Diese Arbeit wurde von der US National Institutes of Health [Zuschuss R01AI-39108 zu ASB] und der American Heart Association [Zuschüsse SDG 0630219N und AID 09GRNT2180065 zu YQX].

1. Vorbereiten der MRSA-Stämme für Infektionsforschung <ol><li> Impfen eine Öse MRSA Kultur aus einem Vorrat Rohr bei -80 ° C zu einer Schafblut Trypticase Soy Agar (TSA) Platte (siehe Tabelle der spezifischen Reagenzien und Ausrüstung), und inkubieren Sie bei 37 ° C über Nacht. </li><li> Überprüfen Sie die Reinheit der Kultur auf das Blut-Agar-Platte (ähnlich Kolonie Phänotypen), um sicherzustellen, gibt es keine Kontamination. </li><li> Wählen Sie eine Kolonie aus dem Schafblut TSA-Platte und beimpfen die Kolonie in 5 ml Trypton-Soja-Bouillon (TSB) in einem 15 ml-Snap-Röhrchen. </li><li> Bei 37 ° C über Nacht unter Schütteln bei 200 UpM. </li></ol> 2. Vorbereiten Chirurgie Katheter Schneiden Sie Polyethylenschlauch (PE10;. Becton Dickinson, Best.-Nr. 427401) bis 10 cm Länge, und schmelzen Sie ein Ende, indem Sie die Spitze mit einer sterilen Pinzette. Der Zweck Abdichten eines Endes des Katheters ist, um eine Blutung während der Katheterisierung zu vermeiden. 3. Pre-enurgery Vorbereitung und Anästhesie <ol><li> Ort Sprague-Dawley Ratten (Harlan, Indianapolis, Ind. weiblich, 250-300 Gramm) in einer Kammer mit Isofluran-Sauerstoff-Gasgemisch (50%: 50%), bis die Betäubung wirksam wird (z. B. Muskeln sind entspannt und Pedal Reflexe fehlen ) und verwalten Tiere in einer anästhesierten Zustand während der Operation mit dem Gasgemisch. </li><li> Reinigen Sie die Hals-Bereich von Kinn bis knapp unterhalb des Brustbeins mit Betadin und 70% Ethanol. </li></ol> 4. Chirurgisches Vorgehen <ol><li> Verwenden steriler Technik in der Chirurgie. Einen Einschnitt (1-1,5 cm) vertikal nur durch die Hautschicht des Halses über dem Brustbein. </li><li> Mit einem stumpfen Instrument trennen die Faszie an der rechten Halsschlagader mit 2 Paar gebogene Hakenpinzette aussetzen. </li><li> Ziehen Sie die Arterie nach oben aus Hals Hohlraum, und Platz zwei 10 cm Länge der Seidenfaden unter der Arterie und bindet von der Arterie an der exponierten Kopfende, platzieren Sie einen Clip auf der artery, um eine Blutung zu verhindern. </li><li> Machen Sie ein kleines Loch in der Spitze der Arterie mit einem Katheter Einführhilfe (Becton Dickinson, Bestell-Nr. 406999), legen Sie einen Katheter mit einer Pinzette durch das Loch in der Arterie, entfernen Sie den Clip und drücken Sie den Katheter zum Herzen hin, bis ein Widerstand erfüllt ist. </li><li> Binden Sie die losen Fäden rund um den kaudalen Ende der Arterie und sichern Sie den Katheter an Ort und Stelle mit dem seidenen Faden, wenn der Katheter ist an Ort und Stelle, wie bestimmt durch: i) die Länge der Katheter eingeführt (4-5 cm), ii) Widerstand gegen eine weitere Förderung und iii) Pulsation des Katheters mit dem Herzschlag. Lassen Sie den Katheter an Ort für den Rest des Experiments. </li><li> Schneiden Sie überschüssige Faden und Katheter Enden, und stellen Sie sicher, dass es keine Blutung aus dem Katheter. Stecken Sie die losen Enden unter die Haut des Halses und schließen Sie die Haut mit Haut-Clips. </li><li> Legen Sie die Ratte im Käfig an einem warmen Ort, bis von der Narkose erholt und bieten Nahrung und Wasser. Überprüfen Sie die Ratte häufig während undnach dem Erwachen aus der Narkose. </li><li> Diese Operation ist eine &quot;Kategorie E&quot;-Verfahren. Die Verfahren produziert Schmerz ist unrelieved von Analgetika seit MRSA-Infektionen begleitende Leiden für die Tiere. </li></ol> 5. MRSA-Infektion <ol><li> Eine Infektion kann zwischen 1 und 7 Tagen nach der Operation durchgeführt werden, aber halten Sie innerhalb von Experimenten konsistent. </li><li> Sauberkeit Rattenschwanz mit 70% Ethanol und 0,5 ml Injektion MRSA an einer gewünschten Zellen (Oktober 4 - Oktober 6 cfu / Tier für den S.-aureus-Stämme). Mit 27 G 1/2 Zoll Nadel intravenös aus der Schwanzvene Achtung : MRSA-Kultur gehört zu Biosicherheitsstufe 2 (BSL2) und hat eine mäßige potenzielle Gefahr für Mensch und Umwelt. </li><li> Halten Sie Druck auf der Baustelle, bis eine Blutstillung vor der Rückkehr der Ratte in den Käfig. </li></ol> 6. Sacrifice Ratten und Kultur Zielgewebe <ol><li> Opfer Ratten durch ip-Injektion von Natriumpentobarbital (200 mg/ Kg) nach 1 bis 6 Tage nach der Infektion. </li><li> Legen Sie die Ratte auf den Rücken und wischen Sie die Brust mit 70% Ethanol. </li><li> Machen Sie einen V-förmigen Schnitt im Brustkorb unterhalb Brustbein, und schneiden Sie die Knorpel der Rippen auf beiden Seiten des Brustbeins, um das Herz freizulegen. </li><li> Ziehen Sie das Herz sanft und Clip durch das Herzgewebe nahe an der Aorta und sezieren bis das Herz zu befreien. </li><li> Legen Sie die Innenstadt, in einer sterilen Petrischale mit 4x4 Zoll Gaze innen, machen Sie einen Schnitt durch die linksventrikuläre Innenwand und öffnen Sie den linksseitigen Kammer. </li><li> Überprüfen Sie visuell auf Katheterplatzierung. Untersuchen und entfernen Vegetationen aus dem Ventil mit Schere und Pinzette. </li><li> Wiegen und Homogenisierung der Vegetation und machen serielle Verdünnungen mit PBS für quantitative Kultur. </li></ol> 7. Repräsentative Ergebnisse Unmittelbar nach dem Einsetzen des Katheters in die Arterie und schiebt den Katheter etwa 4-5 cm in Richtung des Herzens, wird ein Widerstand sein ErfahCED. Wenn kein Widerstand gemessen wird, kann der Katheter nicht erfolgreich in die linke Seite des Herzens Kammer eingeführt worden sind, wozu auch der Platzierung des Katheters zu beeinträchtigen. Eine korrekte Platzierung des Katheters zuvor veröffentlicht 9 ist in Abbildung 1 dargestellt. Ein Teil der MRSA-Infektion Proben müssen quantitative kultivierten die sicherstellen, dass die genaue lebensfähige Keimzahl für die Infektion und die Reinheit der Probe Infektion. Darüber hinaus sollten die Organismen gewonnen aus Vegetationen die gleichen wie die in dem Inokulum verwendet werden. Tabelle 1 zeigt ein Beispiel für die Virulenz eines S. aureus-Stamm in der Ratte Endokarditis-Modell die bisher 9 veröffentlicht wurde. Alle Tiere mit Inokula von 10 5 und 10 6 cfu herausgefordert und geopfert zwischen 3 bis 6 Tage nach der Infektion entwickelt endocardits mit hoher S. aureus Dichten der kardialen Vegetationen sowie Niere und Milz ( <strong&gt; Tabelle 1). Ratten, die mit sterilen Ventil Kulturen gelten als nicht infizierte. <img alt="1" src="/files/ftp_upload/3863/3863fig1.jpg" /> Abbildung 1. Der Katheter ist in einem richtigen Stelle (linke Seite der Herzkammer), und zahlreiche Vegetationen sichtbar sind rund um die Aortenklappen 9. <table border="1"><tbody><tr><td> Inokulum (Anzahl der Animas) </td><td colspan="3"> Mittleren log 10 KBE / g Gewebe ± SD ein in: </td></tr><tr><td></td><td> Vegetation </td><td> Niere </td><td> Milz </td></tr><tr><td> 10 6 KBE / Tier (9) </td><td> 10,36 ± 0,85 </td><td> 7,30 ± 0,64 </td><td> 6,70 ± 0,57 </td></tr><tr><td> 10 5 CFU / Tier (8) </td><td> 9,93 ± 0,53 </td><td> 7,14 ± 0,53 </td><td> 6,44 ± 0,63</td></tr><tr><td> 10 4 KBE / Tier (7) </td><td> 3,46 ± 0,50 * </td><td> 1,81 ± 0,74 * </td><td> 1,58 ± 0,59 * </td></tr></tbody></table> eine SD, Standardabweichung. P &lt;0,001 in Bezug auf 10 5 oder 10 6 CFU-Challenge Tieren. Tabelle 1. S. aureus Dichte der kardialen Vegetationen mit verschiedenen Inokula in der Ratte Endokarditis Modell 9.

<ol>
	<li>Petti, C. A., Fowler, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+bacteremia+and+endocarditis.">Staphylococcus aureus bacteremia and endocarditis.</a> Cardiol. Clin. 21, 219-233 (2003).</li><li>Bashore, T. M., Cabell, C., Fowler, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+infective+endocarditis.">Update on infective endocarditis.</a> Curr. Probl. Cardiol. 31, 274-352 (2006).</li><li>Fowler, V. G., Justice, A., Moore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hematogenous+complications+of+intravascular+catheter-associated+Staphylococcus+aureus+bacteremia.">Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia.</a> Clin. Infect. Dis. 17, 313-320 (1993).</li><li>Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+US+hospitals:+analysis+of+24,179+cases+from+a+prospective+nationwide+surveillance+study.">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clin. Infect. Dis. 39, 309-317 (2004).</li><li>Hoen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+issues+in+the+management+of+infective+endocarditis+caused+by+gram-positive+cocci.">Special issues in the management of infective endocarditis caused by gram-positive cocci.</a> Infect Dis. Clin. North Am. 16, 437-452 (2002).</li><li>Moise, P. A., Hershberger, E., Amodio-Groton, M. I., Lamp, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+clinical+outcomes+when+utilizing+high-dose+(&gt;+or+=8+mg/kg)+daptomycin+therapy.">Safety and clinical outcomes when utilizing high-dose (&gt; or =8 mg/kg) daptomycin therapy.</a> Ann. Pharmacother. 43, 1211-1219 (2009).</li><li>Sakoulas, G., Brown, J., Lamp, K. C., Friedrich, L. V., Lindfield, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+patients+receiving+daptomycin+for+the+treatment+of+Staphylococcus+aureus+infections+and+assessment+of+clinical+factors+for+daptomycin+failure:+a+retrospective+cohort+study+utilizing+the+Cubicin+Outcomes+Registry+and+Experience.">Clinical outcomes of patients receiving daptomycin for the treatment of Staphylococcus aureus infections and assessment of clinical factors for daptomycin failure: a retrospective cohort study utilizing the Cubicin Outcomes Registry and Experience.</a> Clin. Ther. 31, 1936-1945 (2009).</li><li>Xiong, Y. Q., Willard, J., Kadurugamuwa, J. L., Yu, J., Francis, K. P., Bayer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+bioluminescent+imaging+for+evaluating+the+efficacy+of+antibiotics+in+a+rat+Staphylococcus+aureus+endocarditis+model.">Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model.</a> Antimicrob. Agents Chemother. 49, 380-387 (2005).</li><li>Peerschke, E. I., Bayer, A. S., Ghebrehiwet, B., Xiong, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gC1qR/p33+blockade+reduces+Staphylococcus+aureus+colonization+of+target+tissues+in+an+animal+model+of+infective+endocarditis.">gC1qR/p33 blockade reduces Staphylococcus aureus colonization of target tissues in an animal model of infective endocarditis.</a> Infect. Immun. 74, 4418-4423 (2006).</li><li>Ganesh, V. K., Rivera, J. J., Smeds, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+model+of+the+Staphylococcus+aureus+ClfA-fibrinogen+interaction+opens+new+avenues+for+the+design+of+anti-staphylococcal+therapeutics.">A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.</a> PLoS Pathog. 4, e1000226 (2008).</li><li>Boles, B. R., Horswill, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+biofilm+disassembly.">Staphylococcal biofilm disassembly.</a> Trends Microbiol. , (2011).</li><li>Tseng, C. W., Sanchez-Martinez, M., Arruda, A., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+Infection+of+Methicillin+Resistant+Staphylococcus+Aureus+(MRSA).">Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA).</a> J. Vis. Exp. (48), e2528 (2011).</li></ol>

Keine Interessenskonflikte erklärt.

Die Ratte Endokarditis ist eine wichtige und gut charakterisierten Tiermodell für in vivo-Studien in der Pathogenese und Antibiotika in der Behandlung von bakteriellen Infektionen 9-11. Darüber hinaus stellt Ratte Endokarditis Modell eines Verbundes von akuter und sub-akute Infektionen, und ahmt menschlichen Gegenüber und menschlichen nativen Klappe Endokarditis. Darüber hinaus begleitet von einer in Dauerkatheter, stellt sie eine klassische Biofilm-assoziierte Infektionen, eine gemeinsame und sc...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Blutagarplatte, 5% Schafblut in Trypticase Soy Agar (TSA) </td><td> Hardy Diagnostics </td><td> A10BX </td><td></td></tr><tr><td> Trypticase Soy Broth (TSB) </td><td> Becton Dickinson </td><td> 211825 </td><td></td></tr><tr><td> Schüttelinkubator </td><td> Labnet </td><td> I5311-DS </td><td></td></tr><tr><td> Polyethylen-Rohr </td><td> Becton Dickinson </td><td> 427401 </td><td></td></tr><tr><td> Kathetereinführvorrichtung </td><td> Becton Dickinson </td><td> 6999 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Isofluorance </td><td> Western Medical Suppy, Inc. </td><td> 2147 </td><td> Folgen Sie Sicherheit und Handhabung Informationen </td></tr><tr><td> Chirurgische Instrumente </td><td> Fine Science Tools und Biomedical Research Instruments, Inc. </td><td></td><td> Finden Sie die Instrumente aus den beiden Unternehmen für Ihre Bedürfnisse </td></tr></tbody></table>

experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

Endovaskuläre Infektionen, einschließlich Endokarditis, sind lebensbedrohliche Infektionskrankheiten Syndrome 3.1. Staphylococcus aureus ist der häufigste weltweit Ursache solcher Syndrome mit inakzeptabel hohen Morbidität und Mortalität sogar mit geeigneten antimikrobiellen Wirkstoff Behandlungen 4-6. Der Anstieg der Infektionen durch Methicillin-resistente S. aureus (MRSA), haben die hohen Raten von Vancomycin klinischen Therapieversagen und wachsenden Probleme von Linezolid und Daptomycin Widerstand alle weiter kompliziert die Behandlung von Patienten mit solchen Infektionen, und führte zu hohen Kosten im Gesundheitswesen 7, 8. Darüber hinaus ist zu betonen, dass die meisten bisherigen Studien mit Antibiotika Ergebnisse in klinischen Umgebungen zugrunde gelegt wurden, so dass sie möglicherweise auch durch den Host Faktoren variiert von Patient zu Patient beeinflusst werden. Daher ist ein Tiermodell der endovaskulären Infektion in dem Host-Faktoren sind ähnlich von Tier zu Tier mehrentscheidend für mikrobielle Pathogenese als auch die Wirksamkeit neuer antimikrobieller Substanzen zu erforschen. Endokarditis bei Ratte ist ein etabliertes Tiermodell, der nahe menschlichen nativen Klappe Endokarditis. Dieses Modell wurde genutzt, um die Rolle von bestimmten Staphylokokken-Virulenzfaktoren und die Wirksamkeit von Antibiotika-Behandlungsregime für Staphylokokken-Endokarditis zu untersuchen. In diesem Bericht beschreiben wir die experimentellen Modell Endokarditis durch MRSA, die verwendet werden, um bakterielle Pathogenese und Reaktion auf Antibiotika-Behandlung untersucht werden konnte.

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimentelle Ratte Endokarditis Modell durch Methicillin-resistente<em&gt; S. aureus</em&gt;.

Experimentelle Endokarditis Modell von Methicillin-resistenten<em&gt; Staphylococcus aureus</em&gt; (MRSA) in Ratten

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

15.6K Aufrufe.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Experimentelle Ratte Endokarditis Modell durch Methicillin-resistente S. aureus.

Serie: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Die Verwendung von Drip Flow und Rotating Disk-Reaktoren für Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Forschung

Immunologie und Infektion

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Subkutane Infektion von Methicillin-resistenten Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Die Überwindung der nicht mehr reagiert, in der experimentellen autoimmunen Enzephalomyelitis (EAE) Beständig Mausstämmen durch adoptiven Transfer-und Antigen-Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Assay f

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Studieren Wechselwirkungen von<em&gt; Staphylococcus aureus</em&gt; Mit Neutrophilen durch Durchflusszytometrie und Mikroskopie Zeitraffer

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Targeting Biofilm Assoziierte<em&gt; Staphylococcus aureus</em&gt; Verwendung von Resazurin Based Drug-Anfälligkeit Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Genotypisierung<em&gt; Staphylococcus aureus</em&gt; Durch ribosomalen Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Methodik für die Studie des horizontalen Gentransfers in<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Ein Mausmodell zu beurteilen, angeborene Immunantwort auf Staphylococcus Aureus -Infektion

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Experimentelle Endokarditis Modell von Methicillin-resistenten

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Die Verwendung von Drip Flow und Rotating Disk-Reaktoren für Staphylococcus aureus Biofilm Analysis

Subkutane Infektion von Methicillin-resistenten Staphylococcus Aureus (MRSA)

Die Überwindung der nicht mehr reagiert, in der experimentellen autoimmunen Enzephalomyelitis (EAE) Beständig Mausstämmen durch adoptiven Transfer-und Antigen-Challenge

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

Multiplex PCR Assay f

Studieren Wechselwirkungen von

Targeting Biofilm Assoziierte

Genotypisierung

Methodik für die Studie des horizontalen Gentransfers in

Ein Mausmodell zu beurteilen, angeborene Immunantwort auf Staphylococcus Aureus -Infektion