Die Studien wurden durch AdvanDx gesponsert.

1. Probenentnahme und Vorbereitung <ol><li> Sammeln venösen Blut von Patienten mit Sepsis Verdacht und legte sich in zwei BACTEC (BD, Sparks, MD) Blutkulturflaschen, eine aerobe und eine anaerobe, zusammen mit einem Blut Kultur gesetzt. </li><li> Legen Flaschen in die BACTEC 9240 Instrument bei 35 ° C. </li><li> Inkubieren, bis das Gerät in den Alarmzustand wegen des Wachstums der Organismen in der Blutkulturflasche ausgelöst wird. </li><li> Entfernen Flasche aus der Blutkultur Instrument. </li></ol> 2. Vorbereitung der Reagenzien vor dem Färben <ol><li> Bereiten Sie die Waschlösung durch Zugabe von 4 ml 60x Waschlösung von 240 ml frisch destilliertem Wasser direkt auf die Färbung Dish gefolgt. </li><li> Legen Sie die Färbung Gericht in der 55 ° C Wasserbad zu erwärmen. </li><li> Bewahren Sie die restlichen Konzentrat bei 2-8 ° C. </li><li> Entfernen Sie die Eindeckmedium aus dem Kühlschrank und auf Raumtemperatur erwärmt. </li></ol> 3. Gram-Färbung <ol><li> Vorsichtig schwenken die Blutkulturflasche. </li><li> Mit einer Nadel und Spritze oder eine Subkultivierung Apparat, entfernen Sie das Blut (ca. 5 ml) aus der Flasche und in eine sterile Schraube Röhrchen. </li><li> Mit einer sterilen Pipette, stellen Sie einen großen Tropfen Blut auf einen Objektträger. </li><li> An der Luft trocknen schieben und fixieren in Methanol für 10 Sekunden. </li><li> Lassen Sie gleiten an der Luft trocknen. </li><li> Führen Sie eine Gram über die Blut-Film Fleck, um die Morphologie des Organismus wächst in der Blutkulturflasche bestimmen. </li><li> Mit einer Ölimmersion, Diashows. Gram-positive Kokken in Paaren und kurzen Ketten auf die Gram-Färbung zu beobachten ist am ehesten mit Enterococcus Spezies. </li><li> Das Gram-Färbung Ergebnis treibt dann die Auswahl der richtigen PNA FISH-Sonde Kit für Identifizierung von Bakterien verwendet werden. Diese Blutkultur wird gebeizt mit der E. werden faecalis / OE PNA FISH-Färbung. </li></ol> 4. PNA FISH Stain <ol><li> Mischen Sie die Röhrchen mit dem Blut vorsichtig durch Schwenken vor Abstrich Vorbereitung. </li><li> Geben Sie einen Tropfen der Lösung Fixation auf das Wohl eines PNA FISH Objektträger. </li><li> Transfer 10 ul oder einen kleinen Tropfen aus der Tube mit der positiven Blutkultur auf die Fixation Solution und vorsichtig mischen, um zu emulgieren. </li><li> Fix die Flecken durch Erhitzen für 20 min. bei 55 ° C auf einer Heizplatte. </li></ol> 5. Quality Control Material- Eine positive und eine negative Qualitätskontrolle schieben muss jede Charge von Folien für die Färbung getestet werden. <ol><li> Verwenden Sie E. faecalis / OE Objektträger gekauft AdvanDx für diesen Zweck oder bereiten Abstriche aus flüssigen Kulturen Referenz Stämme von E. faecalis und E. faecium als positive Kontrolle entweder auf separaten Folien oder gemischt auf einer Folie und Staphylococcus spp oder Streptococcus spp als negative Kontrolle Material. </li></ol> Die QC-Ergebnisse sollten in der Lage, für eine angemessene Prüfbedingungen, insbesondere in den Hybridisierungsstringenz und Zellwand Eindringen zu überwachen, da PNA-Methodik wurde entwickelt, um Zellwand Eindringen optimieren 6. Hybridisierung <ol><li> Einen Tropfen E. faecalis / OE PNA, die auch auf dem Objektträger mit dem Abstrich. </li><li> Add Deckglas. Vermeiden Sie Luftblasen. </li><li> Inkubieren für 30 ± 5 min. bei 55 ± 1 ° C auf Heizplatte </li><li> Nach der Inkubation Objektträger in Spannplatte </li></ol> 7. Strenge Wash <ol><li> Tauchen Objektträgerhalter in den vorgeheizten Waschlösung bei 55 ° C und entfernen Sie vorsichtig das Deckglas. Oft gleitet das Deckglas ab, indem Sie vorsichtig Schütteln der Folie in der Waschlösung. Gelegentlich muss das Deckglas vorsichtig geschoben werden Sie mit einer Pinzette. </li><li> Inkubieren in der Waschlösung für 30 ± 5 min. bei 55 ± 1 ° C. </li><li> Entfernen Sie die Folien aus der Waschlösung </li><li> Lassen Sie die Folie an der Luft trocknen </li></ol> 8. Montage <ol><li> Einen Tropfen Eindeckmedium auf jeder Folie </li><li> Add Deckglas. Vermeiden Sie Luftblasen. </li><li> Untersuchen Sie die Folie auf einem Fluoreszenzmikroskop innerhalb von 2 Stunden. </li><li> Setzen Sie die Dias dem Sonnenlicht oder anderen starken Lichtquellen direkt, da dies zu Fluoreszenzlöschung führen kann. </li></ol> 9. Interpretation der Ergebnisse Die Fluoreszenz-Mikroskop für Dia-Prüfung verwendet werden, müssen mit dem AdvanDx Dual-Band-Filter und ein 60x oder 100x Öl-Objektiv ausgestattet werden. Die QC Dias sollte zunächst geprüft werden, um zu bestätigen, dass die Hybridisierung entstanden sind. E. faecalis sollte so hell grün fluoreszierende Kokken in mehrere Felder der Ansicht angezeigt werden und die E. faecium wird als leuchtend rote Kokken erscheinen. Non-Enterokokken Steuerschieber erscheinen soll nichtfluoreszierenden. Nach der Bestätigung des Systems in der Kontrollgruppe, kann der Patient gleitet untersucht werden. Auf den ersten Blut film erscheint rötlich, aber die leuchtend rot und grün Kokken werden ganz offensichtlich. <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Repräsentative Beispiele für grün-positive E. faecalis (links), rot positive E. faecium (Mitte) und negative (rechts) Testergebnisse. 10. Repräsentative Ergebnisse Drei Institutionen wurden in einer multizentrischen klinischen Studie zur Evaluierung dieser PNA FISH-Färbung und Vergleich einer 2,5 Stunden Protokoll, um die verkürzte 1,5 Stunden Protokoll, das soeben Bewertung einbezogen. Ein insgesamt 152 Routine grampositive Kokken in Paaren und Ketten (GPC) positive Blutkultur Flaschen wurden in die Studien einbezogen. Es gab 100% (152/152) Vereinbarung zwischen den Ergebnissen der modifizierten und dem ursprünglichen Testverfahren für E. faecalis / OE PNA FISH: 41/41 E. faecalis; 33/33 anderen Enterokokken und 78/78 anderen GPC (Tabelle 1). Diese Färbemethode hatte exquisite Sensitivität und Spezifität in dieser klinischen Studie (Tabelle 2). <table border="1"><tbody><tr><td></td><td> Routine-Methoden </td><td> E. faecalis / OE PNA FISH Standard-Vorgehensweise </td><td> E. faecalis / OE PNA FISH Short Procedure </td></tr><tr><td> E. faecalis </td><td> 41 </td><td> 41 (Grüne Positive) </td><td> 41 (Grüne Positive) </td></tr><tr><td> E. faecium </td><td> 27 </td><td> 27 (Red Positive) </td><td> 27 (Red Positive) </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2 (Red Positive) </td><td> 2 (Red Positive) </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2 (Red Positive) </td><td> 2 (Red Positive) </td></tr><tr><td> Andere Enterococcus spp. </td><td> 2 </td><td> 2 (Red Positive) </td><td> 2 (Red Positive) </td></tr><tr><td> S. pneumoniae </td><td> 17 </td><td> 17 (Negative) </td><td> 17 (Negative) </td></tr><tr><td> S. viridans </td><td> 33 </td><td> 33 (Negative) </td><td> 33 (Negative) </td></tr><tr><td> S. mitis </td><td> 1 </td><td> 1 (Negative) </td><td> 1 (Negative) </td></tr><tr><td> S. pyogenes </td><td> 3 </td><td> 3 (Negative) </td><td> 3 (Negative) </td></tr><tr><td> S. bovis </td><td> 2 </td><td> 2 (Negative) </td><td> 2 (Negative) </td></tr><tr><td> S. salivarius </td><td> 1 </td><td> 1 (Negative) </td><td> 1 (Negative) </td></tr><tr><td> S. sanguinis </td><td> 2 </td><td> 2 (Negative) </td><td> 2 (Negative) </td></tr><tr><td> S. agalagtae </td><td> 7 </td><td> 7 (Negative) </td><td> 7 (Negative) </td></tr><tr><td> Andere Streptococcus spp. </td><td> 7 </td><td> 7 (Negative) </td><td> 7 (Negative) </td></tr><tr><td> Abiotrophia spp. </td><td> 3 </td><td> 3 (Negative) </td><td> 3 (Negative) </td></tr><tr><td> Peptostrepococcus spp. </td><td> 2 </td><td> 2 (Negative) </td><td> 2 (Negative) </td></tr><tr><td> Gesamt </td><td> 152 </td><td> 152/152 100% Zustimmung </td><td> 152/152 100% Zustimmung </td></tr></tbody></table> Tabelle 1. Listing von Bakterien getestet sowohl mit dem Standard (2,5 Stunden) und Rapid (1,5 h)-Protokoll. <table border="1"><tbody><tr><td> Empfindlichkeit E.faecalis </td><td> Empfindlichkeit Andere Enterococcus spp. </td><td> Spezifität </td></tr><tr><td> 100% (41/41) 95% CI (93,0 bis 100) </td><td> 100% (33/33) 33/33 95% CI (91,3 bis 100) </td><td> 100% (78/78) 95% CI (96,2 bis 100) </td></tr></tbody></table> Tabelle 2. Sensitivität und Spezifität der 1,5 Stunden PNA FISH-Protokoll.

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

Die Produktion dieses Video-Artikel wurde von AdvanDx gesponsert. Benjamin Crystal ist ein Mitarbeiter von AdvanDx, die die Werkzeuge und Reagenzien in diesem Artikel verwendet wird.

Die E. faecalis / OE PNA FISH-Assay für Enterokokken können Identifizierung 2-3 Tage früher als Standard-Kultur-Methoden liefern. Die verkürzte Verfahren für den Test (1,5 hourr) ermöglicht eine schnelle Identifikation, die für einen besseren Ansatz, um eine antimikrobielle Therapie und Outcome der Patienten eingesetzt werden können. Eine Studie, dies zu unterstützen wurde von Forrest et al. (6) durchgeführt. In zwei aufeinanderfolgenden Jahren, beginnend im Jahr 2005 die mikrobiologischen Labor identifiziert Gram-positive Kokken in Paaren und Ketten wachsen in Blutkulturflaschen von konventionellen mikrobiologischen Methoden und in 2006 Hinzufügen der E. faecalis / OE PNA FISH. Zusätzlich ist ein Therapie-Algorithmus durch die Organe antimikrobielle Team (AMT) entwickelt, um effektiv zu nutzen die PNA FISH-Daten durch das Labor in zeitnah generiert. Der primäre Endpunkt war die Zeit beurteilt aus Blutkultur auf die Umsetzung von wirksamen antimikrobiellen Therapie ziehen vor und nach der PNA FISH wurde in den Labors Workflow eingeleitet. Die Schwere der Krankheit, Patienten Lage und empirische antimikrobielle Therapie gemessen wurden. Insgesamt 224 Patienten mit im Krankenhaus erworbenen Enterokokken Bakteriämie wurden mit 129 in der Pre-Interventionszeitraum und 95 in der PNA FISH Zeitraum ausgewertet. PNA FISH identifiziert E. faecalis 3 Tage früher als bei herkömmlichen Kulturen (1,1 vs 4,1 Tage, p &lt;0,001). PNA FISH identifiziert E. faecium eine mediane 2,3 Tage früher (1,1 vs 3,4 Tage, p &lt;0,001) und wurde mit statistisch signifikante Reduktion in der Zeit der Einleitung wirksame Therapie (1,3 vs 3,1 Tage, p &lt;0,001) und sank 30 Tage Sterblichkeit (26% vs 45%, p = 0,04). Diese kam zu dem Schluss, dass die E. faecalis / OE PNA FISH-Test in Verbindung mit einem AMT Therapie-Algorithmus führte zu einer frühzeitigeren Einleitung einer entsprechenden empirischen antimikrobiellen Therapie bei Patienten mit im Krankenhaus erworbenen E. faecium Bakteriämie. <ol><li> Reagenzien dürfen nicht verwendet werden, nachdem die Verfallsdaten auf den Etiketten und Reagenzien aufgedruckt ist, soll in keiner Weise modifiziert werden können. Sie sollten in den Kühlschrank, wenn sie nicht in Gebrauch bleiben, oder sie konnten Potenz verlieren. </li><li> Blutkulturflaschen müssen vor der Probenahme vermischt werden. </li><li> Es ist wichtig, nicht zulassen, dass die Tropfflasche Spitze zu einem Blutausstrich zu berühren, da dies eine Kreuzkontamination von Material zwischen den Folien verursachen kann, oder eine Verunreinigung des Reagenz. </li><li> Stellen Sie sicher, das Wasserbad und Heizblock bei einer konstanten 55 ° C gehalten werden </li><li> Verwenden Sie keine Filter als die Dual-Band-Filter oder die Farben nicht klar zu erkennen. </li><li> Verwenden Sie keine Objektträger andere als die Mikropräparate oder Hybridisierung findet nicht statt. </li><li> Es ist wichtig, dass das Mikroskop richtig funktioniert. Stellen Sie sicher, das Mikroskop Lampe ist innerhalb der genehmigten Anzahl von Stunden für die Nutzung. </li></ol><ul><li> Einige seltene vorkommende Enterococcus Spezies sind nicht von der PNA-Sonde hybridisieren anderen Enterococcus Spezies wie E. asinii, E.dispar, E. haemoperoxidus, E. cecorum, E. columbae, E. saccharolyticus, E. solitarius und E erkannt . schwefligen. </li><li> E. moraviensis als E. identifiziert faecalis durch Sequenz Ähnlichkeiten. </li><li> Einige Stämme von Streptococcus anginosus produzieren ein falsches Grün positive Fluoreszenz durch Sequenz Ähnlichkeiten. </li><li> VersaTREK Blutkulturflaschen wurden während der klinischen Studien untersucht. Die Leistung von E. erkennen faecalis und andere Enterococcus spp. mit VersaTREK Blutkulturflaschen ist unbekannt. </li><li> Falsch positive grüne Autofluoreszenz kann auftreten, wenn ein Standard-FITC-Filter wird anstelle des Dual-Band-Filter verwendet. </li><li> Falsch negative Ergebnisse können selten auftreten aufgrund gemischter Wachstum oder aufgrund eines Fehlers in Assay-Technik. </li><li> Die Art und Zustand der Instrumente verwendet werden, Einfluss auf die visuelle Erscheinung des Bildes erhalten. Die Fluoreszenz kann auf die Art von Mikroskop eingesetzt, der Lichtquelle und der Ebene der rRNA in den Zellen varydue </li><li> Isolation auf festen Medien ist notwendig, um gemischte Wachstum mit anderen Organismen zu unterscheiden und die positive Blutkulturen was zu einem negativen Ergebnis zu identifizieren. </li><li> Das Produkt ist nicht mit Exemplaren, die keine Blutkulturen validiert. </li></ul>

Reagenzien E. faecalis / OE PNA FISH besteht aus den folgenden Komponenten des Kits enthalten: <ul><li> Fixation Lösung Fixation Lösung 3 ml Phosphat-gepufferte Kochsalzlösung mit Waschmittel. </li><li> E. faecalis / OE PNA E. faecalis / OE PNA 1,5 ml PNA-Sonden bei der Hybridisierung. Lösung. Enthält 30% Formamid. </li><li> 60x Waschlösung 60x Waschlösung 50 ml Tris-gepufferte Kochsalzlösung mit Detergenz </li><li> Eindeckmedium Eindeckmedium 3 mL photobleaching Inhibitor in Glycerol </li><li> Qualitätskontrolle Dias </li><li> <a href="http://www.advandx.com">AdvanDx</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

Enterokokken sind eine häufige Ursache von Bakteriämie mit E. faecalis wird die vorherrschende Spezies von E. faecium gefolgt. Da die Resistenz gegen Ampicillin und Vancomycin in E. faecalis noch selten auf Widerstand in E. faecium verglichen wird, ist die Entwicklung von Schnelltests erlauben die Differenzierung zwischen Enterokokken-Spezies für eine angemessene Therapie und Überwachung von Resistenzen gegen wichtig. Die E. faecalis OE PNA FISH-Test (AdvanDx, Woburn, MA) verwendet Spezies-spezifische Peptid-Nukleinsäure (PNA)-Sonden in einem Fluoreszenz-in situ-Hybridisierung Format und bietet eine Zeit, um Ergebnisse von 1,5 Stunden und das Potenzial, wichtige Informationen für artspezifische Behandlung. Multicenter-Studien wurden durchgeführt, um die Leistung des 1,5 Stunden E. bewerten faecalis / OE PNA FISH-Verfahren auf den ursprünglichen 2,5 Stunden Testdurchführung und Standard Bakteriologie Methoden zur Identifizierung von Enterokokken direkt von einer positiven Blutkultur Flasche verglichen.

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Eine schnelle Protokoll für die direkte Identifizierung von Enterococcus faecalis und Enterococcus anderen Arten aus eine positive Blutkultur mit einem Peptidnucleinsäuren Fluoreszenz in situ Hybridisierung Assay (PNA FISH).

Ein 1,5 Stunden-Verfahren für die Identifizierung von Enterococcus Spezies direkt aus Blutkulturen

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.8K Aufrufe.  Cedars-Sinai Medical Cente. Eine schnelle Protokoll für die direkte Identifizierung von Enterococcus faecalis und Enterococcus anderen Arten aus eine positive Blutkultur mit einem Peptidnucleinsäuren Fluoreszenz in situ Hybridisierung Assay (PNA FISH).

Serie: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Depletion spezifischer Zellpopulationen durch Ergänzung Depletion

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Forschung

Immunologie und Infektion

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Ein-Tages-Workflow-Schema für bakterielles Pathogen-Erkennung und Resistenz gegen antimikrobielle Tests aus Blutkulturen

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generierung von Organotypische Floßkulturen aus primären humanen Keratinozyten

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolierung von Precursor B-Zell-Subsets aus Nabelschnurblut

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Gesamtproteinextraktion und 2-D Gelelektrophorese-Methoden für<em&gt; Burkholderia</em&gt; Arten

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Verfahren zur Identifizierung von Infektions Prionen Nach Passage durch das Verdauungssystem von eine Vogelart

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Schnelle Identifizierung von Gram-negativen Bakterien aus Blutkulturbrühe unter Verwendung von MALDI-TOF-Massenspektrometrie

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Vorbereitung einer Blutkultur Pellet Rapid Identifizierung von Bakterien und Antibiotika Empfindlichkeitsprüfung

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...