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• W2574165910 abstract "Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and 18F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development. Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and 18F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development. With the increasing prevalence of cardiovascular diseases, the application of vascular grafts has increased substantially during the last decade. However, these vascular grafts do not come without complications because they are accompanied with a 1% to 6% chance of infection.1Hasse B. Husmann L. Zinkernagel A. Weber R. Lachat M. Mayer D. Vascular graft infections.Swiss Med Wkly. 2013; 143: w13754PubMed Google Scholar, 2Erb S. Sidler J.A. Elzi L. Gurke L. Battegay M. Widmer A.F. Weisser M. Surgical and antimicrobial treatment of prosthetic vascular graft infections at different surgical sites: a retrospective study of treatment outcomes.PLoS One. 2014; 9: e112947Crossref PubMed Scopus (59) Google Scholar, 3Legout L. D'Elia P.V. Sarraz-Bournet B. Haulon S. Meybeck A. Senneville E. Leroy O. Diagnosis and management of prosthetic vascular graft infections.Med Maladies Infect. 2012; 42: 102-109Crossref PubMed Scopus (57) Google Scholar, 4Sousa J.V. Antunes L. Mendes C. Marinho A. Gonçalves A. Gonçalves O. Matos A. Prosthetic vascular graft infections: a center experience.Angiol Cirurgia Vasc. 2014; 10: 52-57Crossref Google Scholar These infections are associated with mortality rates of >20%, making them very serious complications.1Hasse B. Husmann L. Zinkernagel A. Weber R. Lachat M. Mayer D. Vascular graft infections.Swiss Med Wkly. 2013; 143: w13754PubMed Google Scholar Staphylococcus aureus is one of the leading causative agents because it readily adheres to the surface of the implanted device and forms thick biofilm layers. These biofilm layers are difficult to detect because they are often unaccompanied by clinical symptoms and are almost impossible to eradicate, even with long-term antibiotic treatment.5Otto M. Staphylococcal biofilms.Curr Top Microbiol Immunol. 2008; 322: 207-228Crossref PubMed Scopus (754) Google Scholar, 6Archer N.K. Mazaitis M.J. Costerton J.W. Leid J.G. Powers M.E. Shirtliff M.E. Staphylococcus aureus biofilms: properties, regulation and roles in human disease.Virulence. 2011; 2: 445-459Crossref PubMed Scopus (619) Google Scholar Within these biofilms the bacteria are protected from host immune responses and antibiotic treatment, using multifactorial mechanisms that are not yet completely unraveled. The clinical consequence is that antibiotic levels up to 1000 times higher than for planktonic bacteria are necessary and these concentrations are not clinically feasible.7McCarthy H. Rudkin J.K. Black N.S. Gallagher L. O'Neill E. O'Gara J.P. Methicillin resistance and the biofilm phenotype in Staphylococcus aureus.Front Cell Infect Microbiol. 2015; 5: 1Crossref PubMed Scopus (247) Google Scholar, 8Singh R. Ray P. Das A. Sharma M. Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms.J Antimicrob Chemother. 2010; 65: 1955-1958Crossref PubMed Scopus (279) Google Scholar, 9Ceri H. Olson M.E. Stremick C. Read R.R. Morck D. Buret A. The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.J Clin Microbiol. 1999; 37: 1771-1776Crossref PubMed Google Scholar, 10Penesyan A. Gillings M. Paulsen I.T. Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities.Molecules. 2015; 20: 5286-5298Crossref PubMed Scopus (233) Google Scholar Much research is being performed to discover ways to prevent the bacteria from adhering to the surface in the first place or to find better delivery methods that would enable antibiotics to eradicate the bacteria hidden in biofilm.11Bisdas T. Beckmann E. Marsch G. Burgwitz K. Wilhelmi M. Kuehn C. Haverich A. Teebken O.E. Prevention of vascular graft infections with antibiotic graft impregnation prior to implantation: in vitro comparison between daptomycin, rifampin and nebacetin.Eur J Vasc Endovasc Surg. 2012; 43: 448-456Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 12Cirioni O. Mocchegiani F. Ghiselli R. Silvestri C. Gabrielli E. Marchionni E. Orlando F. Nicolini D. Risaliti A. Giacometti A. Daptomycin and rifampin alone and in combination prevent vascular graft biofilm formation and emergence of antibiotic resistance in a subcutaneous rat pouch model of staphylococcal infection.Eur J Vasc Endovasc Surg. 2010; 40: 817-822Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 13Schneider F. O'Connor S. Becquemin J.P. Efficacy of collagen silver-coated polyester and Rifampin-soaked vascular grafts to resist infection from MRSA and Escherichia coli in a dog model.Ann Vasc Surg. 2008; 22: 815-821Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 14Kuehn C. Graf K. Mashaqi B. Pichlmaier M. Heuer W. Hilfiker A. Stiesch M. Chaberny I.F. Haverich A. Prevention of early vascular graft infection using regional antibiotic release.J Surg Res. 2010; 164: e185-e191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 15Keeling W.B. Myers A.R. Stone P.A. Heller L. Widen R. Back M.R. Johnson B.L. Bandyk D.F. Shames M.L. Regional antibiotic delivery for the treatment of experimental prosthetic graft infections.J Surg Res. 2009; 157: 223-226Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 16Lehar S.M. Pillow T. Xu M. Staben L. Kajihara K.K. Vandlen R. DePalatis L. Raab H. Hazenbos W.L. Morisaki J.H. Kim J. Park S. Darwish M. Lee B. Hernandez H. Loyet K.M. Lupardus P. Fong R. Yan D. Chalouni C. Luis E. Khalfin Y. Plise E. Cheong J. Lyssikatos J.P. Strandh M. Koefoed K. Andersen P.S. Flygare J.A. Tan M.W. Brown E.J. Mariathasan S. Novel antibody–antibiotic conjugate eliminates intracellular S. aureus.Nature. 2015; 527: 323-328Crossref PubMed Scopus (502) Google Scholar However, progress has been limited, not least because of the lack of adequate in vivo models. Because these biofilms form a prime source for chronic recurrent infections and there is no curative treatment, the implanted device usually needs to be surgically removed, which involves additional invasive interventions for the patient and high costs for the health care system. Thus, it is of high importance to understand the underlying pathogenesis of biofilm formation on vascular grafts within the whole organism to find quick and effective treatment possibilities without having to resort to invasive procedures. To achieve this, it is necessary to have a representative animal model that closely reflects the long-term process of biofilm formation in vivo. Many in vitro models have been set up to study biofilm formation. These models have provided useful information on the genes necessary for biofilm formation and the mechanisms by which a biofilm is maintained.9Ceri H. Olson M.E. Stremick C. Read R.R. Morck D. Buret A. The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.J Clin Microbiol. 1999; 37: 1771-1776Crossref PubMed Google Scholar, 17Merritt J.H. Kadouri D.E. O'Toole G.A. Growing and analyzing static biofilms.Curr Protoc Microbiol. 2005; Chapter 1: Unit-1B.1PubMed Google Scholar, 18Ghigo J.M. Natural conjugative plasmids induce bacterial biofilm development.Nature. 2001; 412: 442-445Crossref PubMed Scopus (510) Google Scholar, 19Woods J. Boegli L. Kirker K.R. Agostinho A.M. Durch A.M. Delancey Pulcini E. Stewart P.S. James G.A. Development and application of a polymicrobial, in vitro, wound biofilm model.J Appl Microbiol. 2012; 112: 998-1006Crossref PubMed Scopus (53) Google Scholar, 20Benoit M.R. Conant C.G. Ionescu-Zanetti C. Schwartz M. Matin A. New device for high-throughput viability screening of flow biofilms.Appl Environ Microbiol. 2010; 76: 4136-4142Crossref PubMed Scopus (136) Google Scholar However, they do not cover the factors that play a role during biofilm formation with regard to the host-pathogen interactions. Within the whole organism the bacteria pass different pathogenic steps, including survival during bacteremia, adherence, and biofilm maturation on foreign material under continuous stress from the host immune system.21Bjarnsholt T. Alhede M. Alhede M. Eickhardt-Sørensen S.R. Moser C. Kühl M. Østrup Jensen P. Høiby N. The in vivo biofilm.Trends Microbiol. 2013; 21: 466-474Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar S. aureus has a complex regulatory network that helps the bacteria cope with stress conditions and also influence the process of biofilm formation.22Tuchscherr L. Bischoff M. Lattar S.M. Llana M.N. Pförtner H. Niemann S. Geraci J. Van de Vyver H. Fraunholz M.J. Cheung A.L. Herrmann M. Völker U. Sordelli D.O. Peters G. Löffler B. Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections.PLoS Pathog. 2015; 11: e1004870Crossref PubMed Scopus (118) Google Scholar Therefore, it is necessary to study biofilm formation in vivo from the initial steps until maturity to detect all virulence and regulatory factors involved in this process. Studies on biofilm in human samples provided first indications that a mature biofilm is built up in complex layers.23Schmiedel D. Kikhney J. Masseck J. Rojas Mencias P.D. Schulze J. Petrich A. Thomas A. Henrich W. Moter A. Fluorescence in situ hybridization for identification of microorganisms in acute chorioamnionitis.Clin Microbiol Infect. 2014; 9: O538-O541Abstract Full Text Full Text PDF Scopus (13) Google Scholar The nature and effect of the biofilm structure formed in vivo are only marginally understood but might have important implications for diagnostics and therapeutic interventions. Up to now the in vivo study of biofilm formation on vascular grafts has mostly been limited to rat models in which the graft is placed subcutaneously and topically infected there.24Arya R. Ravikumar R. Santhosh R.S. Princy S.A. SarA based novel therapeutic candidate against Staphylococcus aureus associated with vascular graft infections.Front Microbiol. 2015; 6: 416Crossref PubMed Scopus (41) Google Scholar, 25De Cremer K. Delattin N. De Brucker K. Peeters A. Kucharíková S. Gerits E. Verstraeten N. Michiels J. Van Dijck P. Cammue B.P.A. Thevissen K. Oral administration of the broad-spectrum antibiofilm compound Toremifene inhibits Candida albicans and Staphylococcus aureus biofilm formation in vivo.Antimicrob Agents Chemother. 2014; 58: 7606-7610Crossref PubMed Scopus (21) Google Scholar, 26Giacometti A. Cirioni O. Ghiselli R. Orlando F. D'Amato G. Kamysz W. Mocchegiani F. Sisti V. Silvestri C. Łukasiak J. Rocchi M. Saba V. Scalise G. Temporin A soaking in combination with intraperitoneal Linezolid prevents vascular graft infection in a subcutaneous rat pouch model of infection with Staphylococcus epidermidis with intermediate resistance to glycopeptides.Antimicrob Agents Chemother. 2004; 48: 3162-3164Crossref PubMed Scopus (7) Google Scholar Although these are straightforward, high-throughput models, they do not accurately represent the dynamic and complex structure of vascular biofilm formation. Some researchers have set up models in dogs and pigs, where the grafts are placed intravascularly to closely mimic the situation in patients.27Gao H. Sandermann J. Prag J. Lund L. Lindholt J.S. Prevention of primary vascular graft infection with silver-coated polyester graft in a porcine model.Eur J Vasc Endovasc Surg. 2010; 39: 472-477Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 28Langerhuus S.N. Tønnesen E.K. Jensen K.H. Damgaard B.M. Heegaard P.M.H. Halekoh U. Lauridsen C. Brief report: biomarkers of aortic vascular prosthetic graft infection in a porcine model with Staphylococcus aureus.Eur J Clin Microbiol Infect Dis. 2010; 29: 1453-1456Crossref PubMed Scopus (4) Google Scholar, 29Javerliat I. Goëau-Brissonnière O. Sivadon-Tardy V. Coggia M. Gaillard J.L. Prevention of Staphylococcus aureus graft infection by a new gelatin-sealed vascular graft prebonded with antibiotics.J Vasc Surg. 2007; 46: 1026-1031Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30Schmacht D. Armstrong P. Johnson B. Pierre K. Back M. Honeyman A. Cuthbertson D. Bandyk D. Graft infectivity of rifampin and silver-bonded polyester grafts to MRSA contamination.Vasc Endovasc Surg. 2005; 39: 411-420Crossref PubMed Scopus (30) Google Scholar However, such an intravascular graft model is still missing in the mouse, offering a high-throughput, cost-effective alternative, having the additional benefit of allowing the use of genetically manipulated, humanized mice.31Schultz L.D. Ishikawa F. Greiner D.L. Humanized mice in translational biomedical research.Nat Rev Immunol. 2007; 7: 118-130Crossref PubMed Scopus (1023) Google Scholar Therefore, the aim of this study was to create a mouse model to realistically study the complex process of biofilm formation on vascular grafts in vivo. This model is based on a polytetrafluoroethylene (PTFE) catheter, a polymer material often used for vascular grafts,32Kapadia M.R. Popowich D.A. Kibbe M.R. Modified prosthetic vascular conduits.Circulation. 2008; 117: 1873-1882Crossref PubMed Scopus (104) Google Scholar placed intravascularly into the right carotid artery (RCA). After recovery from the surgical intervention, animals are systemically infected via the tail vein. Therefore, bacteria were delivered to the catheter via the bloodstream. In addition, to minimize the amount of mice necessary for research and to develop a long-term infection model during several weeks, we followed up the infection noninvasively by positron emission tomography using F18-fluordeoxyglucose (FDG-PET) and magnetic resonance imaging (MRI). For the setup of the infection model, the bacterial strain S. aureus LS1 was used. LS1 is a murine arthritis isolate that has been used in infection models before.33Bremell T. Abdelnour A. Tarkowski A. Histopathological and serological progression of experimental Staphylococcus aureus arthritis.Infect Immun. 1992; 60: 2976-2985PubMed Google Scholar Furthermore, 26 S. aureus strains were collected from patients with vascular infections from the University Hospital in Muenster, Germany, and 33 S. aureus strains were obtained from the nasal swabs of healthy individuals. All collected S. aureus strains were tested for their ability to form biofilm in vitro. The strains Staphylococcus carnosus TM300 and Staphylococcus epidermidis RP62A were used as negative and positive controls, respectively. Bacteria were grown overnight at 37°C in tryptic soy broth plus 0.25% glucose and diluted 1:200 in tryptic soy broth plus 0.25% glucose. Then 200 μL of each strain was inoculated in duplicates into sterile 96-well polystyrene microtiter plates (Greiner, Pleidelsheim, Germany). The plates were incubated for 24 hours at 37°C. The wells were washed twice with 200 μL of sterile phosphate-buffered saline (PBS) after which they were incubated for 15 minutes at room temperature with 100 μL of 1% crystal violet. This was washed three times with 200 μL of PBS, after which 100 μL of an ethanol/aceton (80:20) solution was added to each well and incubated at room temperature until the biofilm was completely resuspended. The absorbance was measured with a Biorad iMark Microplate reader (Bio-Rad Laboratories, Hercules, CA) at 655 nm. CD1 mice were bred and housed in microisolator cages and were fed food and water ad libitum. When they reached 10 to 14 weeks of age, they were used for the vascular graft operations. Mice received an i.p. injection of fentanyl and midazolam (0.04 mg of fentanyl and 4 mg of midazolam per kg of body weight) for sedation and were operated on under 1.5% isoflurane with 100% oxygen at a flow rate of 800 mL/minute−1. A 10-mm incision was made from the manubrium to the chin of the mice, and the RCA was separated from surrounding tissue. Blood flow was temporarily cut off with silk sutures, and a small cut was made into the artery, allowing a 2-mm-long PTFE catheter (inner diameter, 0.3 mm; external diameter, 0.6 mm) to be placed inside the artery. The PTFE catheter was fixed in place using two silk sutures. The silk sutures that were cutting off the blood flow were removed, enabling blood to flow through the artery and catheter. The incision was sewn closed, and the surgical site was disinfected. One week after vascular graft operation, mice were infected with 5 × 106 CFU/mL of S. aureus LS1 in 200 μL of PBS via a lateral tail vein. As uninfected control, mice were injected with 200 μL of PBS via a lateral tail vein. During the infection, mice were measured daily to follow-up weight and disease progression. If a weight loss of 15% occurred within 2 days, mice were euthanized. At 2, 7, 14, 21, or 42 days post infection (d.p.i.), mice were sacrificed, and organs (heart, kidneys, tibia, and brain), intracardiac blood, and catheter samples were collected in 2 mL of sterile PBS for colony-forming unit counts. The organs were homogenized and plated in various dilutions onto blood agar plates. The catheters were sonicated for 10 minutes and vortexed at high speed for 1 minute to ensure full removal of the biofilm from the catheter. This solution was then also plated in various dilutions onto blood agar plates. Mice were infected with 8 different S. aureus strains, with varying capacity to form biofilm in vitro, 1 week after vascular graft operation. The strains and doses used are listed in Supplemental Table S1. At 14 d.p.i., mice were euthanized, and organs (heart, kidneys, tibia, and brain), intracardiac blood, and catheter samples were collected in 2 mL of sterile PBS for colony-forming unit counts. The organs and catheter were processed as mentioned above. Three days after surgery (baseline) and 10 days after infection, mice were investigated by MRI to measure and monitor the blood flow through the LCA and RCA. Measurements were performed at 9.4 T on a BioSpec 94/20 USR small animal MRI system (Bruker BioSpin, Ettlingen, Germany) using ParaVision software version 5.1 (Bruker BioSpin, Ettlingen, Germany). A 35-mm-diameter mouse body quadrature volume coil (Rapid Biomedical, Rimpar, Germany) was used for data acquisition in a 1 T/m gradient insert (BGS-6S, Bruker BioSpin, Ettlingen, Germany). Mice were anesthetized with inhalation anesthesia consisting of a 0.7/0.3 air/oxygen mixture with 1.5% to 2.5% isoflurane. Mice were positioned on a combined MR-PET bed and were connected to a small animal monitoring unit (SA Instruments, Inc., Stony Brook, NY) to measure respiration rate and ECG findings. The position inside the magnet bore was checked by using a self-gated cine FLASH sequence (repetition time/echo time, 85.0/1.7 milliseconds; flip angle, 15°; field of view, 35 × 35 mm2; section thickness, 0.5 mm; acquisition matrix, 296 × 296). Subsequently, blood flow measurements were performed using a section-selective, balanced two-point velocity–encoded phase-contrast MRI sequence, which is based on a respiration and ECG-triggered FLASH gradient echo (Flowmap, Bruker BioSpin) (repetition time/echo time, 15.0 /2.6 milliseconds; flip angle, 30°; field of vision, 35 × 35 mm2; section thickness, 0.8 mm; acquisition matrix, 296 × 296; number of averages: 6) with three parallel sections (placed above and below the catheter as well as on site). After MRI measurements, the MR-PET mouse bed was transferred from the MRI scanner to the PET without changing the position of the mouse. Analysis of velocity-encoded MRI data was performed in Matlab version R2014b (MathWorks, Inc., Natick, MA) as previously described.34Bovenkamp P.R. Brix T. Lindemann F. Holtmeier R. Abdurrachim D. Kuhlmann M.T. Strijkers G.J. Stypmann J. Hinrichs K.H. Hoerr V. Velocity mapping of the aortic flow at 9.4 T in healthy mice and mice with induced heart failure using time-resolved three-dimensional phase-contrast MRI (4D PC MRI).MAGMA. 2015; 28: 315-327Crossref PubMed Scopus (12) Google Scholar Three days after surgery but before infection (baseline) and 10 days after infection with S. aureus or PBS injection, mice were subjected to PET using 18F-fluorodeoxyglucose (18F-FDG) to measure bacteria-induced inflammation. After MRI acquisition, the animal bed was transferred into the PET scanner. PET studies were performed on a high-resolution small animal PET scanner (32-module quadHIDAC, Oxford Positron Systems Ltd., Oxford, UK) with uniform spatial resolution (>1 mm full width at half maximum) over a large cylindrical field of view (165-mm diameter, 280-mm axial length). The PET data were reconstructed using one-pass list mode expectation maximization algorithm with resolution recovery. 18F-FDG PET images were acquired 70 to 85 minutes after intravenous injection of 10 MBq 18F-FDG (150 μL). The MR images were used for anatomical co-registration with the PET images using a landmark-based approach by filling a contrast media/radiotracer mixture into canals with sphere-shaped cavities in the animal bed. The PET and MR images were then co-registered using the in-house developed image analysis software MEDgical version 0.7.3185 (European Institute for Molecular Imaging, Muenster, Germany). To analyze the PET data, volumes of interest (VOIs) were defined based on MR images, with the examiner (S.H.) blinded to the PET imaging results. One VOI was drawn around the catheter in the RCA, and another VOI placed around the left carotid artery (LCA) was used as a reference. 18F-FDG uptake was expressed as standardized uptake values (SUVs), calculated by dividing the VOI radioactivity concentration by the injected activity multiplied by the body weight of the mouse [SUV = (c)VOI/injection dose × body weight]. Uptake ratios (SUV RCA/SUV LCA) were calculated. Catheters were removed from mice 14 days d.p.i., and 4 catheters were analyzed using electron microscopy to visualize the biofilm formation. In brief, catheters were fixed in 4% formaldehyde and 0.25% glutaraldehyde in 100 mmol/L sodium cacodylate buffer, pH 7.4, at 4°C. Afterward, samples were rinsed in PBS, dehydrated in ethanol up to 70%, embedded in LR White medium (London Resin Company, London, UK), and polymerized using UV light according to the manufacturer's instructions. Ultrathin sections were cut on an ultramicrotome and collected on copper grids. For visualization of biofilms, ultrathin sections were incubated for 1 hour at room temperature with a lectin from Triticum vulgaris (wheat) (L 9640, Sigma-Aldrich, Munich, Germany) diluted 1:2000 in PBS. After 5 washing steps with PBS, sections were incubated with rabbit antibodies directed against the T. vulgaris lectin (T4144, Sigma-Aldrich) diluted 1:5000 in PBS followed by incubation for 1 hour with goat antibodies directed to rabbit immunoglobulins conjugated to 18-nm colloidal gold particles (Jackson ImmunoResearch Laboratories, West Grove, PA). Control experiments were performed in which the lectin was preincubated with N-acetyl-d-glucosamine (A8625, Sigma-Aldrich). Finally, sections were rinsed with water and stained with 4% (w/v) uranyl acetate. Electron micrographs were taken at 60 kV with a Philips EM-410 transmission electron microscope (Philips, Eindhoven, the Netherlands). Four catheters isolated from mice 14 d.p.i., fixed in 4% formaldehyde, were longitudinally cut into two halves. After a permeabilization step for 10 minutes in PBS with 0.5% saponin and 0.1% Tween 20, the catheters were incubated in blocking buffer consisting of 5% bovine serum albumin (albumin fraction V from bovine serum, Merck, Darmstadt, Germany) and 300 mmol/L glycine (Sigma-Aldrich) in permeabilization buffer for 1 hour at room temperature. S. aureus was specifically labeled using a rabbit anti–S. aureus polyclonal IgG (generated by Squarix, Marl, Germany) diluted 1:200 in blocking buffer and incubated with the catheter overnight at 4°C. After three washing steps in permeabilization buffer, the catheter were further incubated with a fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories) at 1:200 in blocking buffer for 1 hour at room temperature. Nucleic acid counterstaining was performed using DAPI (AppliChem, Darmstadt, Germany) at a concentration of 1 μg/mL for 10 minutes in blocking buffer. Fluorescence images of stained catheters were acquired with a confocal laser scanning microscope (LSM780, Zeiss, Jena, Germany) using a 20× Plan-Apochromat objective (numerical aperture, 0.8; Zeiss) and ZEN software 2012 black edition (Zeiss). Fluorescence excitation and emission detection were conducted using a 405-nm laser diode and an adjustable bandpass filter set to 410 to 497 nm for DAPI and a 488-nm argon laser and a bandpass filter set at 493 to 634 nm for FITC. The pinhole was set to 34 μm. For spectral images, the emission was scanned from 495 to 691 nm with 8.9-nm spectral resolution. Linear unmixing with the Zeiss implemented algorithm was applied to the obtained spectral image by means of assigning each pixel spectrum to three different reference emission spectra. Reference spectrum for FITC was obtained from a positive control (FITC-labeled S. aureus only), reference spectrum for autofluorescence from a negative control (catheter incubated without anti–S. aureus and with secondary antibody only), and reference spectrum for background without fluorescence from the same image, each by choosing a region of interest displaying the respective fluorescence signal. Four PTFE catheters were incubated with S. aureus LS1 in tryptic soy broth plus 0.25% glucose either in vitro for 24 hours with agitation or in vivo in the vascular graft model mentioned above for 4 weeks. The catheter samples were fixed and processed as described before.35Moter A. Leist G. Rudolph R. Schrank K. Choi B.K. Wagner M. Göbel U.B. 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• W2574165910 title "A Novel Mouse Model of Staphylococcus aureus Vascular Graft Infection" @default.
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