Diese Arbeit wurde von der US Public Health Service Zuschüsse AI69233 und AI073843 aus dem National Institute of Allergy and Infectious Diseases unterstützt. EPS ist ein Burroughs Wellcome Fellow in der Pathogenese von Infektionskrankheiten. KPH wurde von der Cellular and Molecular Microbiology Training Grant Program 5 T32 A107611-10 finanziert.

Ein. Reinigung von Hämoglobin aus frischem Blut <ol><li> Erwerben frisches menschliches Blut mit einem Antikoagulans ergänzt. Halten Sie Blut auf Eis oder bei 4 ° C während der Reinigung. </li><li> Zentrifuge Blut für 20 min bei 1.500 x g beträgt. Die roten Blutkörperchen (Erythrozyten) wird am Boden der Röhre liegen. Vorsichtig den Überstand aspirieren und sanft das Pellet in eiskaltem 0,9% (w / v) NaCl-Lösung. Wiederholen Sie die Zentrifugation und 3 x waschen. </li><li> Das Pellet in 1 Volumen eiskaltem 10 mM Tris-HCl (pH 8,0). Dies induziert die Lyse der Erythrozyten durch osmotischen Druck. Fügen Toluol zu ~ 20% Endvolumen. </li><li> Inkubation bei 4 ° C auf einem Drehspieß Nacht. </li><li> Zentrifuge das Lysat bei 20.000 xg für 1 Stunde. Sammeln Sie die Mitte Hämolysat Bruchteil Verlassen des Toluol (floating on top) und Pellet unberührt. Verwenden Sie einen Langhals-Pipette auf die mittlere Fraktion zu sammeln. </li><li> Fahren Sie durch 0,44 um Spritzenfilter. Enthält die Lösung teilchenförmigenMaterie und nicht durch das Filter geleitet werden, Schritt 1.5. </li><li> Läutern Hämoglobin (Hb) mit einer Hochleistungs-Flüssigkeits-Chromatographie (HPLC) Anionenaustauschersäule (Varian, PL-SAX 1.000 Å 8 um, 150 mm × 4,6 mm). Die mobile Phase A ist 10 mM Tris-HCl (pH 8,0) und die mobile Phase B ist 10 mM Tris-HCl (pH 8,0) + 0,5 M NaCl. A 0% -100% Gradienten von Lösungsmittel B ist über 2 min bei 2,0 ml / min Flussrate laufen. Die Elution wird basierend auf Absorption überwacht (λ: 410 nm und 280 nm). Sammeln nur den Bruch durch eine leuchtend rote Farbe und ein prominenter Absorptionspeak (Abbildung 1). </li><li> Dialysieren die Elution gegen Phosphat-gepufferte Kochsalzlösung (PBS) über Nacht und dann für einige Stunden wieder. Sterilisieren, indem sie durch eine 0,22 um Spritzenfilter. </li><li> Um Hb-Konzentration zu messen, bereiten Standard-Hb-Lösungen bekannter Konzentrationen in PBS. Bestimmung der Konzentration von Hb in der Probe durch Mischen der Standard-Lösungen (siehe Tabelle mit den Reagenzien unsed) oder die Probenlösung mit 2x Drabkin-Reagenz (hergestellt aus Pulver) im Verhältnis 1:1. Beispielsweise mischen 100 ul Hb-Lösung mit 2x 100 ul Drabkin-Reagenz in 96-Well-Platten. Inkubieren für 15 min und Messung der Extinktion bei 540 nm. Plotten einer Standardkurve und bestimmen die Hb-Konzentration in der Probe. Fünf bis fünfzehn mg / ml Erträge sind typisch. </li><li> Führen 15-20 ug gereinigtes Hämoglobin auf einem 15% SDS-PAGE in zweifacher Ausfertigung. Flecken eines der Gele; übertragen die Proteine ​​aus einem anderen Gel auf eine Nitrocellulosemembran und Immunoblot für Hämoglobin (Abbildung 2). </li><li> Einfrieren und lagern 1 ml Aliquots von Hämoglobin in flüssigem Stickstoff. </li></ol> 2. Herstellung von Eisen-Abbau Growth Media <ol><li> Planen Roswell Park Memorial Institute (RPMI) Brühe durch Auflösen des Pulvers in RPMI Wasser, fügt Natriumhydrogencarbonat, wie vom Hersteller und 1% cassamino Säuren (CA) (w / v) empfohlen. Sterilisieren, indem sie durch einen 0,2 um Filter und Speicher refrigerated. </li><li> Planen Metall abgereicherten RPMI (NRPMI) durch Zugabe von 7% (w / v) Chelex 100 und Mischen über Nacht auf einer Rührplatte. Entfernen Chelex 100, indem sie durch einen 0,2 um Filter und gekühlt. Zuschlag Medien mit wesentlichen Nicht-Eisen-Metalle: 25 uM ZnCl 2, 25 uM MnCl 2, 100 mM CaCl 2 und 1 mM MgCl 2 im Voraus als sterile 1.000 x Lösung hergestellt. Verwenden Sie Einweg-Kunststoff-Behältern für diesen Schritt, um Eisen Kontamination von wieder verwendbaren Lieferengpässe zu vermeiden. </li></ol> 3. Staphylococcus aureus Wachstum mit Hämoglobin als einziger Eisenquelle <ol><li> Streak S. aureus zur Isolierung von tryptischen Soja-Agar (TSA) aus einem gefrorenen Lager. Bei 37 ° C für 20-24 Stunden. </li><li> Resuspendieren Ethylendiamin-N, N&#39;-Bis (2-hydroxyphenylessigsäure) (EDDHA) in wasserfreiem Ethanol zu 100 mM. EDDHA nicht in Lösung gehen, aber durch Ethanol sterilisiert. </li><li> Hinzufügen EDDHA zu RPMI bis zu einer Endkonzentration von 0,5 mM. ErlaubenEDDHA für mindestens 30 min, bevor zum nächsten Schritt aufzulösen. Aufgrund von Charge zu Charge Variation kann endgültigen EDDHA Konzentration müssen bis 0,25 mM abgesenkt werden, um das Bakterienwachstum zu ermöglichen. </li><li> Beimpfen einzelne Kolonien von S. aureus in 5 ml RPMI mit EDDHA in 15 ml mit Schraubverschluss konische Röhrchen. Bei 37 ° C unter Schütteln mit 180 Umdrehungen pro Minute (rpm) für 16-20 Stunden. </li><li> Zentrifugieren Sie die Übernacht-Kulturen für 5 min bei 7.500 xg und das Pellet in NRPMI mit 0,5 mM EDDHA. Normalisieren OD 600 bis ~ 3. </li><li> Bereiten NRPMI mit 2,5 ug pro ml Hb und 0,1-1,0 mM EDDHA. Durch Variation zwischen den Losen kann die EDDHA Konzentration erforderlich, um freies Eisen in NRPMI Chelat variieren. </li><li> Subculture 10 ul der Bakteriensuspension von Schritt 3,5 in 1 ml NRPMI + EDDHA + Hb in einem 15 ml mit Schraubverschluss konischen Rohr. </li><li> Inkubieren der Kulturen bei 37 ° C für bis zu 48 Stunden unter Schütteln bei 180 UpM oder einer Rolltrommel. </li><li&gt; Jeder 6-12 hr nehmen OD 600 Lesungen, indem 50 ul der Kultur und Mischen mit 150 ul PBS in 96-Well-Platten. </li></ol>

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+weak+protein-protein+complexes+transfer+heme+across+the+cell+wall+of+Staphylococcus+aureus.">Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.</a> Journal of the American Chemical Society. 133, 14176-14179 (2011).</li><li>Muryoi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+the+iron-regulated+surface+determinant+(Isd)+heme+transfer+pathway+in+Staphylococcus+aureus.">Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.</a> J. Biol. Chem. 283, 28125-28136 (2008).</li><li>Reniere, M. L., Skaar, E. P. <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+haem+oxygenases+are+differentially+regulated+by+iron+and+haem.">Staphylococcus aureus haem oxygenases are differentially regulated by iron and haem.</a> Mol. Microbiol. 69, 1304-1315 (2008).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+hemin+transfer+from+IsdA+to+IsdC+in+the+iron-regulated+surface+determinant+(Isd)+heme+acquisition+system+of+Staphylococcus+aureus.">Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus.</a> J. Biol. Chem. 283, 6668-6676 (2008).</li><li>Pishchany, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+for+human+hemoglobin+enhances+Staphylococcus+aureus+infection.">Specificity for human hemoglobin enhances Staphylococcus aureus infection.</a> Cell Host Microbe. 8, 544-550 (2010).</li><li>Pishchany, G., Dickey, S. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+the+Staphylococcus+aureus+heme+iron+transport+components+IsdA+and+IsdB.">Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.</a> Infect. Immun. 77, 2624-2634 (2009).</li><li>Torres, V. J., Pishchany, G., Humayun, M., Schneewind, O., Skaar, E. P. <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+IsdB+is+a+hemoglobin+receptor+required+for+heme+iron+utilization.">Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization.</a> J. Bacteriol. 188, 8421-8429 (2006).</li><li>Kim, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IsdA+and+IsdB+antibodies+protect+mice+against+Staphylococcus+aureus+abscess+formation+and+lethal+challenge.">IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge.</a> Vaccine. 28, 6382-6392 (2010).</li><li>Cheng, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+requirements+for+Staphylococcus+aureus+abscess+formation+and+persistence+in+host+tissues.">Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues.</a> Faseb J. 23, 3393-3404 (2009).</li><li>Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L., Thornton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ions+in+biological+catalysis:+from+enzyme+databases+to+general+principles.">Metal ions in biological catalysis: from enzyme databases to general principles.</a> J. Biol. Inorg. Chem. 13, 1205-1218 (2008).</li><li>Weinberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+availability+and+infection.">Iron availability and infection.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790, 600-605 (2009).</li><li>Drabkin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+Hemin+Chromoproteins.">Metabolism of the Hemin Chromoproteins.</a> Physiological Reviews. 31, 345-431 (1951).</li><li>Graversen, J. H., Madsen, M., Moestrup, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD163:+a+signal+receptor+scavenging+haptoglobin-hemoglobin+complexes+from+plasma.">CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.</a> The international journal of biochemistry &amp; cell biology. 34, 309-314 (2002).</li><li>Torres, V. J., et al. <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+Fur+regulates+the+expression+of+virulence+factors+that+contribute+to+the+pathogenesis+of+pneumonia.">Staphylococcus aureus Fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia.</a> Infect. Immun. 78, 1618-1628 (2010).</li><li>Hammer, N. D., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Staphylococcus+aureus+Iron+Acquisition.">Molecular Mechanisms of Staphylococcus aureus Iron Acquisition.</a> Annu. Rev. Microbiol. , (2011).</li><li>Hurd, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron-regulated+surface+proteins+IsdA,+IsdB,+and+IsdH+are+not+required+for+heme+iron+utilization+in+Staphylococcus+aureus.">The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus.</a> Fems. Microbiology Letters. 329, 93-100 (2012).</li><li>Boys, B. L., Kuprowski, M. C., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetric+behavior+of+hemoglobin+alpha-+and+beta-+subunits+during+acid-induced+denaturation+observed+by+electrospray+mass+spectrometry.">Symmetric behavior of hemoglobin alpha- and beta- subunits during acid-induced denaturation observed by electrospray mass spectrometry.</a> Biochemistry. 46, 10675-10684 (2007).</li><li>Williams, R. C., Tsay, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+chromatographic+method+for+the+preparation+of+human+hemoglobin.">A convenient chromatographic method for the preparation of human hemoglobin.</a> Analytical Biochemistry. 54, 137-145 (1973).</li><li>Shen, T. 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Wir haben nichts zu offenbaren.

Eisen ist ein wichtiger Nährstoff, die durch Organismen aus allen Reichen des Lebens 15 erforderlich. Bei Wirbeltieren wird Eisen sequestriert, um die Toxizität von diesem Element zu vermeiden. Diese Sequestrierung birgt auch Eisen aus eindringende Mikroben in einem Prozess als Nahrungsergänzungsmittel Immunität 16 bekannt. In Reaktion darauf haben die Erreger Strategien, Ernährung Immunität zu umgehen entwickelt. Ein solcher Mechanismus beruht auf Hämoglobin, das ist der häufigste Quelle v...

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> HPLC Anionenaustauschersäule </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkin-Reagenz </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Hämoglobin-Standard </td><td> Pointe Scientific </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex 100 Natrium-Form </td><td> Sigma </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC Standards GmbH </td><td> ANC 001 </td><td></td></tr><tr><td> Hämoglobin ein Antikörper </td><td> Santa Cruz Biotechnology, Inc. </td><td> SC-21.005 </td><td> &lt;/ Td&gt; </tr><tr><td> CASO-Agar </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus ist ein Bakterium, das Eisen durchzuführen lebenswichtige Stoffwechselfunktionen und Krankheiten verursachen erfordert. Das häufigste Reservoir von Eisen im menschlichen Wirt Häm, das der Cofaktor von Hämoglobin ist. Eisen aus Hämoglobin erwerben, S. aureus nutzt ein ausgeklügeltes System, wie der Eisen-regulierten Oberfläche Determinante (Isd) System 1 bekannt. Komponenten des Isd System zuerst bind Host Hämoglobin, dann extrahieren und importieren Häm, und schließlich befreien Eisen aus Häm in dem bakteriellen Zytoplasma 2,3. Dieser Weg ist durch zahlreiche in-vitro-Studien 4-9 wurden seziert. Ferner hat der Beitrag des Isd-System auf eine Infektion immer wieder in Mausmodellen 8,10-14 demonstriert. Gründung der Beitrag des Isd System Hämoglobin-abgeleiteten Eisenaufnahme und das Wachstum hat sich als größere Herausforderung. Growth Assays mit Hämoglobin als einziger Eisenquelle kompliziert by die Instabilität von handelsüblichen Hämoglobin, kontaminierenden freiem Eisen im Nährmedium und Toxizität mit Eisenchelatoren verbunden. Hier präsentieren wir eine Methode, die diese Beschränkungen überwindet. Hochqualitative Hämoglobin wird aus frischem Blut hergestellt und in flüssigem Stickstoff gelagert. Gereinigtes Hämoglobin in ergänzt Eisen-Abbau Mediums imitiert die Eisen-armen Umgebung von Krankheitserregern in der Wirbeltier-Wirt gestoßen. Von hungernden S. aureus von freiem Eisen und Ergänzung mit einer minimal manipulierte Form von Hämoglobin zu induzieren wir Wachstum in einer Weise, die völlig abhängig von der Fähigkeit, Hämoglobin binden, zu extrahieren Häm, Häm passieren durch die bakterielle Zellwand und degradieren Häm im Zytoplasma ist. Dieser Test wird für Forscher, die die Mechanismen der hemoglobin-/heme-derived Eisenaufnahme in S. aufzuklären nützlich aureus und möglicherweise auch andere bakterielle Erreger.

Wir haben gereinigtes humanes Hämoglobin aus Hämolysat mit HPLC (Protocol Schritt 1,7). Abbildung 1 zeigt aufgezeichnete Absorption des Eluats bei 280 und 410 nm Wellenlänge. Fraktion 5 wurde gesammelt und anderen Fraktionen wurden verworfen. Ausbeuten von fünf bis fünfzehn Milligramm Hämoglobin pro Milliliter des Eluats werden typischerweise erworben. Gereinigtes Hämoglobin wurde durch SDS-PAGE analysiert und in doppelter Ausführung wurden die Gele entweder für Proteine ​​gefärbt oder auf...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Hier beschreiben wir ein Wachstum Assay für<em&gt; Staphylococcus aureus</em&gt; Mit Hämoglobin als die alleinige Quelle der verfügbaren Nährstoff Eisen. Dieser Test stellt die Rolle der bakteriellen Faktoren in Hämoglobin-derived Eisenaufnahme beteiligt.

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 ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K Aufrufe.  Vanderbilt University Medical School. Hier beschreiben wir ein Wachstum Assay für Staphylococcus aureus Mit Hämoglobin als die alleinige Quelle der verfügbaren Nährstoff Eisen. Dieser Test stellt die Rolle der bakteriellen Faktoren in Hämoglobin-derived Eisenaufnahme beteiligt.

Serie: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Kostengünstige Methode für Mikrobielle Source Tracking mit spezifischen menschlichen und tierischen Viren

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

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 ...

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 ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Quantifizierung der Zytotoxizität von Staphyloccus aureus gegen humane polymorphonukleare Leukozyten

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...