Questo lavoro è stato supportato da una Burroughs-Wellcome Premio alla Carriera e da National Institutes of Health a concedere AI074832 GY Liu.

1. Preparazione del MRSA per infezione (due giorni prima infezione) <ol><li> Inoculare un&#39;ansata di MRSA da una coltura madre ad un agar sangue (soia-agar (TSA)) piastra. </li><li> Controllare il fenotipo emolisi (una zona chiara intorno ad ogni colonia) sulla piastra di agar sangue. </li><li> Scegli una colonia che ha un fenotipo emolitica che è coerente con altri esperimenti. </li><li> Inoculare la colonia in 3 ml di Todd Hewitt Broth (THB), con l&#39;antibiotico appropriato quando necessario, in un mL 15 snap-provetta. </li><li> Incubare a 37 ° C per una notte in agitazione a 220 rpm. </li></ol> 2. Preparazione del Mice per infezione (un giorno prima infezione) <ol><li> A seconda delle dimensioni dei topi, accorciare il pelo fuori uno x 3 4 cm sulla superficie posteriore di topi. </li><li> Applicare ~ 5 mm 3 crema Epilatore (acquistato da un negozio locale di droga) per l&#39;area rasata. </li><li> Lasciare la crema Epilatore per incubare sulla superficie della pelle per circa 1 min. </li><li> Tovaglioli di carta bagnati con DDH 2 O. </li><li> Togliere la crema Epilatore con il tovagliolo di carta bagnato. </li></ol> 3. Preparazione del MRSA per infezione (nel giorno di infezione) <ol><li> Diluire la coltura batterica durante la notte a 1:500 a 1:1000 con 10 ml di pre-riscaldato THB, in una da 50 ml con tappo a vite del tubo. </li><li> Incubare a 37 ° C per circa 2,5 ore con agitazione a 220 giri al minuto, fino a raggiungere 540 2.5. </li><li> Raccogliere MRSA per centrifugazione a 3.225 xg per 10 minuti a 4 ° C. </li><li> Scartare il surnatante. </li><li> Risospendere il pellet batterico in 10 ml di fosfato salina tamponata Dulbecco (DPBS). </li><li> Ripetere i passaggi 3 e 4. </li><li> Risospendere il pellet batterico in DPBS ad una concentrazione desiderata. </li><li> Serie diluire la sospensione batterica da 10 Gennaio - 10 AGOSTO. </li><li> Piastra la sospensione diluita batterica su piastre di agar sangue. </li><li> Incubare le piastre a 37 ° C durante la notte. </li><li> Osservare e registrare il fenotipo emolitica del inoculi. </li><li> Contare le unità formanti colonia (CFU) il numero sul piatto. </li><li> Calcola il inoculi in base al numero UFC sul piatto. </li></ol> 4. Sottocutaneo infezione di MRSA (nel giorno di infezione e 3 giorni dopo l&#39;infezione) <ol><li> Iniettare per via sottocutanea 100 microlitri della sospensione batterica nella zona rasata. </li><li> Osservare gli animali da 3 a 5 ore dopo l&#39;iniezione per assicurarsi che i topi sono vivi. </li><li> Osservare la lesione sul dorso degli animali e registrare ogni giorno la zona della lesione ogni giorno quando è necessario. Osservazione lesione dovrebbe includere la registrazione di dimensione della lesione e morfologia. Sia la pelle e le lesioni muscolari sono quantificata moltiplicando la lunghezza e la larghezza della lesione. Lesioni di forma irregolare devono essere suddivisi in piccoli pezzi simmetrici, e ogni pezzo misurato con lo stesso metodo. Misure lesione può anche utilizzare un computer-assistita del programma di valutazione istomorfometrica (ImageJ; open-source disponibile presso il NIH di <a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij/</a> ) 2. </li><li> Sacrificare gli animali al giorno 3 dopo l&#39;infezione per inalazione dei isoflurano seguita da dislocazione cervicale. </li><li> Osservare e registrare la lesione sulla pelle. </li><li> Nick la pelle con un paio di forbici sterili. </li><li> Sbucciare la pelle fuori e osservare attentamente e registrare la lesione del muscolo. </li><li> Accise della lesione e di circa 2 - 5 mm della zona circostante. </li><li> Raccogliere milza e reni. </li><li> Omogeneizzare i tessuti utilizzando un omogeneizzatore tessuto o dalla ripetuta applicazione di un pistone di una siringa da 1 ml in una provetta da microcentrifuga contenente il tessuto e 100 ml di DPBS. </li><li> Aggiungere 900 ml di DPBS ad ogni provetta. </li><li> Vortex i campioni per 5 minuti. </li><li> Serie diluire la sospensione con DPBS 10 gennaio-10 giugno. </li><li> Piastra le sospensioni diluito su piastre THA. </li></ol> 5. Rappresentante dei risultati: <ol><li> Subito dopo ogni iniezione sottocutanea, una bozza verrà osservato sulla superficie della pelle quando l&#39;infezione è fatto correttamente (Figura 1A). Se non bleb si osserva sulla superficie della pelle, l&#39;iniezione può essere troppo profondo, e questo può influenzare l&#39;esito dell&#39;infezione (dimensione della lesione, CFU). </li><li> La sospensione batterica deve essere diluito in serie e la CFU devono essere esaminati su piastre di agar sangue per ogni esperimento. Questo passo porterà importanti informazioni: 1) se l&#39;inoculo hanno fenotipo omogeneo; 2) l&#39;esatto numero di organismi vitali per l&#39;infezione batterica. </li><li> Le lesioni e la sopravvivenza dei batteri può essere valutato in vari momenti dopo l&#39;infezione. Qui, abbiamo esaminato l&#39;infezione al giorno 3 dopo l&#39;infezione. Le dimensioni della lesione vengono analizzati sulla superficie della pelle (Figura 1B: 1x 10 9; 1D Figura: 1 x 10 7 CFU) e sulla superficie del muscolo (Figura 1C: 1x 10 9 </sfino&gt;; Figura 1E: 1 x 10 7 CFU). L&#39;area di lesione uguale lunghezza (mm) x larghezza (mm) può essere misurato per valutare il danno tissutale (Fig. 2 A e B). Questo risultato può aiutare a determinare l&#39;entità del danno tissutale causato da un fattore di virulenza dato. Inoltre, le lesioni sono escisse e omogeneizzati in DPBS, e placcato per quantificare il numero di CFU (Figura 2C), che indica la vitalità dei batteri nella sede di infezione. </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="Figura 1" /> Figura 1. La pelle e le lesioni muscolari. CD1 topi sono stati inoculati per via sottocutanea su un fianco con MRSA (LAC). Le foto sono state scattate a 0 ora e il giorno 3 post-infezione. (A) durante il giorno 0 post-infezione (pi), (B) - (G) 3 ° giorno pi, B, D e F: lesioni cutanee, C, E e G: lesioni muscolari, B e C: 1 x 10 9 CFU infetti, D ed E: 1 x 10 7 CFU infetti, F e G: finto. <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="Figura 2" /> Figura 2. Dimensioni della lesione cutanea e batteri vitali contare nella sede di infezione al giorno 3 dopo l&#39;infezione. Lesioni della pelle e muscoli sulla MRSA (LAC) infettati topi CD1. (A) dimensioni lesione della pelle. (B), dimensione della lesione muscolare. (C) Totale CFU tessuto. H: 1 x 10 9 inoculi CFU; L: 1 x 10 7 CFU inoculi. *: P &lt;0,05, test di Mann-Whitney.

<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>

Nessun conflitto di interessi dichiarati.

<ol><li> La pelle morbida modello murino e l&#39;infezione dei tessuti è un potente strumento per la valutazione di virulenza in vivo di un agente patogeno. La patogenicità di S. aureus in infezioni della pelle e dei tessuti molli può variare in funzione una serie di parametri. Queste includono la dimensione dell&#39;inoculo, fase di crescita batterica, la profondità di inoculazione, all&#39;età di topi, e lo sfondo del mouse genetico 7, 8. Nell&#39;esaminare la funzione di virulenza utilizzando questo...

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<td>THB</td>
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<td>DPBS</td>
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<td>Invitrogen</td>
<td>21-031-CV</td>
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<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309602</td>
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<td>27G1/2 needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
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<td>Sheep Blood Agar (TSA)</td>
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subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

MRSA è una minaccia mondiale per la salute pubblica, e la pelle MRSA e dei tessuti molli ora rappresentano più della metà di tutti i tessuti molli negli Stati Uniti. Fra tessuti molli, miosite, pyomyositis, e fascite necrotizzante sono state aumentate le segnalazioni in associazione con MRSA derivanti dalla comunità. Per capire l&#39;interazione tra MRSA e immunità dell&#39;ospite che porta a più gravi infezioni, la disponibilità di modelli animali è fondamentale, permettendo lo studio dei fattori di accoglienza e batterica. Diversi modelli di infezione sono stati introdotti per valutare la patogenesi di S. aureus durante l&#39;infezione superficiale della pelle. Qui, descriviamo un modello di infezione per via sottocutanea che esamina la pelle, sottocutaneo, muscolare e patologie.

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

Murino e morbida pelle modello di infezione dei tessuti viene utilizzato per valutare la funzione di virulenza di S. aureus meticillino-resistente (MRSA) e le risposte immunologiche ospite. Qui, abbiamo presentato un modello di infezione per via sottocutanea per la pelle e le infezioni dei tessuti molli.

Sottocutaneo infezione di Staphylococcus aureus meticillino resistente (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 Views.  Cedars-Sinai Medical Center. Murino e morbida pelle modello di infezione dei tessuti viene utilizzato per valutare la funzione di virulenza di S. aureus meticillino-resistente (MRSA) e le risposte immunologiche ospite. Qui, abbiamo presentato un modello di infezione per via sottocutanea per la pelle e le infezioni dei tessuti molli.

Video: 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.

L&#39;uso del flusso gocciolamento e Reattori disco rotante per Staphylococcus aureus Analisi Biofilm

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

Ricerca

Immunologia e infezioni

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.

Endocardite modello sperimentale di meticillino resistente<em&gt; Staphylococcus aureus</em&gt; (MRSA) in Rat

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 crescita con emoglobina umana come fonte di ferro

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 per la tipizzazione di stafilococco cassette cromosomiche Mec Tipi I a V, resistente alla meticillina<em&gt; Staphylococcus aureus</em

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.

Lo studio delle interazioni di<em&gt; Staphylococcus aureus</em&gt; Con neutrofili mediante citometria a flusso e 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. ...

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.

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione 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.

Istituzione e Ottimizzazione di un programma di installazione High Throughput per studiare<em&gt; Staphylococcus epidermidis</em&gt; E<em&gt; Mycobacterium marinum</em&gt; Infezione come modello per 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 Associated<em&gt; Staphylococcus aureus</em&gt; Uso resazurina base Assay farmaco-sensibilità

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.

La genotipizzazione di<em&gt; Staphylococcus aureus</em&gt; Per ribosomiale 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.

Metodologia per lo studio del orizzontale Gene Transfer 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 ...