Name: 'bioluminescent' reporter phage for the detection of category a bacterial pathogens
Uploaded: 2011-07-08T12:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

Diese Arbeit wurde von der Small Business Innovation Research-Programm des National Institutes of Health (NIAID, 1R43AI082698-01) und der USDA National Institute of Food and Agriculture (NIFA, 2009-33610-20028) unterstützt.

1. Y. pestis Platte Impfung <ol><li> Streak Y. pestis A1122 (BeiResources # NR15) Stammkulturen auf Luria-Bertani (LB)-Agar (Miller). Verwenden Sie sterile Technik und erfüllen alle Y. pestis Manipulationen in einer Klasse-II-Typ A Biosicherheitswerkbank. Y. pestis A1122 ist ein Biosafety Level (BSL) 2 ausgeschlossen wählen Agent Belastung. Es fehlt sowohl die 75 kb Paar Low-Calcium-Reaktion (LCR) Virulenzplasmid und pgm locus, dass für die Virulenz erforderlich sind. Wachsen Sie Y. pestis bei 28 ° C für 48 h in einer statischen temperaturgeregelten Luft Inkubator. Die Wachstumsrate von Y. pestis ist langsam mit einer Generationszeit von 1,25 h 9. </li></ol> 2. Y. pestis flüssigen Medien Impfung <ol><li> Übertragen einer einzigen Y. pestis Kolonie in eine sterile 17 x 100 mm Kultur Röhrchen mit 2 ml LB-Medium mit einer sterilen Impföse Metall. Wählen Sie eine Kolonie, die eine einheitliche Größe ist, ist bezeichnend für die anderen Kolonien, und sich deutlich von dem Rest der Kolonien auf der Platte getrennt. Vortex Tube kurz und wachsen bei 28 ° C für ca. 20 h in einem Schüttelinkubator (225 rpm). </li></ol> 3. Y. pestis Auswuchs, Reporter Phagen hinaus und Biolumineszenz-Erkennung <ol><li> Verdünnen Sie die Nacht Y. pestis Kultur 1:20 in frischem LB-Medium in einem 50 ml Falcon-Röhrchen (z. B. 500 ul der Zellen in 9,5 ml Medium) und wachsen bei 28 ° C unter Schütteln (225 rpm). Wachsen, bis eine optische Dichte bei 600 nm (A 600) von 0,2 erreicht ist (ca. 5 h). Aktiv wachsende Zellen sind zu bevorzugen für den Nachweis, da die Fähigkeit des Phagen zu transduzieren einem biolumineszierenden Reaktion auf die Lebensfähigkeit und die Fitness des Wirtes Bakterien korreliert ist. Dennoch hat das Detektionssystem konnte gezeigt werden, kompatibel mit Zellen in allen Phasen des Wachstumszyklus 7 geerntet. </li><li> Generieren Sie das Substrat, die für die Biolumineszenzreaktion durch Herstellung einer 2% n-Decanal Lösung. Mix 200 ul n-Decanal mit 9,8 ml LB-Brühe. Vortex kräftig 5 s. Prime der Mikroplatten-Luminometer Injektor mit 2 ml der 2%-n-Decanal Lösung. Pre-set das Luminometer auf 67 ul der Decanal Lösung für jede Mikrotiterplatten und dann autoinject sofort lesen Sie die Probe für 10 s. </li><li> Dispense 1 mL Aliquots von LB-Medium in 3 Kulturröhrchen. Add 20 uL des Reporters Phagen-Stammlösung (stock von 5 x 10 9 Plaque-bildenden Einheiten [PFU] / ml) in jedes Röhrchen, die Phagen-und Medien-alone-Proben dient als negative Kontrolle. In Abwesenheit von Y. pestis-Zellen, sollte die Zugabe des Reporters Phagen nicht entlocken eine Biolumineszenz Antwort. </li><li> Dispense 1 mL Aliquots der Y. pestis Kultur (aus Schritt 3.1) in 6 Kulturröhrchen. Add 20 uL des Reporters Phagenstock bis 3 der Kulturen (Testkulturen). Die restlichen 3 Kulturen dienen als &quot;Zellen allein&quot; Negativ-Kontrolle (Hintergrund autobioluminescence). Mix Kulturen durch Vortexen kurz, und die Ernte 200 ul von jeder Probe (für die Zeit 0 zu lesen) und verzichten in eine weiße 96-well Mikrotiterplatten. Inkubieren Sie die verbleibenden Kultur bei 28 ° C unter Schütteln (225 rpm). </li><li> Messen Sie die Zeit 0 Muster für Biolumineszenz (relative light units, RLU) mit dem Mikrotiterplatten-Luminometer. </li><li> Ernte 200 ul jeder Probe nach 10, 20, 30, 40, 50 und 60 min post-Reporter Phagen hinaus und messen Sie die Probe für die Biolumineszenz. Wenn Y. pestis Zellen vorhanden sind, wird der Reporter Phagen spezifisch an die Zellen binden, injizieren ihre DNA, und die Gastgeber der Transkription und Translation Maschinen zu transkribieren und übersetzen die luxAB Reportergene (Abbildung 1). </li><li> Die Stärke des Signals und der Signal-Response-Zeit ist proportional zu der Anzahl der Zellen zu präsentieren. Bei hohen Konzentrationen von Zellen (10 5 -10 8 CFU / ml), einen signifikanten Anstieg in RLU im Vergleich zu den Kontrollen sollten innerhalb von 20 min deutlich. Am unteren Zelle Konzentrationen (10 2 -10 4 KBE / ml), sollte eine deutliche Steigerung in RLU werden innerhalb von 40-60 min deutlich. </li><li> Alternativ zur Erkennung ohne die Notwendigkeit für Y. beschleunigen pestis Auswuchs, eine Kolonie von einer frisch gewachsenen Platte gemischt (durch Vortexen) direkt in ein 1,5 ml Eppendorf-Röhrchen mit 200 ul LB-Brühe, Beherbergung der Reporter Phagen werden. Dosieren Sie die Zelle / Phagen Mischung in eine Vertiefung einer Mikrotiterplatte. Inkubieren Sie die Mikrotiterplatte bei 28 ° C und lesen Sie die Probe für die Biolumineszenz nach 60 min (single Zeitpunkt nur). </li></ol> 4. Repräsentative Ergebnisse: Ein Vertreter Zeitverlauf Experiment Reporter Phagen-vermittelte Erkennung von Y. pestis ist in Abbildung 2 dargestellt. Die negativen Kontrollen von 1) Phagen allein (keine Zellen), oder 2)-Zellen allein (ohne Phagen) bieten Baseline-Wertes der Biolumineszenz von ca. 20 RLU während der 60 min inkl.ubation (Ausgangswerte sind Luminometer spezifisch). Im Gegensatz dazu ist eine Zunahme der Biolumineszenz für die Testproben (reporter Phagen und Zellen) bei 15 min nach dem Phagen Neben evident. Die Signalstärke sollte stetig über 60 min. Inkubationen für längere Zeit (über 80 min), wird in ein Signal, das von der Spitze Signal aufgrund vermittelte Lyse der Wirtszellen Phagen Rückgang führen wird. Ähnliche Ergebnisse werden bei einer Inkubationstemperatur von 37 ° C gewonnen wurde, obwohl die optimale Temperatur für Y. pestis Wachstum beträgt 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Phage-vermittelte Biolumineszenz Detektion von Y. pestis. Zum Zeitpunkt 0, Reporter Phagen und Zellen wurden gemischt, bei 28 ° C, und Biolumineszenz (RLU) wurde im Laufe der Zeit überwacht. Ein signifikanter Anstieg in RLU (* Studenten t-Test, p &lt;0,05) liegt auf der Hand innerhalb von 15 min.

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. 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+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

Diese Methode zeigt die Fähigkeit des Reporters Phagen schnell erkennen Y. pestis, da die Reporter Phagen können transduzieren eines Biolumineszenz-Signal Antwort auf kultivierten Y. pestis-Zellen innerhalb von 20 min nach dem Phagen hinaus. Der Reporter Phagen ist auch in der Lage direkt nachzuweisen Y. pestis in klinischen Matrizen, ohne die Voraussetzung der Isolation und der anschließende Kultivierung 7. Im Vergleich zu den Standard-Phagen-Lyse-Assays die in der Regel 48 h f?...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Difco LB-Agar, Miller </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB Brühe, Miller </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 x 100 mm Kulturröhrchen </td><td> USA Scientific </td><td> 1485-0810 </td></tr><tr><td> n-Decanal </td><td> Sigma </td><td> D7384 </td></tr><tr><td> Veritas Mikroplatten-Luminometer </td><td> Turner Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite Mikrotiterplatten 96-Well-Platte </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

Yersinia pestis und Bacillus anthracis sind Kategorie A bakterielle Erreger, die die Erreger der Pest und Milzbrand sind jeweils 1. Obwohl das natürliche Vorkommen beider Krankheiten &quot;ist jetzt relativ selten sind, ist die Möglichkeit terroristischer Gruppen mit diesen Erregern als Biowaffe Echtzeit. Wegen der Krankheit innewohnende Kommunikationsfähigkeit, schnelle klinischen Verlauf und eine hohe Sterblichkeitsrate, ist es entscheidend, dass ein Ausbruch schnell erkannt werden. Deshalb Methoden, die eine schnelle Erkennung und Diagnose stellen sind unerlässlich, um eine sofortige Umsetzung der Maßnahmen des Gesundheitswesens und die Aktivierung des Krisenmanagements gewährleisten. Rekombinante Phagen-Reporter kann einen schnellen und spezifischen Ansatz für den Nachweis von Y. pestis und B. anthracis. Die Centers for Disease Control and Prevention nutzen derzeit das klassische Phagen Lyse-Assays für die bestätigten Identifizierung dieser bakteriellen Erregern 2-4. Diese Assays profitieren von natürlich vorkommenden Phagen, die spezifisch und lytisch sind für ihre bakteriellen Wirten. Nach Wachstum über Nacht der kultivierten Bakterien in Gegenwart der spezifischen Phagen, bietet die Bildung von Plaques (bakterielle Lyse) eine positive Identifikation der bakteriellen Ziel. Obwohl diese Tests robust sind, leiden sie unter drei Mängel: 1) sie sind im Labor basiert, 2) die sie benötigen bakterielle Isolierung und Kultivierung von den verdächtigen Probe, und 3) nehmen sie 24-36 h in Anspruch nehmen. Um diese Probleme anzugehen, wurden rekombinante &quot;light-Tag&quot;-Reporter Phagen genetisch durch die Integration der Vibrio harveyi luxAB Gene in das Genom von Y. entwickelt pestis und B. anthracis spezifische Phagen 5-8. Die daraus resultierende luxAB Reporter Phagen konnten ihre spezifische Ziel von schnell zu erkennen (in Minuten) und sensibel Übertragung einer Biolumineszenz Phänotyp an den Empfänger-Zellen. Wichtig ist, war die Detektion entweder mit kultivierten Zellen oder Empfänger mit Mock-infizierte klinische Proben 7 erhalten. Zu Demonstrationszwecken hier beschreiben wir das Verfahren für die Phagen-vermittelte Erkennung eines bekannten Y. pestis isolieren mit einem luxAB Reporter Phagen von der CDC Pest diagnostische Phagen ΦA1122 6,7 (Abb. 1) konstruiert. Eine ähnliche Methode, mit geringfügigen Änderungen (zB Änderung des Wachstums Temperatur und Medien), kann für den Nachweis von B. verwendet werden anthracis-Isolate mit der B. anthracis Reporter Phagen Wβ: luxAB 8. Die Methode beschreibt die Phagen-vermittelte Transduktion eines biolumescent Phänotyp angebaut Y. pestis Zellen, die anschließend gemessen werden mit einem Mikrotiterplatten-Luminometer. Die wesentlichen Vorteile dieser Methode gegenüber der traditionellen Phagen Lyse-Assays ist die einfache Handhabung, die schnelle Ergebnisse, und die Möglichkeit, mehrere Proben gleichzeitig Test in einer 96-well Mikrotiterplatten-Format. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Erkennung schematisch. Die Phagen werden mit der Probe vermischt, infiziert der Phage die Zelle, luxAB ausgedrückt sind, und die Zelle bioluminesces. Probenvorbereitung ist nicht notwendig, die Phagen und Zellen vermischt und anschließend für Licht gemessen.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Eine einfache Methode zur Identifizierung der prioritären bakteriellen Erregern ist gentechnisch Reporter Phagen verwenden. Diese Reporter-Phagen, die spezifisch für ihre besonderen Wirtsarten sind, sind in der Lage schnell Transduktion eines Biolumineszenz-Signal Antwort auf Wirtszellen. Hier beschreiben wir die Verwendung von Reporter Phagen für den Nachweis von<em&gt; Yersinia pestis</em&gt;.

&quot;Bioluminescent &#39;Reporter Phage für den Nachweis von Kategorie A bakteriellen Erregern

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.4K Aufrufe.  Guild Associates, Inc.. Eine einfache Methode zur Identifizierung der prioritären bakteriellen Erregern ist gentechnisch Reporter Phagen verwenden. Diese Reporter-Phagen, die spezifisch für ihre besonderen Wirtsarten sind, sind in der Lage schnell Transduktion eines Biolumineszenz-Signal Antwort auf Wirtszellen. Hier beschreiben wir die Verwendung von Reporter Phagen für den Nachweis von Yersinia pestis.

Serie: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Mit Luciferase to Image Bakterielle Infektionen bei Mäusen

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Forschung

Immunologie und Infektion

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

In Situ Messung und Korrelation der Zelldichte und Lichtemission von Biolumineszenten Bakterien

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Biochemie

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensor zum Nachweis von Antibiotika-resistenten Staphylococcus Bakterien

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Biotechnik

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Eine Fluoreszenz basierende Methode, bakterielle Genregulation in infizierten Gewebe zu studieren

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetik

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Nachweis von Bakterien mit fluorogenen DNAzyme

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakterielle Imaging<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

A Step Beyond BRET: Fluoreszenz von Unbound Anregung von Lumineszenz (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Erkennung von Enterohemorrhagic Escherichia Coli Besiedlung in murinen Host durch nicht-invasive In Vivo Biolumineszenz-System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Mit Cokultur zur Erkennung chemisch vermittelte Interspezies-Interaktionen

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Biologie

Verfolgung von Krankheitserregern im ganzen Organ durch Quantifizierung von fluoreszierenden Reportern

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Autor im Rampenlicht: Entschlüsselung der bakteriellen Reaktionen auf Antibiotika und Immunsystem in Geweben

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Hochauflösende dreidimensionale Ganzorgantomographie von mikrobiellen Infektionen

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

"Bioluminescent 'Reporter Phage für den Nachweis von Kategorie A bakteriellen Erregern

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