1. Bakterienkultur <ol><li> Gram-negative P. aeruginosa Stamm PAO1 wurden in Luria Broth (LB) bei 37 ° C für 18 Stunden gezüchtet. </li><li> Die Kultur der Zellen wurde dann bei 7500 rpm (Umdrehungen pro Minute) für 5 min zentrifugiert und der Überstand wurde entfernt. </li><li> Die Bakterien wurden in 10% LB resuspendiert und für weitere 2 Stunden wieder gewachsen, um die Kultur in der stationären Phase erneut einzugeben. </li><li> Die Bakteriensuspension wurde dann in zwei Gruppen unterteilt: in der ersten Gruppe (2 Tuben) kein Antibiotikum zugegeben, in der zweiten Gruppe wir Gentamycin Antibiotikum (50 ug / ml) zugegeben. </li></ol> 2. Bestimmung der Kolonie-bildenden Einheiten (KBE) <ol><li> Um die Lebensfähigkeit der Zellen zu bestimmen 20 ul-Proben wurden aus dem Experiment etwa alle 2 Stunden innerhalb des Zeitrahmens von 24 Stunden gemacht. Serielle Verdünnungen der Proben wurden hergestellt und auf LB-Agarplatten ausplattiert und über Nacht bei 37 ° C inkubiert, </li><li> Für jede Behandlung, die KBE pro plate wurde bestimmt und ein Vergleich zwischen den Zeiträumen und verschiedenen Behandlungen gemacht. Die Log-Reduktion in CFU wurde berechnet, wie in Gl. (1): Log Reduktion = Logu-LogC [KBE / ml] Wobei U die koloniebildenden Einheiten Wert zu jedem Zeitpunkt; CFU die koloniebildende Einheit, während die Einheiten der CFU / ml, entsprechend: CFU / ml = (Anzahl an Kolonien x Verdünnungsfaktor) / (Volumen beimpft) Und c die CFU in der Kontrollprobe zur Startzeit gefunden. Beachten Sie, dass U die Kolonie bildenden Faktor Messzeitpunkt bezeichnet. </li><li> Der Verdünnungsfaktor ist die Anzahl der Verdünnungen während in jedem von ihnen die Konzentration der Bakterien um den Faktor 10 reduziert wurde. Das Volumen geimpft war immer 200 Mikroliter und es wird auf die Größe unseres Reagenzglas zusammen. </li></ol> Deshalb, um die Konzentration Sicht zusammenzufassen, war das Antibiotikum Gentamycin in einer Konzentration von 50 ug / ml. In Bezug auf die Bakterien, bis zum Ende derdas Verfahren hatten wir insgesamt 8 Verdünnungen. Jede Verdünnung wurde mit einem Faktor von 10 und es wurde in Röhren von 200 ul getan. Der Ausgangspunkt war 20 ul Proben in die 200 ul Röhrchen gegeben (und damit die anfängliche Konzentration war 20/200C 0 = 0.1C0 mit C 0 die anfängliche Konzentration in der 20 ul Proben) und die endgültige Konzentration wurde durch 8 reduziert Größenordnungen aufgrund der 8 Verdünnungen. 3. Beleuchtung <ol><li> CW Nd: YAG-Lasers (Wellenlänge von 532 nm und der durchschnittlichen optischen Energie von 200 mW) in zwei optische Wege geteilt durch optische 50% / 50% Strahlteiler. Der Strahldurchmesser betrug etwa 10 mm. Die Expositionsdauer betrug 24 Stunden. </li><li> Gütegeschalteten Nd: YAG-Lasers (Wellenlänge von 532 nm, mittlere Leistung von 300 mW und optische Spitzenleistung von 2,5 MW) wurde ebenfalls in zwei Pfade aufgeteilt, die optische 50% / 50% Strahlteiler. Der Punktdurchmesser betrug 6 mm. Die Impulsbreite des Q-Switch-Laser betrug 6 ns und die Wiederholungsrate war 15Hz. Diemittlere Leistung betrug 106 mW / cm 2 und die Spitzenleistung betrug 8,83 kW / mm 2. Die Expositionsdauer betrug 24 Stunden. </li></ol> Beachten Sie, dass die Bakteriensuspension während der Bestrahlung gerührt und es wurde unter geeigneten Kulturbedingungen für das bakterielle Wachstum (in allen Röhren gab es Luria Broth Medium, damit die Bakterien wachsen) gehalten.

<ol>
	<li>Feuerstein, O., Persman, N., Weiss, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxic+Effect+of+Visible+Light+on+Porphyromonas+gingivalis+and+Fusobacterium+nucleatum:+An+In+Vitro+Study.">Phototoxic Effect of Visible Light on Porphyromonas gingivalis and Fusobacterium nucleatum: An In Vitro Study.</a> Photochemistry and Photobiology. 80, 412-415 (2004).</li><li>Enwemeka, C. S., Williams, D., Enwemeka, S. K., Hollosi, S., Yens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+470-nm+light+kills+methicillin-resistant+Staphylococcus+aureus+(MRSA)+in+vitro.">Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro.</a> Photomed. Laser Surg. 27, 221-226 (2009).</li><li>Guffey, J. S., Wilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+bactericidal+effects+of+405-nm+and+470-nm.">In vitro bactericidal effects of 405-nm and 470-nm.</a> Photomed. Laser Surg. 24, 684-688 (2006).</li><li>Lipovsky, A., Nitzan, Y., Friedman, H., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Staphylococcus+aureus+strains+to+broadband+visible+light.">Sensitivity of Staphylococcus aureus strains to broadband visible light.</a> Photochem. Photobiol. 85, 255-260 (2008).</li><li>Lipovsky, A., Nitzan, Y., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+Mechanism+for+visible+light+induced+wound+healing.">A possible Mechanism for visible light induced wound healing.</a> Lasers in Surgery and Medicine. 40, 509-514 (2008).</li><li>Lipovsky, A., Nitzan, Y., Gedanken, A., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+light-induced+killing+of+bacteria+as+a+function+of+wavelength:+Implication+for+wound+healing.">Visible light-induced killing of bacteria as a function of wavelength: Implication for wound healing.</a> Lasers in Surgery and Medicine. 42, 467-472 (2010).</li><li>Feuerstein, O., Ginsburg, I., Dayan, E., Veler, D., Weiss, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Visible+Light+Phototoxicity+on+Porphyromonas+gingiwalis+and+Fusobacferium+nucleaturn.">Mechanism of Visible Light Phototoxicity on Porphyromonas gingiwalis and Fusobacferium nucleaturn.</a> Photochemistry and Photobiology. 81, 1186-1189 (2005).</li><li>Nussbaum, E. L., Lilge, L., Mazzulli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+630-,+660-,+810-,+and+905-nm+laser+irradiation+delivering+radiant+exposure+of+1-50+J/cm2+on+three+species+of+bacteria+in+vitro.">Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm2 on three species of bacteria in vitro.</a> J. Clin. Laser Med. Surg. 20, 325-333 (2002).</li><li>Dadras, S., Mohajerani, E., Eftekhar, F., Hosseini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+Photoresponses+of+Staphylococcus+aureus+and+Pseudomonas+aeruginosa+to+514,+532,+and+633+nm+Low+Level+Lasers+In+Vitro.">Different Photoresponses of Staphylococcus aureus and Pseudomonas aeruginosa to 514, 532, and 633 nm Low Level Lasers In Vitro.</a> Current Microbiology. 53, 282-286 (2006).</li><li>Stover, C. K., Pham, X. Q., Erwin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+Pseudomonas+aeruginosa+PAO1,+an+opportunistic+pathogen.">Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.</a> Nature. 406, 952-964 (2000).</li><li>Hamblin, M. R., Demidova, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+low+level+light+therapy.">Mechanisms of low level light therapy.</a> Proc. SPIE. 6140, 1-12 (2006).</li><li>Krespi, Y. P., Stoodley, P., Hall-Stoodley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+disruption+of+biofilm.">Laser disruption of biofilm.</a> Laryngoscope. 118, 1168-1173 (2008).</li><li>Reznick, Y., Banin, E., Lipovsky, A., Lubart, R., Zalevsky, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+laser+light+enhancement+of+susceptibility+of+bacteria+to+gentamycin+antibiotic.">Direct laser light enhancement of susceptibility of bacteria to gentamycin antibiotic.</a> Opt. Commun. 284, 5501-5507 (2011).</li></ol>

Keine Interessenskonflikte erklärt.

Lichttherapie ist ein Gebiet der modernen multidisziplinären Forschung in den letzten Jahren Schwellenländern als vielversprechender Ansatz für die Behandlung zahlreicher Krankheiten. In diesem Zusammenhang ist die Verwendung von Licht im sichtbaren Bereich ist eingehend untersucht worden. Zum Beispiel wurde gefunden, dass infizierte Wunden effektiver, indem sie sie intensive sichtbare Licht entkeimt geheilt werden. Der Wirkmechanismus für diesen Ansatz wurde nachgewiesen, dass durch die Induktion von Licht-induzier...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Lauria Broth</td>
 <td>Difco</td>
 <td>241420</td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamycin</td>
 <td>Sigma </td>
 <td>G1914</td>
 <td></td>
 </tr>
 <tr>
 <td>Bacto Agar</td>
 <td>Difco </td>
 <td>231710 </td>
 <td></td>
 </tr>
 </tbody>
</table>

the synergistic effect of visible light and gentamycin on pseudomona aeruginosa microorganisms

Kürzlich gab es mehrere Veröffentlichungen über die bakterizide Wirkung des sichtbaren Lichts, die meisten von ihnen behaupten, dass blaue Teil des Spektrums (400 nm-500 nm) ist verantwortlich für die Tötung verschiedene Pathogene 1-5. Die phototoxische Wirkung von blauem Licht wurde vorgeschlagen, eine Folge der durch Licht induzierten reaktiven Sauerstoffspezies (ROS) Bildung durch endogene bakterielle Photosensibilisatoren, die meist absorbieren Licht im blauen Bereich 4,6,7 sein. Es gibt auch Berichte von Biozid-Wirkung der roten und nahen infraroten 8 sowie 9 grünes Licht. In der vorliegenden Studie haben wir eine Methode, die uns um die Wirkung der hohen Leistung Grün (Wellenlänge 532 nm) kontinuierlich (CW) und gepulste Q-switched (QS) Licht auf Pseudomonas aeruginosa charakterisieren erlaubt. Mit dieser Methode untersuchten wir auch die Wirkung von grünem Licht mit einer Antibiotika-Behandlung (Gentamycin) an der Lebensfähigkeit Bakterien kombiniert. P. aeruginosa ist acommon noscomial opportunistische Erreger verschiedener Erkrankungen. Der Stamm ist ziemlich resistent gegen verschiedene Antibiotika und enthält viele vorhergesagt AcrB / Mex-Art RND Multidrug Effluxsysteme 10. Das Verfahren verwendet freilebenden stationären Phase Gram-negative Bakterien (P. aeruginosa Stamm PAO1) in Luria Broth (LB)-Medium ausgesetzt Q-switched und / oder CW-Laser mit und ohne Zugabe des Antibiotikums Gentamycin. Die Lebensfähigkeit der Zellen wurde zu unterschiedlichen Zeitpunkten bestimmt. Die erhaltenen Ergebnisse zeigten, dass Laser-Behandlung allein nicht reduzieren die Lebensfähigkeit der Zellen im Vergleich zu unbehandelten Kontrolle und dass Gentamycin Behandlung allein nur in einer 0,5 log Reduktion der Keimzahl für P. führte aeruginosa. Die kombinierte Laser-und Gentamycin Behandlung führte jedoch zu einem synergistischen Effekt und die Lebensfähigkeit der P. aeruginosa wurde von 8 LOG reduziert. Das vorgeschlagene Verfahren kann weiter umgesetztmentiert über die Entwicklung des Katheters ähnliches Gerät in der Lage Einspritzen einer Antibiotika-Lösung in den infizierten Organ gleichzeitig beleuchtet den Bereich mit Licht.

Der Laser basierten Setup ist schematisch in Abbildung 1 dargestellt. Die ersten experimentellen Bedingungen verwendete einen CW Nd: YAG-Laser mit einer Wellenlänge von 532 nm ist (der zweiten Harmonischen des Nd: YAG) und der durchschnittlichen optischen Energie von 200 mW. Dieser Strahl wurde in zwei optische Wege geteilt durch optische 50% / 50% Strahlteiler so dass jeder Teilstrahl von 100 mW hat. Der Strahldurchmesser von ca. 10 mm und damit die Leistungsdichte von etwa 100 mW / cm 2. D...

Watch this Scientific Journal Video about The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms at JoVE.com

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Wir zeigen, dass eine entwickelte biomedizinischen Vorrichtung mit kontinuierlicher oder gepulster sichtbaren Laser basierende Behandlung, die mit Antibiotika (Gentamycin), führt zu einer statistisch signifikanten synergistischen Effekt führt zu einer Verringerung der Lebensfähigkeit kombiniert<em&gt; P. aeruginosa</em&gt; PAO1, um 8 log ist eine antibiotische Behandlung allein verglichen.

Die synergistische Wirkung von sichtbarem Licht und Gentamycin auf<em&gt; Pseudomona aeruginosa</em&gt; Mikroorganismen

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

the-synergistic-effect-visible-light-gentamycin-on-pseudomona

Research

JoVE Journal

Immunology and Infection

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

13.3K Aufrufe.  Bar-Ilan University. Wir zeigen, dass eine entwickelte biomedizinischen Vorrichtung mit kontinuierlicher oder gepulster sichtbaren Laser basierende Behandlung, die mit Antibiotika (Gentamycin), führt zu einer statistisch signifikanten synergistischen Effekt führt zu einer Verringerung der Lebensfähigkeit kombiniert P. aeruginosa PAO1, um 8 log ist eine antibiotische Behandlung allein verglichen.

Serie: The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-Kultur Modelle Pseudomonas aeruginosa Biofilme auf lebenden menschlichen Zellen gezüchtet Airway

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Forschung

Immunologie und Infektion

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA Isolierung von Pseudomonas aeruginosa Kolonisierung der Murine Gastrointestinaltrakt

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Mit Klick-Chemie, um die Wirkung von Viral Infection auf Host-Zell-RNA Synthese Messen

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Nach der in Echtzeit die Auswirkungen der Pneumokokken-Virulenzfaktoren in einer akuten Lungenentzündung Maus-Modells mit Biolumineszenz-Bakterien

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa induzierte Lungenschädigung-Modell

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Makrophagen-Cholesterin Depletion und Auswirkungen auf die Phagozytose<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Generierung von lymphatische Mikropartikel und Nachweis ihrer pro-apoptotische Wirkung auf die Epithelzellen der Atemwege

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protokolle für die Untersuchung die Host-Gewebeverteilung, Transmission-Modus und Effekt auf der Host-Fitness eines Densovirus im Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Besuchen Sie die Licht-Seite: In Vivo Monitoring von Pseudomonas Aeruginosa Biofilms Infektionen bei chronischen Wunden in einer diabetischen haarlose Mausmodell

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Replikation von bestellt, Nonredundant Bibliothek von Pseudomonas Aeruginosa Stamm PA14 Transposon Insertion Mutanten

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...