NIAID gewähren K22AI081748.

1. Die Drip Flow-Biofilm-Reaktor <ol><li> Die Tropf-Flow Biofilmreaktor (erhältlich bei Biosurface Technologies oder kundenspezifische Versionen können in der Regel von der Universität Werkstätten vorgenommen werden, siehe Abbildung 1) montiert ist und autoklaviert. Montage beinhaltet das Anbringen Coupons in Kammern und Sicherung Kammer Deckel. Die Kammer, zusammen mit Biofilm Medium (CASO-Bouillon 2 g / L und Glucose 2 g / L) und Zulauf Nährstoff-Rohren werden durch Autoklavieren sterilisiert. </li><li> Beimpfung des Tropf Reaktor wird erreicht, indem der Reaktor auf eine flache Oberfläche, Klemmung des Abwassers Schlauchleitungen, Abfüllung jeder Kammer mit 10 ml CASO-Bouillon und Zugabe von 10 ul einer S. vorgeformten aureus Kultur über Nacht in CASO-Bouillon gewachsen. Die beimpften Reaktor wird dann in einem 37 ° C Inkubator Platz für 18 Stunden. </li><li> Nach 18 Stunden Inkubationszeit wird das Abwasser Rohr abgeklemmt und der Reaktor auf einen Holzblock geschnitten, um eine 10 °-Winkel angeordnet. </li><li> Aseptisch verbinden Zulauf Nährstoff Schlauch an der Flasche mit der kontinuierlichen Nährlösung. Feed the Rohrleitung durch die Pumpe und prime die Rohre, indem Sie die Pumpe mit einer maximalen Geschwindigkeit (variiert je nach Pumpentyp). </li><li> Sobald der Zulauf-Schlauch ist grundiert Stopp der Pumpe und befestigen Sie verbinden Nadeln (22 Gauge, 1 Zoll) bis zum Ende jeder Röhre. Wischen Sie die Kammereinlass Stopfen mit einem Ethanol-Wisch-und aseptisch legen Sie die Nadeln durch den Einlass Stopper. </li><li> Schalten Sie die Pumpe und lassen Sie die Medien langsam tropft (Durchfluss bis 125 ul / Minute) über die Coupons. Die Medien sollten nach unten entlang der Kupon vom Einlass Anschlag Anschluss an das Abwasser-Anschluss. Betreiben Sie den Reaktor in kontinuierlichen Fluss für 2-5 Tage (je nach Anwendung), gelegentlich die Überprüfung der Reaktor für eine ordnungsgemäße Entwässerung. </li><li> Zur Ernte der Tropf Reaktor Biofilmen, die Pumpe stoppen und entfernen Sie vorsichtig die Nadeln aus dem Reaktor. Der Reaktor kann dann auf einer ebenen Fläche aufgestellt werden und die Coupons können aseptisch entfernt werden mit einer sterilen Pinzette. Wenn Mikroskopie ist erwünscht, die Coupons können nun entsprechend verarbeitet werden (Abbildung 2B ist eine rasterelektronenmikroskopische Aufnahme einer S. aureus Biofilm in einen Tropf Biofilmreaktor gewachsen). Wenn die Quantifizierung des Biofilms Biomasse oder Physiologie Studien das Ziel der Studie sind, kann der Biofilm aus den Coupon mit einem Zellschaber entfernt werden. Halten Sie die Coupons mit einer Pinzette vorsichtig abkratzen der Biofilm aus den Coupon in einem konischen Rohr mit Phosphat-gepufferter Kochsalzlösung mit einem Zellschaber. Hinweis: Zur Quantifizierung der Kolonie-bildenden Einheiten in den Biofilmen, ist es notwendig, die geerntet Biofilmen mit einem Gewebe-Homogenisator zu Klumpen disaggregate und bilden eine homogene Suspension zu homogenisieren. Verschiedene Modelle von Gewebe Homogenisatoren werden für diese Anwendung geeignet. Wir verwenden eine Fisher Scientific Tissuemiser Homogenisator (Produkt # 15-338-420) bei voller Geschwindigkeit für 1 Minute bis Biofilm Proben zu homogenisieren. Die Nichtbeachtung der Biofilm homogenisieren wird in einer Unterschätzung der Kolonie bildenden Einheiten in der Probe vorhanden führen. </li></ol> 2. Die rotierende Scheibe Biofilm Reactor <ol><li> Die rotierende Scheibe Biofilmreaktor (erhältlich bei erhältlich Biosurface Technologies oder können nach Maß, siehe Abbildung 3) montiert ist und autoklaviert. Montieren Sie den Reaktor durch den ersten Platz die sich drehende Scheibe Coupons in die Schlitze der drehenden Scheibe und legte sie in einen 1-Liter Becherglas mit einem Überlauf-Anschluss. Eine Reihe 15-Gummistopfen mit Löchern darin gebohrt, um Fördermedien-und Entlüftung zu ermöglichen, da der Reaktor Kappe verwendet. Der Reaktor, Biofilm Medien (CASO-Bouillon 2 g / L und Glucose 2 g / L) und Zulaufschlauch werden dann durch Autoklavieren sterilisiert. </li><li> Die rotierende Scheibe Biofilmreaktor ist, indem 250 ml des sterilen Mediums in den Reaktor geimpft und Zugabe von 0,5 ml einer Übernachtkultur S. aureus Kultur in CASO-Bouillon gewachsen. Der Reaktor wird dann auf einer Rührplatte auf 250 rpm gestellt und über Nacht bei der gewünschten Temperatur. </li><li> Nach 16 Stunden Inkubationszeit verbinden Sie den Zulaufschlauch mit dem Anschluss auf mittlere Reservoir und verbinden Sie es mit der Schlauchpumpe. Die Pumpe wird dann auf Turn und Fluss auf ~ 0,25 ml / min (Durchfluss kann je nach Wachstum gewünscht verändert werden). </li><li> Nach 24 Stunden beenden den Reaktor und aseptisch entnehmen Sie die Disc, ohne den Biofilm-Coupons. Entfernen Sie die Coupons mit einer sterilen Pinzette und tauchen jeweils in Phosphat-gepufferter Kochsalzlösung keine lose Bakterien zu entfernen. </li><li> Für antimikrobielle Verbindung testen, können die Chips in die einzelnen Vertiefungen einer 96-Well-Platte mit Verbindungen von Interesse platziert werden. Nach der Inkubation werden Chips in 1,5 ml Reaktionsgefäßen mit 1 mL Phosphat-Puffer-Kochsalzlösung überführt und homogenisiert mit einem Gewebe-Homogenisator auf den intakten Biofilm zu zerstreuen. </li><li> Die Zellen werden dann seriell verdünnt und ausplattiert auf Nähragar Medium, um lebensfähige Kolonie Formi bestimmenng Einheiten. </li><li> Hinweis: viele Varianten finden Sie auf der oben genannten Protokoll vorgenommen werden. Zum Beispiel kann sich drehende Scheibe Coupons mit beschichtet werden oder ausgefertigten potenzieller Anti-Biofilm-Verbindungen, um die Wirksamkeit zu testen. Biofilm Dispergiermittel können auch ausgewertet werden Inkubation Coupons in Dispergiermittel Verbindungen und Quantifizierung von Bakterien angebracht gegenüber abgenommen werden. </li></ol> 3. Repräsentative Ergebnisse Ein Beispiel für eine Einrichtung Tropf Reaktor ist, wenn Abbildung 1 dargestellt. Nach drei Tagen fließen reichlich Biofilm wird auf dem Coupon Oberfläche, 2A akkumulieren. Die gesamte Biomasse ist abhängig von Bakterienstämmen und präzise Wachstumsbedingungen. Eine rasterelektronenmikroskopische Aufnahme eines S. aureus Biofilm in den Tropf Reaktor aufgewachsen ist in Abbildung 2B gezeigt. Eine rotierende Scheibe Reaktor ist in Abbildung 3A gezeigt. Das Protokoll beschrieben, kann auf die spezifischen Anforderungen in der Regel keine Mikroorganismus, der Bildung eines Biofilms angepasst werden. Abbildung 3B zeigt die sich drehende Scheibe mit 18 Kunststoff-Platten angebracht. Diese Platte gewachsenen Biofilmen sind besonders gut für antimikrobielle Tests geeignet und liefern gut reproduzierbare Ergebnisse 11. <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Der Tropf Reaktor-Setup. Wichtige Komponenten sind beschriftet. <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Beispiel für einen Tropf flow S. aureus Biofilm. A) Dieser Biofilm wurde drei Tage nach dem beschriebenen Protokoll gewachsen. Deckel aus den ersten beiden Kammern des Reaktors entfernt werden, um die gelbe S. zeigen aureus Biofilm Biomasse. B) Sacnning elektronenmikroskopische Aufnahme eines S. aureus Biofilm in den Tropf Reaktor aufgewachsen. <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Die rotierende Scheibe Reaktor. A) Beispiel für ein laufendes Spinning-Disk-Reaktor. Key-Komponente gekennzeichnet sind. B) Nahaufnahme einer sich drehenden Scheibe.

<ol>
	<li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Biofilms.">Microbial Biofilms.</a> Annu. Rev. Microbiol. 49, 711-745 (1995).</li><li>Costerton, J. W., Cheng, K. J., Gessey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., Marrie, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+nature+and+disease.">Bacterial biofilms in nature and disease.</a> Ann. Rev. Microbiol. 41, 435-464 (1987).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signaling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis.</a> Mol. Micro. 28, 449-461 (2002).</li><li>Boles, B. R., Horswill, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agr-mediated+dispersal+of+Staphylococcus+aureus+biofilms.">Agr-mediated dispersal of Staphylococcus aureus biofilms.</a> PLoS Pathog. 4, e1000052-e1000052 (2008).</li><li>Goeres, D. M., Haamilton, M. A., Beck, N. A., Buckingham-Meyer, K., Hilyard, J., Loetterle, L. A., Walker, D. K., Stewart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor.">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nature Protocols. 4, 783-788 (2009).</li><li>Fu, W., Forster, T., Mayer, O., Curtin, J. J., Lehman, S. M., Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+cocktail+for+the+prevention+of+biofilm+formation+by+Pseudomonas+aeruginosa+on+catheters+in+an+in+vitro+model+system.">Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system.</a> Antimicrob Agents Chemother. 54, 397-404 (2010).</li><li>Xu, K. D., McFeters, G. 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Biofilme in verschiedenen Reaktoren gezüchtet wird, haben oft unterschiedliche Eigenschaften und jeden Reaktor verfügt über verschiedene Anwendungen. In dieser Arbeit beschreiben wir die Verwendung von zwei Biofilmreaktoren: einen Tropf flow Biofilmreaktor und einer rotierenden Scheibe Reaktor. Drip Strömungsreaktoren sind nützlich für den Anbau von geringer Scherung Biofilmen bei einem Luft-Flüssigkeit-Grenzfläche und sind für eine Vielzahl von Bedingungen. Wir finden sie sehr bequem für Studien, in denen ein...

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

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<td>Drip Flow Reactors</td>
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<td>BioSurface Technologies Corporation</td>
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<td>Rotating Disk Reactors</td>
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<td>BioSurface Technologies Corporation</td>
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the use of drip flow and rotating disk reactors for staphylococcus aureus biofilm analysis

Die meisten Mikroben in der Natur gedacht, als Aufputz-assoziierten Gemeinschaften in Biofilmen existieren. 1 Bakterielle Biofilme sind in einer Matrix umgeben und an einer Oberfläche. 2 Biofilmbildung und Entwicklung sind häufig im Labor mit der Batch-Systeme, wie Mikrotiterplatten oder Durchfluss studiert Systeme, wie zB Flow-Zellen. Diese Methoden sind nützlich für das Screening von Mutanten und chemischen Bibliotheken (Mikrotiterplatten) 3 oder wachsende Biofilme zur Visualisierung (flow-Zellen) 4. Hier präsentieren wir Ihnen detaillierte Protokolle für den Anbau von Staphylococcus aureus in zwei weitere Arten von Flow-System Biofilme: die Tropf-Flow Biofilmreaktor und der rotierenden Scheibe Biofilmreaktor. Drip flow Biofilm Reaktoren sind für die Untersuchung von Biofilmen unter geringer Scherung Bedingungen angebaut konzipiert. 5 Der Tropf Reaktor besteht aus vier parallelen Test-Kanäle, einer Kapazität von jeweils einem Standard-Glasobjektträger großen Coupon oder eine Länge von Katheter oder Stint. Die Tropf-Flow-Reaktor ist ideal für Mikrosensorik Überwachung, allgemeine Biofilm Studien, Biofilm Kryoschneiden Proben, hohe Produktion von Biomasse, medizinisches Material Auswertungen und innewohnenden medical device testing. 6,7,8,9 Die rotierende Scheibe Reaktor besteht aus einem Teflon-Platte mit Aussparungen für herausnehmbare Coupons. 10 Die herausnehmbare Coupons können von jedem zerspanbaren Material. Der Boden der rotierenden Scheibe enthält einen Stabmagneten, damit Plattenrotation zu Flüssigkeitsoberfläche Scher über flächenbündige Coupons zu erstellen. Die gesamte Festplatte mit 18 Coupons ist in einem 1000 mL Glas Seitenarm Reaktor vorgelegt. Eine flüssige Nährmedien wird durch den Behälter zirkuliert, während die Festplatte durch einen Magnetrührer gedreht wird. Die Coupons werden aus dem Reaktor entnommen und dann abgeschabt, um den Biofilm Probe für weitere Untersuchungen oder Mikroskopie zu sammeln. Rotating Disc Reaktoren sind für Laboruntersuchungen von Biozid-Wirksamkeit, Biofilmentfernung und Leistung von Anti-Fouling-Materialien entwickelt. 9,11,12,13

Watch this Scientific Journal Video about The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis at JoVE.com

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

Protokolle für die Nutzung offenes System fließen Biofilmen mit Tropf Strömungsreaktoren und rotierenden Scheibe Reaktoren sind im Detail vorgestellt.

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 ...

the-use-drip-flow-rotating-disk-reactors-for-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

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

17.0K Aufrufe.  University of Michigan. Protokolle für die Nutzung offenes System fließen Biofilmen mit Tropf Strömungsreaktoren und rotierenden Scheibe Reaktoren sind im Detail vorgestellt.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa und Saccharomyces cerevisiae Biofilm im Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

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 ...

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. ...

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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Charakterisierung der menschliche Monocyte Teilmengen von Vollblut Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...