NIAID concedere K22AI081748.

1. La goccia di flusso reattore Biofilm <ol><li> Il flusso del reattore a goccia biofilm (disponibile da Technologies Biosurface o versioni progettate su misura di solito può essere fatta da officine meccaniche università, vedi figura 1) è assemblato e autoclave. Assemblea coinvolge coupon apposizione in camere e coperchi della camera di fissaggio. La camera, insieme con il mezzo biofilm (brodo di soia trittico 2 grammi / L di glucosio e 2 grammi / L) e influente tubi nutrienti sono sterilizzati in autoclave. </li><li> Inoculazione del reattore flusso goccia è preformato posizionando il reattore su una superficie piana, le linee di serraggio tubi effluenti, riempiendo ogni camera con 10 ml di brodo di soia trittico e l&#39;aggiunta di 10 L di una S. cultura aureus cresciute overnight in brodo di soia trittico. Il reattore viene inoculato svolge in un incubatore a 37 ° C per 18 ore. </li><li> Dopo 18 ore di incubazione, il tubo emissario è unclamped e il reattore è posto su un blocco di legno tagliato ad un angolo di 10 °. </li><li> Asetticamente collegare il tubo influente nutriente per la bottiglia contenente il brodo di coltura in continuo flusso. Alimentare la linea di tubazione attraverso la pompa e il primo dei tubi eseguendo la pompa ad una velocità massima (varia a seconda del modello della pompa). </li><li> Una volta che il tubo influente è innescato fermare la pompa e collegare collegare aghi (22 gauge e da 1 pollice) alla fine di ogni provetta. Pulire il tappo di ingresso camera con etanolo pulire in modo asettico e inserire gli aghi attraverso il tappo di aspirazione. </li><li> Accendere la pompa e lasciare i media a sgocciolare lentamente (portata ~ 125 microlitri / minuto) sui coupon. I media dovrebbero flusso verso il basso lungo la cedola dal porto tappo di ingresso al porto effluenti. Far funzionare il reattore in flusso continuo per 2-5 giorni (a seconda dell&#39;applicazione), controllando di tanto in tanto il reattore per il drenaggio adeguato. </li><li> Per raccogliere il flusso di biofilm reattore a goccia, fermare la pompa e rimuovere con attenzione gli aghi dal reattore. Il reattore può essere collocato su una superficie piana ed i tagliandi possono essere rimosse in modo asettico con pinza sterile. Se microscopia è desiderato, i tagliandi possono essere trattati di conseguenza (Figura 2B è un microscopio elettronico a scansione di un biofilm S. aureus cresciuto in un reattore a biofilm goccia a goccia). Se la quantificazione della biomassa biofilm o studi fisiologia sono l&#39;obiettivo dello studio, il biofilm può essere rimosso dalla cedola con un raschietto cellulare. Tenendo il coupon con una pinza, raschiare delicatamente il biofilm fuori il buono in una provetta conica contenente tampone fosfato con un raschietto cellulare. Nota: per quantificare l&#39;unità formanti colonia nel biofilm, è necessario omogeneizzare il biofilm raccolte con un tessuto omogeneizzatore di disaggregare grumi e formare una sospensione omogenea. Diversi modelli di omogeneizzatori tessuto sono adatti per questa applicazione. Utilizziamo un Fisher Scientific Tissuemiser Omogeneizzatore (prodotto # 15-338-420) alla massima velocità per 1 minuto per omogeneizzare campioni di biofilm. Il mancato omogeneizzare il biofilm si tradurrà in una sottostima delle unità formanti colonie presenti nel campione. </li></ol> 2. La rotazione del disco reattore Biofilm <ol><li> La rotazione del reattore biofilm disco (disponibile da disponibile da Technologies Biosurface o possono essere realizzati su misura, vedi figura 3) è assemblato e autoclave. Montare il reattore dal primo posto il disco rotante tagliandi nelle fessure del disco filatura e inserirlo in una da 1 litro bicchiere di vetro di una porta di overflow. Un numero 15-tappo di gomma con fori in esso per consentire il flusso dei media e aerazione è usato come il tappo del reattore. Il reattore, biofilm media (brodo di soia trittico 2 g / L di glucosio e 2 g / L) e quello in ingresso sono poi sterilizzati in autoclave. </li><li> La rotazione del reattore biofilm disco viene inoculato mettendo 250 ml di medium sterile all&#39;interno del reattore, e l&#39;aggiunta di 0,5 ml di una notte di S. cultura aureus coltivato in brodo di soia trittico. Il reattore è quindi posto su un piatto mescolate insieme a 250 giri al minuto e incubato per una notte alla temperatura desiderata. </li><li> Dopo 16 ore di incubazione collegare il tubo di ingresso alla porta sul serbatoio medio e collegarlo alla pompa peristaltica. La pompa è quindi accendere e il flusso impostato ~ 0,25 ml / min (flusso può essere modificato a seconda del tasso di crescita desiderato). </li><li> Dopo 24 ore fermare il reattore e asetticamente rimuovere il disco senza toccare i tagliandi biofilm. Rimuovere i tagliandi con pinza sterile e immergere ognuno in tampone fosfato di rimuovere i batteri debolmente collegato. </li><li> Per il test composto antimicrobico, i chip possono essere inseriti in singoli pozzetti di una piastra a 96 pozzetti contenenti composti di interesse. Dopo l&#39;incubazione, i chip vengono trasferiti in tubi da 1,5 ml microcentrifuga contenente 1 ml di soluzione salina tampone fosfato e omogeneizzati con un fazzoletto omogeneizzatore per disperdere il biofilm intatto. </li><li> Le cellule sono poi diluiti seriale e placcato su terreno di agar nutriente per determinare microbismo Formiunità ng. </li><li> Nota: molte varianti possono essere effettuate sul protocollo di cui sopra. Per esempio, buoni disco rotante può essere rivestito con o fatti di potenziale anti-biofilm composti per testare l&#39;efficacia. Disperdenti biofilm possono essere valutati anche in incubazione tagliandi in composti disperdente e quantificazione dei batteri attaccati contro distaccato. </li></ol> 3. Rappresentante Risultati Un esempio di un set up reattore a goccia flusso viene visualizzato se Figura 1. Dopo tre giorni di flusso abbondante biofilm si accumulano sulla superficie della cedola, figura 2A. La biomassa totale varia a seconda ceppi batterici e condizioni di crescita precisa. Un microscopio elettronico a scansione di un S. biofilm aureus cresciuto nel reattore flusso goccia è mostrato nella Figura 2B. Un reattore disco rotante è mostrato in figura 3A. Il protocollo descritto può essere adattato alle specifiche esigenze di generale qualsiasi microrganismo in grado di formare un biofilm. Figura 3B mostra il disco rotante con 18 dischi di plastica apposta. Questi disco-grown biofilm sono particolarmente adatti per i test antimicrobici e portare a risultati altamente riproducibili 11. <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="Figura 1" /> Figura 1. Il flusso di setup a goccia reattore. Componenti importanti sono etichettati. <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="Figura 2" /> Figura 2. Esempio di un flusso goccia S. aureus biofilm. A) Il biofilm è stato coltivato per tre giorni, seguendo il protocollo descritto. Coperchi dai primi due camere del reattore vengono rimossi per mostrare il giallo S. aureus biofilm biomassa. B) Sacnning microscopio elettronico di una S. biofilm aureus cresciuto nel reattore flusso gocciolamento. <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="Figura 3" /> Figura 3. Il reattore disco rotante. A) Esempio di funzionamento di un reattore disco rotante. Componente chiave sono etichettati. B) Primo piano vista di un disco rotante.

<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. 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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. A., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+resistance+to+antimicrobial+agents.">Biofilm resistance to antimicrobial agents.</a> Microbiology. 146, 547-549 (2000).</li><li>Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T., McFeters, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+physiological+heterogeneity+in+Pseudomonas+aeruginosa+biofilm+is+determined+by+oxygen+availability.">Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.</a> Appl. Environ. Microbiol. 64, 4035-4039 (1998).</li><li>Boles, B. R., Thoendel, M., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-generated+diversity+produces+"insurance+effects"+in+biofilm+communities.">Self-generated diversity produces "insurance effects" in biofilm communities.</a> Proc. Natl. Acad. Sci. USA. 101, 16630-16635 (2004).</li><li>Pitts, B., Willse, A., McFeters, G. A., Hamilton, M. A., Zelver, N., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+repeatable+laboratory+method+for+testing+the+efficacy+of+biocides+against+toilet+bowl+biofilms.">A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms.</a> J. Appl. Microbiol. 91, 117-11 (2001).</li><li>Boles, B. R., Thoendel, M., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhamnolipids+mediate+detachment+of+Pseudomonas+aeruginosa+from+biofilms.">Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms.</a> Mol. Microbiol. 57, 1210-1223 (2005).</li><li>Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+overproduction+affects+Pseudomonas+aeruginosa+biofilm+structure+and+function.">Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function.</a> J. Bacteriol. 183, 5395-5401 (2001).</li><li>Lin, H. Y., Chen, C. T., Huang, C. 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Nessun conflitto di interessi dichiarati.

Biofilm cresciuto nei reattori differenti spesso hanno caratteristiche diverse e ogni reattore ha applicazioni diverse. In questo lavoro, si descrive l&#39;uso di due reattori biofilm: un flusso reattore a goccia biofilm e un reattore disco rotante. Drip reattori di flusso sono utili per la coltivazione a basso biofilm taglio in un aria-liquido e sono adattabili ad una varietà di condizioni. Li troviamo estremamente conveniente per gli studi in cui una grande quantità di biomassa biofilm è auspicabile. Questa configu...

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>
<td>DFR 110</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

La maggior parte dei microbi in natura si pensa di esistere come superficie associata comunità in biofilm 1. Biofilm batterici sono racchiusi all&#39;interno di una matrice e collegato a una superficie di 2. Formazione di biofilm e lo sviluppo sono comunemente studiate in laboratorio utilizzando sistemi batch come micropiastre o di flusso sistemi, come il flusso delle cellule. Queste metodologie sono utili per lo screening di librerie di mutanti e chimiche (micropiastre) 3 o crescente biofilm per la visualizzazione (celle di flusso) 4. Qui vi presentiamo protocolli dettagliati per Staphylococcus aureus in crescita in due ulteriori tipi di biofilm sistema di flusso: il flusso del reattore a goccia biofilm e la rotazione del reattore biofilm disco. Drip reattori biofilm flusso sono progettati per lo studio dei biofilm cresciuto in condizioni di taglio basso. 5 Il reattore di flusso a goccia si compone di quattro canali di test in parallelo, ciascuno in grado di tenere uno standard microscopio cedola di vetro di dimensioni diapositiva o una lunghezza del catetere o stint. Il reattore di flusso a goccia è ideale per il monitoraggio microsensor, generale studi biofilm, i campioni di biofilm criosezionamento, la produzione di biomassa ad alta, valutazioni materiale medico, e dimora test del dispositivo medico. 6,7,8,9 Il reattore disco rotante è costituito da un disco di teflon che contiene recessi per cedole rimovibile. 10 Il coupon staccabile può in fatto di qualsiasi materiale lavorabile. La parte inferiore del disco rotante contiene una barra magnetica per consentire la rotazione del disco di creare taglio liquido sulla superficie-superficie a filo coupon. L&#39;intero disco contenente 18 tagliandi è posto in un recipiente di vetro 1000 ml reattore braccio laterale. Un supporto crescita liquido viene fatto circolare attraverso la nave mentre il disco viene ruotato da un agitatore magnetico. Le cedole vengono rimossi dal contenitore del reattore e poi raschiato per raccogliere il campione biofilm per ulteriori studi o immagini di microscopia. Reattori disco rotante sono progettate per le valutazioni di laboratorio di efficacia biocida, rimozione biofilm, e le prestazioni di anti-fouling dei materiali. 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

Protocolli per l&#39;utilizzo aperto biofilm flusso sistema con reattori a goccia e flusso di rotazione del disco reattori sono presentati in dettaglio.

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 ...

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 Views.  University of Michigan. Protocolli per l'utilizzo aperto biofilm flusso sistema con reattori a goccia e flusso di rotazione del disco reattori sono presentati in dettaglio.

Video: 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 e biofilm Saccharomyces cerevisiae in celle di flusso

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

Ricerca

Immunologia e infezioni

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.

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 ...

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. ...

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 ...

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).

Caratterizzazione dei sottoinsiemi di monociti umani da sangue intero flusso Cytometry analisi

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