Diese Arbeit wurde durch ein Burroughs-Wellcome Career Award und von den National Institutes of Health gewähren AI074832 zu GY Liu unterstützt.

1. Vorbereiten des MRSA für Infektionsforschung (zwei Tage vor der Infektion) <ol><li> Impfen einer Öse MRSA aus einer Stammkultur eine Blut-Agar (Trypticase Soy Agar (TSA)) Platte. </li><li> Überprüfen Sie die Hämolyse Phänotyp (eine klare Zone um jede Kolonie) auf das Blut-Agar-Platte. </li><li> Wählen Sie eine Kolonie, die eine hämolytische Phänotyp, die im Einklang mit anderen Experimenten wurde. </li><li> Impfen der Kolonie in 3 ml Todd Hewitt Broth (THB), mit dem angeeigneten Antibiotikum, wenn nötig, in einer 15 ml-Snap-Röhrchen. </li><li> Bei 37 ° C über Nacht unter Schütteln bei 220 Umdrehungen pro Minute. </li></ol> 2. Vorbereitung der Mäuse für Infektionsforschung (einen Tag vor der Infektion) <ol><li> Je nach Größe der Mäuse, rasieren das Fell ab einer 3 x 4 cm-Bereich auf der Rückseite von Mäusen. </li><li> Bewerben ~ 5 mm 3 Haarentferner Creme (gekauft von einer örtlichen Drogerie) auf die rasierte Fläche. </li><li> Lassen Sie die Haarentfernung Creme auf der Haut für ca. 1 min inkubieren. </li><li> Wet Papierhandtücher mit ddH 2 O. </li><li> Wischen Sie die Haarentfernung Creme mit dem nassen Papiertuch. </li></ol> 3. Vorbereiten des MRSA für Infektionsforschung (am Tag der Infektion) <ol><li> Verdünnen Sie die Nacht Bakterienkultur bei 1:500 bis 1:1000 mit 10 ml vorgewärmten THB, in einem 50 mL Röhrchen mit Schraubverschluss. </li><li> Bei 37 ° C für ca. 2,5 Stunden unter Schütteln bei 220 rpm, bis A 540 erreicht 2,5. </li><li> Sammeln MRSA durch Zentrifugation bei 3.225 xg für 10 min bei 4 ° C. </li><li> Überstand verwerfen. </li><li> Resuspendieren Sie das Bakterienpellet in 10 ml Dulbecco phosphatgepufferte Kochsalzlösung (DPBS). </li><li> Wiederholen Sie die Schritte 3 und 4. </li><li> Resuspendieren Sie das Bakterienpellet in DPBS auf eine gewünschte Konzentration. </li><li> Serienmäßig verdünnen Bakteriensuspension von Januar 10 - August 10. </li><li> Platte der verdünnten Bakteriensuspension auf Blut-Agar-Platten. </li><li> Die Inkubation bei 37 ° C über Nacht. </li><li> Beobachtung und Aufzeichnung der hämolytischen Phänotyp der Inokula. </li><li> Zählen Sie die Kolonie bildenden Einheiten (KBE) Nummer auf der Platte. </li><li> Berechnen Sie die Inokula auf die CFU-Nummer auf der Platte basiert. </li></ol> 4. Subkutane Infektion von MRSA (am Tag der Infektion und 3 Tage nach der Infektion) <ol><li> Inject subkutan 100 ul Bakteriensuspension in die rasierte Fläche. </li><li> Beobachten Sie die Tiere für 3 bis 5 Stunden nach der Injektion sicherstellen, dass die Mäuse am Leben sind. </li><li> Beachten Sie die Läsion auf dem Rücken der Tiere täglich und notieren Sie die Läsion Bereich täglich, wenn es gebraucht wird. Läsion Beobachtung sollte die Aufnahme der Läsion Größe und Morphologie. Beide Haut und Muskel Läsionen sind durch Multiplikation der Länge und Breite der Läsion quantifiziert. Unregelmäßig geformte Läsionen müssen in kleinere symmetrische Stücke zerbrochen werden, und jedes Stück mit dem gleichen Verfahren gemessen. Läsion Messungen können auch eine EDV-gestützte histomorphometrische Assessment Program (ImageJ, Open-Source zur Verfügung von der NIH in <a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij/</a> ) 2. </li><li> Sacrifice die Tiere am Tag 3 nach der Infektion durch Inhalation von Isofluran durch Genickbruch folgte. </li><li> Beobachtung und Aufzeichnung der Läsion auf der Haut. </li><li> Nick die Haut mit einem Paar sterile Schere. </li><li> Ziehen Sie die Haut vorsichtig ab und beobachten und aufzeichnen der Läsion auf den Muskel. </li><li> Excise der Läsion und ca. 2 bis 5 mm von der Umgebung. </li><li> Sammeln Milz und Nieren. </li><li> Homogenisieren des Gewebes mit einem Gewebe-Homogenisator oder durch wiederholte Anwendung eines Kolbens aus einer 1 ml-Spritze ein Mikrozentrifugenröhrchen mit dem Gewebe und 100 ul DPBS. </li><li> Add 900 ul DPBS in jedes Röhrchen. </li><li> Vortex die Proben für 5 min. </li><li> Serienmäßig verdünnt die Suspension mit DPBS 1 Oktober - 6. Oktober. </li><li> Platte der verdünnten Suspensionen auf THA-Platten. </li></ol> 5. Repräsentative Ergebnisse: <ol><li> Unmittelbar nach jeder subkutane Injektion wird eine Blase an der Hautoberfläche beobachtet werden, wenn die Infektion richtig eingestellt ist (Abbildung 1A) durchgeführt. Wenn keine Bläschen auf der Haut beobachtet wird, kann die Injektion zu tief sein, und dies kann das Ergebnis der Infektion (Größe der Läsion, CFU) zu beeinflussen. </li><li> Die Bakteriensuspension muss seriell verdünnt und die CFU müssen auf Blutagarplatten für jedes Experiment untersucht werden. Dieser Schritt wird liefern wichtige Informationen: 1) ob die Inokula homogenen Phänotyp haben, 2) die exakte praktikable Keimzahl für die Infektion. </li><li> Die Läsionen und bakteriellen Überleben kann zu verschiedenen Zeitpunkten nach der Infektion beurteilt werden. Hier haben wir die Infektion an Tag 3 nach der Infektion. Die Läsion Größen sind auf der Hautoberfläche (: 1x 10 9; Abbildung 1D: 1B 1 x 10 7 CFU) analysiert und auf den Muskel (Abbildung 1C: 1x 10 9 </sbis&gt;; Abbildung 1E: 1 x 10 7 CFU). Die Läsion Bereich gleicher Länge (mm) x Breite (mm) kann für die Beurteilung der Gewebeschädigung (Abb. 2 A und B) gemessen werden. Dieses Ergebnis könnte helfen, bestimmen das Ausmaß der Gewebeschädigung durch einen bestimmten Virulenzfaktor verursacht. Darüber hinaus sind die Läsionen exzidiert und homogenisiert in DPBS und beschichtet, um die CFU-Nummer (Abbildung 2C), welche die Lebensfähigkeit der Bakterien am Ort der Infektion zeigt quantifizieren. </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Haut und Muskel Läsionen. CD1 Mäusen wurden subkutan an einer Flanke mit MRSA (LAC) geimpft. Die Bilder wurden zum Zeitpunkt 0 und Tag 3 nach der Infektion aufgenommen. (A) Zeit 0 nach der Infektion (pi), (B) - (G) Tag 3 pi; B, D. und F: Hautläsionen; C, E und G: Muskel-Läsionen; B und C: 1x 10 9 CFU infiziert, D und E: 1x 10 7 CFU infiziert, F und G: Mock. <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Hautläsion Größe und lebensfähigen Bakterien am Ort der Infektion zählen am Tag 3 nach der Infektion. Haut-und Muskel-Verletzungen auf MRSA (LAC) infiziert CD1-Mäusen. (A) Hautläsion Größe. (B) Muscle Läsionsgröße. (C) Total Gewebe CFU. H: 1 x 10 9 KBE Inokula; L: 1 x 10 7 CFU Inokula. *: P &lt;0,05, Mann-Whitney-Test.

<ol>
	<li>Ahn, J. Y., Song, J. Y., Yun, Y. S., Jeong, G., Choi, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+Staphylococcus+aureus-infected+septic+mice+by+suppression+of+early+acute+inflammation+and+enhanced+antimicrobial+activity+by+ginsan.">Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan.</a> FEMS Immunol Med Microbiol. 46, 187-197 (2006).</li><li>Girish, V., Vijayalakshmi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affordable+image+analysis+using+NIH+Image/ImageJ.">Affordable image analysis using NIH Image/ImageJ.</a> Indian J Cancer. 41, 47-47 (2004).</li><li>Hahn, B. L., Onunkwo, C. C., Watts, C. J., Sohnle, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+dissemination+and+cutaneous+damage+in+a+mouse+model+of+staphylococcal+skin+infections.">Systemic dissemination and cutaneous damage in a mouse model of staphylococcal skin infections.</a> Microb Pathog. 47, 16-23 (2009).</li><li>Hruz, P., Zinkernagel, A. S., Jenikova, G., Botwin, G. J., Hugot, J. P., Karin, M., Nizet, V., Eckmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD2+contributes+to+cutaneous+defense+against+Staphylococcus+aureus+through+alpha-toxin-dependent+innate+immune+activation.">NOD2 contributes to cutaneous defense against Staphylococcus aureus through alpha-toxin-dependent innate immune activation.</a> Proc Natl Acad Sci U S A. 106, 12873-12878 (2009).</li><li>Ji, Y., Zhang, B., Van, S. F., Horn, P. W. a. r. r. e. n., Woodnutt, G., Burnham, M. K., Rosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+critical+staphylococcal+genes+using+conditional+phenotypes+generated+by+antisense+RNA.">Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.</a> Science. 293, 2266-2269 (2001).</li><li>Somerville, G. A., Beres, S. B., Fitzgerald, J. R., DeLeo, F. R., Cole, R. L., Hoff, J. S., Musser, 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=In+vitro+serial+passage+of+Staphylococcus+aureus:+changes+in+physiology,+virulence+factor+production,+and+agr+nucleotide+sequence.">In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.</a> J Bacteriol. 184, 1430-1437 (2002).</li><li>Tseng, C. W., Kyme, P., Low, J., Rocha, M. A., Alsabeh, R., Miller, L. G., Otto, M., Arditi, M., Diep, B. A., Nizet, V., Doherty, T. M., Beenhouwer, D. O., 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=Staphylococcus+aureus+Panton-Valentine+leukocidin+contributes+to+inflammation+and+muscle+tissue+injury.">Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.</a> PLoS One. 4, e6387-e6387 (2009).</li><li>von Kockritz-Blickwede, M., Rohde, M., Oehmcke, S., Miller, L. S., Cheung, A. L., Herwald, H., Foster, S., Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+mechanisms+underlying+the+genetic+predisposition+to+severe+Staphylococcus+aureus+infection+in+the+mouse+model.">Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.</a> Am J Pathol. 173, 1657-1668 (2008).</li></ol>

<ol><li> Die murine Haut-und Weichteilinfektionen Infektionen Modell ist ein leistungsfähiges Werkzeug für die in vivo Virulenz Beurteilung eines Erregers. Die Pathogenität von S. aureus in Haut-und Weichteilinfektionen kann je nach einer Reihe von Parametern. Dazu gehören Inokulum Größe, das Wachstum von Bakterien Phase, Tiefe der Impfung, Alter der Mäuse, und die Maus genetischen Hintergrund 7, 8. Bei der Prüfung der Virulenz-Funktion mit diesem Modell ist es wichtig, für diese Parameter werden, um sicher...

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<td>THB</td>
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subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

MRSA ist eine weltweite Bedrohung für die öffentliche Gesundheit und MRSA Haut-und Weichteil-Infektionen mittlerweile mehr als die Hälfte aller Weichteil-Infektionen in den Vereinigten Staaten. Unter Weichteilinfektionen, haben Myositis, pyomyositis und nekrotisierende Fasziitis zunehmend in Verbindung mit MRSA aus der Gemeinde berichtet. Um zu verstehen, das Wechselspiel zwischen MRSA-und Host-Immunität führt zu schweren Infektion ist die Verfügbarkeit von Tiermodellen kritisch, erlaubt die Untersuchung von Host-und bakterielle Faktoren. Mehrere Infektion Modelle wurden eingeführt, um die Pathogenese von S. bewerten aureus während oberflächliche Infektion der Haut. Hier beschreiben wir eine subkutane Infektion Modell, das die Haut, Unterhaut und Muskulatur Pathologien untersucht.

Watch this Scientific Journal Video about Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA at JoVE.com

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine Haut-und Weichteilinfektionen Infektionen Modell ist für die Beurteilung der Virulenz Funktion von Methicillin-resistenten Staphylococcus aureus (MRSA) und dem Host-Immunreaktionen eingesetzt. Hier präsentierten wir eine subkutane Infektion Modell für Haut-und Weichteilinfektionen.

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 ...

subcutaneous-infection-methicillin-resistant-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

22.8K Aufrufe.  Cedars-Sinai Medical Center. Murine Haut-und Weichteilinfektionen Infektionen Modell ist für die Beurteilung der Virulenz Funktion von Methicillin-resistenten Staphylococcus aureus (MRSA) und dem Host-Immunreaktionen eingesetzt. Hier präsentierten wir eine subkutane Infektion Modell für Haut-und Weichteilinfektionen.

Serie: Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

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

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 ...

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. ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Der Nachweis eines multiresistenten<em&gt; Mycobacterium tuberculosis</em&gt; Verstärkung Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Aufbau und Optimierung eines Hochdurchsatz-Setup zum Studium<em&gt; Staphylococcus epidermidis</em&gt; Und<em&gt; Mycobacterium marinum</em&gt; Infektion als Modell für Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

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 ...