Verena Hoerr

A Novel Mouse Model of Staphylococcus aureus Chronic Osteomyelitis That Closely Mimics the Human Infection: An Integrated View of Disease Pathogenesis

Osteomyelitis is a serious bone infection typically caused by Staphylococcus aureus. The pathogenesis of osteomyelitis remains poorly understood, mainly for lack of experimental models that closely mimic human disease. We describe a novel murine model of metastatic chronic osteomyelitis initiated after intravenous inoculation of S. aureus microorganisms. The bacteria entered bones through the bloodstream and, after an acute phase with progressive growth (first 2 weeks after infection), they remained at constant numbers for up to 56 days (chronic phase). Clinical signs of illness and systemic inflammation were apparent only during the acute phase. Bone destruction and remodeling processes were readily detectable by magnetic resonance and X-ray imaging 3 weeks after infection, and high levels of bone deformation were observed during the chronic phase. Histological examination of infected bones demonstrated suppurative inflammation with foci of intense bacterial multiplication and necrosis during acute infection and osteoclastic resorption accompanied by new woven bone formation during chronic infection. Transmission electron microscopy revealed S. aureus microorganisms forming microcolonies within the nonmineralized collagen matrix or located intracellularly within neutrophils. In summary, our mouse model of staphylococcal hematogenous osteomyelitis precisely reproduces most features of the human disease. Although the extent of lesions in the chronic phase was subject to variation, this model is ideal for testing and monitoring novel treatment modalities via noninvasive imaging.

Bacteria tracking by in vivo magnetic resonance imaging

BackgroundDifferent 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.ResultsWe 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.ConclusionLabeled 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.

Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

Tang et al. report that dietary restriction increases hematopoietic stem cell quiescence and their repopulation capacity during aging but impairs hematopoietic stem cell differentiation into lymphoid lineages and inhibits proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function.

Characterization and prediction of the mechanism of action of antibiotics through NMR metabolomics

BackgroundThe emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs.ResultsIn an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted.ConclusionThe metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.

Diabetic db/db mice do not develop heart failure upon pressure overload:A longitudinal in vivo PET, MRI, and MRS study on cardiac metabolic, structural, and functional adaptations

Aims Heart failure is associated with altered myocardial substrate metabolism and impaired cardiac energetics. Comorbidities like diabetes may influence the metabolic adaptations during heart failure development. We quantified to what extent changes in substrate preference, lipid accumulation, and energy status predict the longitudinal development of hypertrophy and failure in the non-diabetic and the diabetic heart. Methods and results Transverse aortic constriction (TAC) was performed in non-diabetic (db/+) and diabetic (db/db) mice to induce pressure overload. Magnetic resonance imaging, 31P magnetic resonance spectroscopy (MRS), 1H MRS, and 18F-fluorodeoxyglucose-positron emission tomography (PET) were applied to measure cardiac function, energy status, lipid content, and glucose uptake, respectively. In vivo measurements were complemented with ex vivo techniques of high-resolution respirometry, proteomics, and western blotting to elucidate the underlying molecular pathways. In non-diabetic mice, TAC induced progressive cardiac hypertrophy and dysfunction, which correlated with increased protein kinase D-1 (PKD1) phosphorylation and increased glucose uptake. These changes in glucose utilization preceded a reduction in cardiac energy status. At baseline, compared with non-diabetic mice, diabetic mice showed normal cardiac function, higher lipid content and mitochondrial capacity for fatty acid oxidation, and lower PKD1 phosphorylation, glucose uptake, and energetics. Interestingly, TAC affected cardiac function only mildly in diabetic mice, which was accompanied by normalization of phosphorylated PKD1, glucose uptake, and cardiac energy status. Conclusion The cardiac metabolic adaptations in diabetic mice seem to prevent the heart from failing upon pressure overload, suggesting that restoring the balance between glucose and fatty acid utilization is beneficial for cardiac function.

In vivo visualization of single native pancreatic islets in the mouse

The purpose of this study was to investigate the potential of a novel targeted contrast agent (CA) for the in vivo visualization of single native pancreatic islets, the sites of insulin production, in the pancreas of mice using magnetic resonance imaging (MRI). The CA for intravenous administration was composed of the β-cell-specific single-chain antibody fragment, SCA B1, and ferromagnetic carbon-coated cobalt nanoparticles. MRI experiments were performed at 7, 9.4 and 16.4 T in excised organs (pancreas, liver, kidney, spleen), at 7 T in mice fixed in formalin and at 9.4 and 16.4 T in living mice. Image contrast in untreated control animals was compared with images from mice treated with unspecific and specific CA. For the validation of MRI results, selected pancreases were subjected to immunohistochemical staining and numerical contrast simulations were performed. Ex vivo results and the outcome of immunohistochemistry suggest that islets are marked only by the CA containing SCA B1. Strong accumulation of particles was found also in other investigated organs owing to the uptake by the reticuloendothelial system, but the contrast in the MR images is clearly distinguishable from the islet specific contrast in pancreases and numerical predictions. In vivo experiments based on averaged dynamic sampling with 66 × 66 × 100 µm³ and triggered acquisition with 90 × 90 × 200 µm³ nominal resolution resulted in similar particle contrast to in in vitro measurements. The newly developed CA and MRI strategies have the potential to be used for studying mouse diabetes models by visualizing single native pancreatic islets.

NMR Separation of Intra- and Extracellular Compounds Based on Intermolecular Coherences

NMR spectroscopy is a powerful tool for detection and characterization of chemical compounds in biological systems. Its application in pharmaceutical studies in cell cultures, however, has been hampered by the enormous technical challenges in separating intra- from extracellular amounts of one substance. We introduce a novel approach to separate intra- from extracellular NMR signal based on the detection of intermolecular zero-quantum coherences in presence of a chemical shift agent. In a sample of large cells in culture, the investigation of cellular uptake of pharmacological substances becomes feasible. The addition of 10 mM Tm-DOTP to a suspension of 100 Xenopus laevis oocytes resulted in sufficient separation of resonance frequencies between intra- and extracellular water. Upon selective excitation of either intra- or extracellular water signal, only intra- or extracellular components were observed, respectively. The presented localization technique provides intrinsic averaging over a large number of cells, resulting in a significant signal gain. The method works on standard NMR spectrometers, which are available at most scientific research institutions today. On a high-resolution NMR system with a cryoprobe, a 20-fold sensitivity gain was observed as compared to conventionally localized NMR spectroscopy of a single X. laevis oocyte on dedicated NMR microscopes.

MRI visualization of Staphyloccocus aureus-induced infective endocarditis in mice.

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.

Magnetic resonance imaging characterization of microbial infections

The investigation of microbial infections relies to a large part on animal models of infection, if host pathogen interactions or the host response are considered. Especially for the assessment of novel therapeutic agents, animal models are required. Non-invasive imaging methods to study such models have gained increasing importance over the recent years. In particular, magnetic resonance imaging (MRI) affords a variety of diagnostic options, and can be used for longitudinal studies. In this review, we introduce the most important MRI modalities that show how MRI has been used for the investigation of animal models of infection previously and how it may be applied in the future.

S. aureus endocarditis: Clinical aspects and experimental approaches

Infective endocarditis (IE) is a life-threatening disease, caused by septic vegetations and inflammatory foci on the surface of the endothelium and the valves. Due to its complex and often indecisive presentation the mortality rate is still about 30%. Most frequently bacterial microorganisms entering the bloodstream are the underlying origin of the intracardiac infection. While the disease was primarily restricted to younger patients suffering from rheumatic heart streptococci infections, new at risk categories for Staphylococcus (S.) aureus infections arose over the last years. Rising patient age, increasing drug resistance, intensive treatment conditions such as renal hemodialysis, immunosuppression and long term indwelling central venous catheters but also the application of modern cardiac device implants and valve prosthesis have led to emerging incidences of S. aureus IE in health care settings and community. The aetiologic change has impact on the pathophysiology of IE, the clinical presentation and the overall patient management. Despite intensive research on appropriate in vitro and in vivo models of IE and gained knowledge about the fundamental mechanisms in the formation of bacterial vegetations and extracardiac complications, improved understanding of relevant bacterial virulence factors and triggered host immune responses is required to help developing novel antipathogenic treatment strategies and pathogen specific diagnostic markers. In this review, we summarize and discuss the two main areas affected by the changing patient demographics and provide first, recent knowledge about the pathogenic strategies of S. aureus in the induction of IE, including available experimental models of IE used to study host-pathogen interactions and diagnostic and therapeutic targets. In a second focus we present diagnostic (imaging) regimens for patients with S. aureus IE according to current guidelines as well as treatment strategies and surgical recommendations.