activated tracers
Recently, activatable fluorescent tracers show a promising development that allows fast and specific testing of the metabolic processes of specific strains of bacteria . These tracers allow detection of bacteria expressing nuclease, reductase or hydrolyse activities.
The advantage of enzyme-activatable tracers is that it has potency in imaging of infections with drug-resistant bacteria .
Imaging of bacterial proteins
Bacteria cell walls and membranes contains various proteins that are essential for maintenance of their external structure and integrity. As some of these proteins are solely expressed by bacteria, they are of interest in targeting by fluorescent tracers.
The carbohydrate-binding protein (lectin) concanavalin A, has a high affinity for mannose residues present in bacterial cell walls. The plant-derived lectin concanavalin A binds α-d-mannosyl and α-d-glucosyl residues of B-glycans. When concanavalin A was conjugated to poly(N-isopropylacrylamide microspheres PNIPAM-co-St which were functionalised with the NIR dye IR750 (chemical structure unknown) , this compound has shown to allow for imaging in a murine wound and catheter infection model with S. aureus. Following topical application and washing in live mice, the tracer rapidly displayed the presence of bacteria in the wounds and catheters already from 2 × 106 viable bacteria with the infection-to-background ratios ranging between 2 and 5. Unfortunately, concanavalin A interacts with all mannose-containing proteins and receptors,  e.g., those present in erythrocytes and various cancer cells [54, 55].
Imaging of bacterial metabolic activity
Other bacteria-imaging tracers
Discussion and future perspectives
Differentiation between infectious and non-infectious causes of inflammatory processes is of crucial importance for clinicians. For this purpose, progress has been directed towards specific targeting of pathogens. In the current review, we updated earlier reviews with new findings on fluorescent and even multimodal radiopharmaceuticals for imaging of bacterial infections. Fluorescence imaging alone holds particular promise for the inspection of human body surfaces of, e.g., surgical wounds [12, 13]. Hybrid radiopharmaceutical analogues, on the other hand, provide outcome and help connect fluorescence tracers with their translationally more advanced radionuclear counterparts. With hybrid radiopharmaceuticals, the radioactive imaging is the best total body-imaging modality for detecting and localizing deep infections, whereas fluorescence imaging allows real-time imaging and has a high spatial resolution [61, 62]. This innovative concept has already been applied in oncological surgery where it provides full-body nuclear imaging to roughly locate the lesions and aids the surgeon during inspection of the primary tumour and searching for microscopical metastasis guided by a fluorescent signal [63, 64, 65, 66, 67]. In imaging of infections, there is a need to combine nuclear and optical imaging radiopharmaceuticals as well as ideally, one single dual-modal compound could be administered to (i) assess total disease and infection burden using nuclear imaging techniques, followed by (ii) identification of the extent of the infection, and prevention of sampling errors. For example, in wound and trauma surgery, infections often occurs and in this respect, the optical modality can be used to visualize the localization of the bacteria and thus facilitates complete resection during surgical debridement of infected areas. A similar approach is feasible, after a surgical intervention and inspection, persistent infections of transplants can be detected using the visual signal. Such a dual-modal compound could be especially attractive to minimize patient burden and healthcare costs; as imaging is immediately followed by image-guided surgery within a short time frame, it can accelerate improvement of health status and costs on healthcare. Recent data shows that bacterial imaging is slowly finding its way into ex vivo human applications. As this type of progression is similar to what has been observed for infection-specific radiopharmaceuticals , this illustrates that fluorescence-based bacterial imaging is also steadily moving towards medical implementation and can ultimately be used for guiding surgical interventions.
Tissue attenuation, e.g., absorption of scattering of the light is a restrictive factor during the pharmacological evaluation of fluorescent tracers. Although it is known that fluorescent labels can influence tracer pharmacokinetics [69, 70], this effect cannot be quantified without having a radioisotope for quantitative imaging/biodistribution assessments.
Next to the traditional use of organic dyes, inorganic dyes may also be used in the future as these may differ in pharmacokinetics or the number of attached reporter groups. The introduction of inorganic medicinal chemistry is making rapid progress, with enormous impact, e.g., with platinum-based compounds in antitumor chemotherapy, and iron oxides and gadolinium(III) compounds in MRI contrast agents for noninvasive diagnostics . Several transition metal complexes (in particular those with ruthenium(II)) have been explored for targeted fluorescent imaging , including lifetime imaging [73-76]. Uniquely, these inorganic dyes may also support theranostic applications due to their ability to create reactive singlet oxygen [75, 77]. Within the context of multimodal imaging, it is interesting to note that transition metal ions such as ruthenium can also be replaced with radioactive isotopes, e.g., 97Ru , further extending the scope of inorganic medicinal compounds in which, uniquely, one and the same metal ion could act as the photophysically and/or photochemically active centre, as well as the radioactive nuclide for tracing or therapy. Importantly, combining multiple properties into a single molecular (sub)unit, dramatically alleviates the negative effects of multiple bulky molecular sub-units on targeting and uptake properties.
Developments in imaging of bacterial infections with fluorescent, radioactive  or multimodality radiopharmaceuticals are still in progress and besides generic tracers that can image the site of infections, improvements in sensitivity were also made that allows to assess the bacterial burden. Also, imaging of specific bacterial strains can be very helpful as a tool for imaging of pathogens in environments which colonized with other bacteria as in the gut or the lungs. In our view, the highest translational potential lies with tracers that are based on targeting vectors that are specific for bacteria: e.g., fluorescently labelled UBI29–41, polymyxin B, vancomycin, ZnDPA and a M. tuberculosis-specific β-lactamase-cleavable linker CNIP800. Multimodal concepts using dually labelled UBI29–41, vancomycin, and ZnDPA help connect optical imaging to the more traditional use of radiopharmaceuticals in infectious diseases. Multimodal bacterial imaging is a promising strategy not only to diagnose bacterial infections but also to evaluate the effectivity of surgical treatment for infections. Although the recent developments are promising regarding imaging of specific bacterial species (Gram-positive or Gram-negative strains), it must be considered that bacterial imaging tracers as we described in this review cannot discriminate between antibiotic-sensitive or -resistant bacterial strains. Therefore, further research in exploiting molecular targets may support the discrimination between bacterial species and those that are unique to resistance properties.
Altogether, pre-clinical development and evaluation of dual-labelled bacteria imaging radiopharmaceuticals for SPECT/PET and optical imaging is still in progress and the first results shows great promise, but as for every new compound, further studies concerning specificity, sensitivity, and safety assessments including toxicity and dosimetry are required.
The research leading to these results was funded with grants from the Netherlands Organization for Scientific Research (VIDI Grant—STW BGT11272 to F. W. B. van Leeuwen and 864.10.003 to Wiep Klaas Smits), Meta Roestenberg was supported by a VENI Grant from ZONMW and a Gisela Thier fellowship from the LUMC.
Compliance with ethical standards
Conflict of interest
The authors have declared that no competing interest exists.
In the selected clinical literature manuscripts were only included when all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008.
Informed consent was obtained from all patients for being included in these studies.
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