In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation

Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Nature medicine (Impact Factor: 27.36). 08/2011; 17(9):1142-6. DOI: 10.1038/nm.2423
Source: PubMed


Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

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    • "Although cardiac masses in the human heart are detectable by MRI, a specific MR diagnosis of bacteria-induced endocarditis is often not possible, since contrast-enhanced MR for detection of bacteria has not yet been developed. In experimental preclinical imaging approaches, optical and nuclear techniques have succeeded in imaging IE in mice [10]–[12]. However, MR has only been reported to be capable of imaging infiltration of immune cells in response to bacterial infections of other organs [13]–[19]. "
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    ABSTRACT: Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy.
    PLoS ONE 09/2014; 9(9):e107179. DOI:10.1371/journal.pone.0107179 · 3.23 Impact Factor
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    • "Non-invasive in vivo imaging techniques, such as magnetic resonance imaging (MRI), computed tomography (CT), fluorescence/bioluminescence imaging (BLI) or positron emission tomography (PET), have been employed to visualize infections over the time course [8-12]. Although different strategies for specific detection of bacteria have been developed [13-15], most imaging studies target host factors [16] or detect immune cells [17]. Due to its high spatial resolution and excellent soft tissue contrast, especially MRI is a versatile method to image inflammatory processes upon bacterial infections non-invasively. "
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    ABSTRACT: Background Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria. Results We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response. Conclusion Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.
    BMC Biology 05/2013; 11(1):63. DOI:10.1186/1741-7007-11-63 · 7.98 Impact Factor
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    • "One study found that maltodextrin-based imaging probes (MDPs), which are composed of a fluorescent dye conjugated to maltohexaose and are internalized by bacteria via a bacterial-specific maltodextrin transport pathway, can be used in combination with similar noninvasive fluorescent in vivo imaging to detect bacteria during a thigh infection in vivo [31]. Another study found that fluorescent-labeled prothrombin derivatives that bind to staphylocoagulase in combination with fluorescence molecular tomography fused to X-ray computed tomography (FMT-CT) could be used to detect the presence of a S. aureus infection in endocarditic vegetations [32]. Our model provides an opportunity to evaluate whether these or other bacterial-specific tracers and probes could identify the presence of the infection/biofilms on the metal implants, which may lead to the development of new and accurate diagnostic methods to detect the presence of an orthopaedic implant infection in humans. "
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    ABSTRACT: Recent advances in non-invasive optical, radiographic and μCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure. An orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×10(3) CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and μCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with μCT images. To quantify the anatomical bone changes on the μCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection. Bioluminescent and fluorescent optical imaging was combined with X-ray and μCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.
    PLoS ONE 10/2012; 7(10):e47397. DOI:10.1371/journal.pone.0047397 · 3.23 Impact Factor
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