We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. Biofilm Initiation <ol><li> Wachsen einer Kultur des Biofilms produzierenden Organismus in einem nährstoffreichen Medium. Impfen Staphylococcus aureus - Stamm Newman in 10 ml Mueller-Hinton - Medium aus einem Glycerin Lager. Führen Sie alle Arbeiten , die mit der Handhabung von S. aureus mit Handschuhen und in einem Biosicherheitsschrank. </li><li> bei 37 ° C für 16 Stunden inkubieren auf einem Rotationsschüttler (100-200 rpm). </li><li> Bestimmen Sie OD 600 der Kultur eines Spektrometers und eine Standardküvette mit (Weglänge: 1cm). </li><li> Berechne das Volumen (V) der Kultur benötigt 20 herzustellen ml des Inokulums mit OD 600 (Inokulum) von 0,1 in chelexed Roswell Park Memorial Institute Medium (cRPMI) 9 mit der Formel: [V = 20 ml * 0,1 / OD 600 (Kultur )] Hinweis: Referenz 9 enthält Einzelheiten über cRPMI Vorbereitung. <ol><li> Die berechnete Kulturvolumen (von oben) in ein Zentrifugenröhrchen und Pelletzellen durch bei 10.000 x g für 1 min dreht. Um prepadas verbrauchte Nährmedium und resuspendieren Zellpellet in 20 ml cRPMI re Biofilm Inokulum absaugen. </li></ol></li><li> Gießen Inokulum in einen 25-ml-Reservoir. </li><li> Entfernen Sie vorsichtig den Deckel aus einer sterilen 96-Well-Platte peg diese gerade nach oben ziehen. Hinweis: Bitte achten Sie dürfen nicht die Heringe zu verunreinigen nach unten aus dem Deckel hängen. Der Deckel kann in einem Biosicherheitsschrank auf eine saubere Oberfläche den Kopf gestellt werden. </li><li> Verwendung eines 200 &amp; mgr; l Mehrkanalpipette, fügen 125 ul Impfgut zu jeder Vertiefung der Reihen A bis G der Bodenplatte. </li><li> Füllen Sie 125 ul sterile cRPMI Medium in jeder Vertiefung der Reihe H als Sterilitätskontrolle. </li><li> Nehmen Sie die peg-Deckel und halten Sie ihn mit den Zapfen über dem Plattenboden nach unten. sorgfältig ausrichten, um die einzelnen Stifte mit ihren jeweiligen Brunnen. Vermeiden Sie Körperkontakt zwischen der Platte und den Zapfen. Nach der Ausrichtung senken vorsichtig nach unten den Deckel. Vermeiden Sie Fehlstellungen wie ausgeführt Nachjustierungen nach den Zapfen der Bodenplatte berührt haben cont kannaminate Sterilität Kontrollen in Reihe H. </li><li> Verschließen Sie den Deckel auf die Platte mit Paraffin Filmverdampfung und in einen verschließbaren Plastikbeutel zu verhindern, Abdichtung fest. Halten Sie das Luftvolumen in der Tasche so gering wie möglich Verdunstung zu minimieren. </li><li> Inkubieren ohne für 48 Stunden in einem 5% CO 2 Inkubator bei 37 ° C schüttelnd. Hinweis: 48 Stunden des Wachstums von S. optimal gewesen aureus, sondern wird auf den Organismus verwendet , variieren. Hinweis: Obwohl statische Inkubation ein guter Ausgangspunkt ist der Test zu optimieren, sanft bei 150 Umdrehungen pro Minute auf einem Schüttler Schütteln ist eine mögliche Variante , die die Biofilmbildung von einigen S. verbessern kann aureus - Isolaten. </li></ol> 2. Herstellung der Biofilm Challenge-Platte <ol><li> Am Tag des Biofilms Herausforderung nehmen eine frische 96-Well-Platte und fügen 100 ul cRPMI, auf RT ins Gleichgewicht gebracht, in jede Vertiefung der Zeilen B bis H. inkonsistente Ergebnisse zu vermeiden, verwenden eine 96-Well-Platte, die 200 &amp; mgr; l aufnehmen kanninnerhalb der Medien, wenn die Heringe, und verfügt über Vertiefungen mit Abmessungen identisch mit denen des Biofilms Einleitung Platte. </li><li> Verdünne bis zu 4 Testverbindungen getrennt in 1,0 ml cRPMI die gewünschte Endkonzentration, um zu 2x für eine Endverdünnung in Schritt 2.7 zu berücksichtigen. Hinweis: Ein guter Ausgangspunkt ist 8-fach über die hemmende Konzentration von planktonischen Zellen. </li><li> In 200 ul der Verbindung 1 in den Vertiefungen A1 bis A3, die Verbindung 2 in Brunnen A4 bis A6, Verbindung 3 in Brunnen A7 bis A9 und Verbindung 4 in Brunnen A10 bis A12. </li><li> Abwechselnd verdünnen die Testverbindungen mit einer Mehrkanalpipette mit und 100 &amp; mgr; l aus Reihe A übertragen B und mischen zu rudern. </li><li> Auf diese Weise weiterhin 100 ul Vertiefung zu Vertiefung zu übertragen. Mischen Sie nach jeder Übertragung und stoppen in der Reihe F. </li><li> Nach dem Mischen vertreiben 100 &amp; mgr; l aus Reihe F in einen Abfallbehälter. Verwenden Sie keine Flüssigkeit in Reihen G und H. übertragen </li><li> 100 l cRPMI zu jeder Vertiefung der Platte mit einer Mehrkanalpipettebringen das Volumen auf 200 ul. Siehe Abbildung 1 für die endgültige Plattenlayout. </li></ol> 3. Biofilm Herausforderung <ol><li> Mit 200 &amp; mgr; l phosphatgepufferter Salzlösung (PBS) auf eine frische sterile 96-Well-Platte, die Vertiefungen mit Abmessungen identisch mit denen des Biofilms Initiationsplatte verfügt. </li><li> Entfernen Sie den Plastikbeutel und Paraffinfilm aus inkubiert Biofilm Initiationsplatte. </li><li> Heben Sie den Deckel gerade nach oben. Vermeiden Sie den Kontakt zwischen den Zapfen und dem Plattenboden, da dies den Biofilm beschädigen können. Halten Sie Unterteil für die nachfolgende OD 600 - Analyse (siehe 3.8). </li><li> Wegspülen planktonischen Zellen durch den Stift-Deckel auf die Platte legen enthält PBS (aus Schritt 3.1). Versenken Pflöcke für 5 Sekunden, heben Sie aus PBS, und tauchen noch einmal, man aufpassen, nicht die Zapfen gegen die Seiten gut zu treffen. </li><li> Setzen Sie den abgespült peg-Deckel in die Herausforderung Platte. Vermeiden Sie unnötigen Kontakt zwischen Zapfen und Bodenplatte, da dies der Biofilm führt zu Schäden aninkonsistente Ergebnisse. </li><li> Die Dichtungsplatte mit Paraffinfilm und in einen verschließbaren Beutel aus Kunststoff, wie in Schritt 1.10 beschrieben. </li><li> Inkubiere die Platte für 24 Stunden bei 37 ° C in einem 5% CO 2 Inkubator ohne Schütteln. </li><li> Nehmen Unterseite des Biofilms Initiationsplatte von 3,3. Resuspendieren der Bakterien in jeder Vertiefung einer Mehrkanalpipette mit und die OD 600 mit einem geeigneten Mikroplatten - Reader messen Wachstum zu bestätigen in allen Vertiefungen , aber die Sterilität Kontrollen in Reihe H. vorkommenden </li></ol> 4. Biofilm Bestimmung <ol><li> Verwenden Leser eine Platte mit einer Inkubationskabine ausgestattet und in der Lage nehmen kinetische Fluoreszenzmesswerte zur Erfassung von Resazurin Umwandlung. Legen Sie eine 24-Stunden-kinetische Fluoreszenzmessung unter Verwendung eines 530 nm Anregung und 590 nm Emission auf. </li><li> Stellen Sie die Raumtemperatur auf 37 ° C und haben die Platte alle 20 Minuten lesen. Stellen Sie den Leser Platte von der Unterseite der Platte zu messen, nach Herstelleranweisunggen. </li><li> Bereiten Waschplatte durch Zugabe von 200 ul PBS mit einer Mehrkanalpipette in eine sterile 96-Well-Platte. </li><li> Bereiten Biofilm Wiederherstellungsmedium durch Zugabe von 400 &amp; mgr; l von 0,8 mg / ml Stammlösung zu 20 ml cRPMI Medium auf eine Endkonzentration von 16 &amp; mgr; g / ml Resazurin Resazurin. Hinweis: Resazurin ist eine bekannte hautreizend. Verwenden Sie einen Laborkittel, Schutzbrille und Handschuhe bei der Handhabung. CRPMI als Biofilm Recovery-Medien wurde verwendet, da es den niedrigsten Hintergrundsignal liefert, aber es kann von Mueller-Hinton-Medien gegebenenfalls ersetzt werden. </li><li> In 150 ul Biofilm Recovery Medium mit einer Mehrkanalpipette in jede Vertiefung einer frischen 96-Well-Platte. </li><li> Transfer Herausforderung Platte aus Inkubator in einem Biosicherheitsschrank und sorgfältig Plastiktüte und Siegelfolie entfernen. </li><li> Entfernen Sie die peg-Deckel von der Herausforderung Platte und abzuspülen planktonischen Zellen in PBS, wie in 3.4 beschrieben. Halten Sie den Boden der Herausforderung Platte für nachfolgende OD 600 anaLyse (siehe 4.10). </li><li> Nach dem Spülen, legen Sie den zapfen Deckel in den Plattenboden des Biofilms Recovery-Medium enthält. </li><li> Wickeln Sie die Seite der Platte mit Paraffinfilm, wobei darauf geachtet, nicht den Boden der Umfang Brunnen zu behindern. Unmittelbar nach der Platte Verpackung, legen Sie sie auf dem Plattenleser und eine kinetische Lese über einen Zeitraum von 24 h beginnen, indem die bisherigen Resazurin kinetische Protokoll von Schritt 4.1 zu initiieren. </li><li> Um das Wachstum von planktonischen Zellen zu quantifizieren , die während der Herausforderung auftreten, lesen Sie die OD 600 der gesamten Herausforderung Plattenboden kann oder auch nicht ein Mikroplattenleser. Folgen Sie die Anweisungen in Schritt 3.8. Hinweis: vorsichtig resuspendieren den Inhalt jeder Vertiefung als Shedding Zellen aus den Biofilm neigen dazu, in Klumpen am Boden der Vertiefung zu wachsen. Alle Blasen innerhalb des Schachtes wird stark mit OD 600 Lesungen stören und müssen vor dem Lese vermieden oder entfernt werden. </li></ol>

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The authors have nothing to disclose.

Hier haben wir ein abgewandeltes Biofilm - Assay zur Bestimmung der Aktivität der getesteten Inhibitoren auf S. beschrieben aureus Biofilm auf den Stoffwechselzustand von Biofilm assoziierten Zellen konzentriert. Während die beschriebene Biofilm Initiierung und Widerspruchsverfahren meist nachgeahmten Herstellerempfehlungen, die Verwendung von Resazurin Farbstoff Biofilm-assoziierte Zellen, die eine 24-Stunden-Inhibitor Herausforderung überleben dramatisch vereinfacht die empfohlene Verfahren der Ze...

Pathogene Keime können 1 Biofilmen in vivo führt zu nicht akuten chronischen Infektionen bilden. Biofilm-assoziierte Infektion ist eine ernste Risikofaktor für medizinische Verfahren , die Implantation von Fremdkörpern (zB künstliche Knochenersatz, Brustimplantate) oder die Installation von Trachealtuben oder Urinkatheter 2 beinhalten. In diesen Zusammenhängen ist eine antiinfektive Therapie fast immer notwendig , als Biofilm-basierte Infektionen auf ihren eigenen selten gelöscht werden, selbst in immunkompetenten Individuen. Staphylococcus aureus eine der am häufigsten beobachteten Krankheitserreger in Biofilm-Komplikationen beteiligt ist , während der Verwendung auftreten , von invasive medizinische Geräte 3. Sie mehr resistent gegen die Behandlung als planktonischen Zellen 1,4, und die Bewertung der vorhergesagten klinische Wirksamkeit Leider macht das Wesen von Biofilmen als Schutzbarriere ist ein wichtiger Bestandteil der beiden AnfangsArzneimittelentwicklung sowie Arzneimittelresistenzüberwachung. Es gibt immer mehr Anerkennung , dass Laborbedingungen auf planktonischen Kulturen konzentriert, kann nicht treu reale Krankheit 5 darstellen. Weiterhin das Problem Compoundierung, ist die Replikation von Biofilm - Phänotypen schwierig, und vorhandene Biofilm - Modelle sind langwierig und leiden unter hohen inter- und intra Assay Variabilität 6. viele Forscher, durch die Notwendigkeit, Standard zu planktonischen Zellassays von Medikamentenempfindlichkeit, wodurch möglicherweise ein wichtiger Aspekt der bakteriellen Virulenz zu vernachlässigen und Krankheit So. Hier beschreiben wir das Protokoll für Bakterien Testen, insbesondere S. aureus, in pre-grown Biofilmen eine 96-Well - basierten Biofilm System 7,8 verwendet. Während das Protokoll für die Biofilmbildung und die Herausforderung im Wesentlichen die Empfehlungen des Herstellers folgt präsentieren wir eine alternative informationsreiche Methode, um die Lebensfähigkeit des bioF zur Quantifizierungilm nach Herausforderung. Kurz gesagt, sind Bakterien in der Stange Platte kultiviert, wo Biofilmen an der Platte des Deckels angebracht an den vorstehenden Zapfen bilden. Nach der Biofilmbildung sind die Zapfen in die Vertiefungen einer frischen Platte mit PBS gefüllt zu entfernen planktonischen Zellen vorsichtig getaucht. Der PEG-Deckel mit Biofilmen angebracht ist, wird auf eine neue Herausforderung Platte übertragen, verschiedene Konzentrationen der Antibiotika enthält, untersucht werden. Nach einer zweiten Inkubation werden Deckel wieder entfernt, gewaschen und in ein Rückgewinnungsplatte Resazurin Farbstoff enthält, überführt, wo sie einer abschließenden Inkubation unterzogen werden. Resazurin Umwandlung kann nach einer definierten Erholungszeit als Endpunkt Lesen kinetisch oder verbucht. Dieser Farbstoff-basierte Methode , um die Lebensfähigkeit von Biofilmen zu quantifizieren , unterscheidet sich erheblich von den langweiligen KBE (Kolonie bildende Einheiten) zählen basierte Methodik im ursprünglichen Protokoll 7 beschrieben. OD 600 Messungen der Droge Herausforderung Platte und Resazurin Umwandlungskinetik dienen als Lebensfähigkeit Leseouts von planktonischen und Biofilm-Zellen, die jeweils eine schnelle, zuverlässige, informationsreiche und technisch einfachen Test zur Biofilm Überleben anbietet.

<table><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></td></tr></tbody></table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Der Resazurin-Assay ist empfindlich genug, um zuverlässig sehr wenige lebensfähige Zellen replizieren in drogenfreien Medium nach der Herausforderung der Lage zu erkennen. In dieser Methode definieren wir die minimale Biofilm auszurotten Konzentration als die niedrigste Konzentration, bei der keine Resazurin Umwandlung innerhalb von 24 h zu sehen ist. Der Resazurin-Assay beruht auf oxidativen Moleküle in metabolisch aktiven Zellen gefunden, die den Farbstoff Farbe von blau zu rosa kon...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Targeting Biofilm Assoziierte<em&gt; Staphylococcus aureus</em&gt; Verwendung von Resazurin Based Drug-Anfälligkeit Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

13.0K Aufrufe.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

Serie: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

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) ...

Forschung

Biotechnik

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Einsatz von künstlichen Sputum Mittlere bis antibiotische Wirkung gegen die getestet<em&gt; Pseudomonas aeruginosa</em&gt; AGB in einen engeren Bezug zu den Mukoviszidose-Lunge

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Immunologie und Infektion

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Stressinduzierte Antibiotika-Anfälligkeitstests auf einem Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Eine Plattform von Assays Anti-Biofilm auf die Erforschung der natürlichen Substanzbibliotheken Geeignet

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

System zur Wirksamkeit und Zytotoxizität Screening von Inhibitoren Targeting intrazellulärer<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Antibiotika-Wirksamkeitstests in einem Ex-vivo-Modell von Pseudomonas aeruginosa und Staphylococcus aureus Biofilmen in der Mukoviszidose-Lunge

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

Standardisierte In-vitro-Assays zur Visualisierung und Quantifizierung von Interaktionen zwischen humanen Neutrophilen und Staphylococcus aureus-Biofilmen

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Ein löslicher Tetrazolium-basierter Reduktionstest zur Bewertung der Wirkung von Antikörpern auf Candida tropicalis-Biofilme

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Verbesserter Enzymschutzassay zur Untersuchung der Internalisierung von Staphylococcus aureus und der intrazellulären Wirksamkeit antimikrobieller Verbindungen

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biologie

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Source: Schaaf, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55453">A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

Ein Assay zur Bewertung der Wirksamkeit von Medikamenten gegen ein Mycobacterium tuberculosis-Infektionsmodell

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain ...

Targeting Biofilm Assoziierte

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