This work was supported by grants from the Academic Research Fund of the Ministry of Education, and the National Medical Research Council and the National Research Foundation, Singapore.

1. Kristallviolett Quantifizierung von Biofilm-Bildung in A. baumannii S1 <ol><li> Wachsen Sie eine 5-ml-Kultur von A. baumannii S1 in LB-Medium (LB) (Trypton 10 g / L, Hefeextrakt 5 g / L) bei 30 ° C in einem Schüttelinkubator (220 UpM) für 16 Stunden. </li><li> Stellen Sie die Kultur von A. baumannii S1 auf eine gewünschte OD 600 von 0,8 auf. Verwendung eines 96-Well-Platte, zu inokulieren Bakterienkultur (1: 100 Verdünnung) in frisches LB, das 10 &amp; mgr; l gereinigtes GKL-Enzym (40 mg / ml); Endvolumen der neuen Kultur ist 100 ul. </li><li> Bereiten Sie eine Kontrollkultur als auch, Dies hat jedoch keinerlei Enzym enthalten. Wiederholen ähnlichen Bedingungen, um die gewünschte Anzahl an Wiederholungen ergeben. </li><li> Die Platte mit einem Deckel und legen Sie sie in einem verschlossenen 10 l Kunststoffbehälter. Die Platte bei 30 ° C für 3 Stunden, bevor sanft Entfernen der Medien. </li><li> Fügen Sie eine weitere 100 ul frischem LB-Medium zu der Vertiefung und inkubieren Sie die Platte für 21 Stunden bei 30 ° C. </li&gt; <li> Nach der zweiten Inkubationszeit, entfernen Sie vorsichtig alle Medien. Waschen Sie die planktonischen Bakterien-Zelle mit 200 ul sterilem Wasser. Sicherzustellen, dass es nur eine minimale Störung der Zellen während des Waschvorgangs. </li><li> Fügen Sie 100 ul von 1% Kristallviolett-Lösung in jede Vertiefung und Inkubation für 15 min bei RT. Entfernen Sie die Kristallviolett-Lösung durch Waschen der gut mit 200 ul sterilem Wasser. Wiederholen Sie den Waschgang für zwei weitere Male. </li><li> 100 l 33% Essigsäure in jede Vertiefung und inkubieren Sie für 15 min unter leichtem Schütteln; Dies wird den Farbstoff aufzulösen. </li><li> Quantifizieren der Menge des Biofilms durch Messung der Absorption des Kristallviolett bei 600 nm ausgebildet. Die Menge an Kristallviolett, ist proportional zu der Menge an Biofilm gebildet. </li></ol> 2. konfokale Laser Scanning Mikroskopie von A. baumannii S1 Biofilm <ol><li> Wachsen Sie eine 5-ml-Kultur von A. baumannii S1 in LB bei 30 ° C in einem Schüttelinkubator (220 UpM) für 16 Stunden. </li><li> Anzeigenur die Kultur von A. baumannii S1 auf eine gewünschte OD 600 von 0,8 auf. Mit Hilfe eines 35-mm-Glasboden μ-Dish, zu impfen die Bakterienkultur (1: 100 Verdünnung) in frischem LB, das 30 &amp; mgr; l des gereinigten GKL Enzym (40 mg / ml); Endvolumen der neuen Kultur ist 1 ml. </li><li> Decken Sie die μ-Dish mit Deckel und legen Sie sie in einem verschlossenen 10 l Kunststoffbehälter. Inkubieren Sie die μ-Dish bei 30 ° C für 3 Stunden, bevor sanft Entfernen der Medien. Werden 30 &amp; mgr; l gereinigtes GKL Enzym und frisches Medium, das bis zu einem Gesamtvolumen von 1 ml zu bringen. Inkubation für weitere 21 h bei 30 ° C. </li><li> Wiederholen Sie Schritt 2.3 und inkubieren Sie die μ-Dish für einen weiteren 24 Stunden bei 30 ° C. Dann entfernen Sie die Medien vorsichtig. </li><li> Gib 500 ul 5 ug / ml Alex Fluo 488 konjugiertem Weizenkeim-Agglutinin (WGA) in Hanks ausgewogener Salzlösung (HBSS) mit dem μ-Geschirr gelöst und bei 37 ° C für 30 min; Dies wird das gebildete Biofilm färben. Entfernen Sie die Farblösung und waschen ter u-Teller mit 2 ml HBSS. Wiederholen Sie den Waschvorgang noch einmal. </li><li> Gib 500 &amp; mgr; l von 3,7% igem Formaldehyd in HBSS gelöst und bei 37 ° C für 30 min; Dies wird den Biofilm auf den μ-Dish beheben. Einmal Waschen Sie die μ-Dish mit 2 ml HBSS und entfernen Sie die Lösung vollständig. Die μ-Schale mit Biofilm fixiert kann in der Dunkelheit bei 4 ° C vor dem CLSM Abbildungs ​​gespeichert werden. </li><li> Für CLSM Bildgebung und Analyse sind 63-facher Vergrößerung und 97 Stapel pro Bild mit einem Abstand von 0,21 um pro Stapel zu erzeugen. </li></ol>

<ol><li>Alanis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resistance+to+antibiotics%3A+are+we+in+the+post-antibiotic+era%3F%5BTitle%5D)+AND+%22Archives+of+medical+research%22%5BJournal%5D)">Resistance to antibiotics: are we in the post-antibiotic era?</a> Archives of medical research. 36, 697-705 (2005).</li>
<li>Cegelski, L., Marshall, G. R., Eldridge, G. R., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biology+and+future+prospects+of+antivirulence+therapies%5BTitle%5D)+AND+%22Nature+reviews.+Microbiology%22%5BJournal%5D)">The biology and future prospects of antivirulence therapies.</a> Nature reviews. Microbiology. 6, 17-27 (2008).</li>
<li>LaSarre, B., Federle, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploiting+quorum+sensing+to+confuse+bacterial+pathogens%5BTitle%5D)+AND+%22Microbiology+and+molecular+biology+reviews+%3A+MMBR%22%5BJournal%5D)">Exploiting quorum sensing to confuse bacterial pathogens.</a> Microbiology and molecular biology reviews : MMBR. 77, 73-111 (2013).</li>
<li>Waters, C. M., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quorum+sensing%3A+cell-to-cell+communication+in+bacteria%5BTitle%5D)+AND+%22Annual+Review+of+Cell+and+Developmental+Biology%22%5BJournal%5D)">Quorum sensing: cell-to-cell communication in bacteria.</a> Annual Review of Cell and Developmental Biology. 21, 319-346 (2005).</li>
<li>Rasko, D. A., Sperandio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-virulence+strategies+to+combat+bacteria-mediated+disease%5BTitle%5D)+AND+%22Nature+reviews.+Drug+discovery%22%5BJournal%5D)">Anti-virulence strategies to combat bacteria-mediated disease.</a> Nature reviews. Drug discovery. 9, 117-128 (2010).</li>
<li>Lazar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quorum+sensing+in+biofilms--how+to+destroy+the+bacterial+citadels+or+their+cohesion%2Fpower%3F%5BTitle%5D)+AND+%22Anaerobe%22%5BJournal%5D)">Quorum sensing in biofilms--how to destroy the bacterial citadels or their cohesion/power?</a> Anaerobe. 17, 280-285 (2011).</li>
<li>Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+Biofilms%3A+A+Common+Cause+of+Persistent+Infections%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Bacterial Biofilms: A Common Cause of Persistent Infections.</a> Science. 284, 1318-1322 (1999).</li>
<li>Perez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+challenge+of+multidrug-resistant+Acinetobacter+baumannii%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Global challenge of multidrug-resistant Acinetobacter baumannii.</a> Antimicrob Agents Chemother. 51, 3471-3484 (2007).</li>
<li>Tay, S. B., Yew, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+quorum-based+anti-virulence+therapeutics+targeting+Gram-negative+bacterial+pathogens%5BTitle%5D)+AND+%22International+journal+of+molecular+sciences%22%5BJournal%5D)">Development of quorum-based anti-virulence therapeutics targeting Gram-negative bacterial pathogens.</a> International journal of molecular sciences. 14, 16570-16599 (2013).</li>
<li>Igarashi, J., Suga, H. Ch. 19. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aIgarashi%2C+J+%22Quorum+Sensing%22">Quorum Sensing</a>. Rumbaugh, K. P. 692, Methods in Molecular Biology. Humana Press. 265-274 (2011).</li>
<li>Ng, F. S., Wright, D. M., Seah, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+phosphotriesterase-like+lactonase+from+Sulfolobus+solfataricus+and+its+immobilization+for+disruption+of+quorum+sensing%5BTitle%5D)+AND+%22Applied+and+environmental+microbiology%22%5BJournal%5D)">Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing.</a> Applied and environmental microbiology. 77, 1181-1186 (2011).</li>
<li>Stacy, D. M., Welsh, M. A., Rather, P. N., Blackwell, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attenuation+of+quorum+sensing+in+the+pathogen+Acinetobacter+baumannii+using+non-native+N-Acyl+homoserine+lactones%5BTitle%5D)+AND+%22ACS+chemical+biology%22%5BJournal%5D)">Attenuation of quorum sensing in the pathogen Acinetobacter baumannii using non-native N-Acyl homoserine lactones.</a> ACS chemical biology. 7, 1719-1728 (2012).</li>
<li>Chow, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+evolution+of+a+thermostable+quorum-quenching+lactonase+from+the+amidohydrolase+superfamily%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.</a> The Journal of biological chemistry. 285, 40911-40920 (2010).</li>
<li>Chow, J. Y., Yang, Y., Tay, S. B., Chua, K. L., Yew, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+biofilm+formation+by+the+human+pathogen+Acinetobacter+baumannii+using+engineered+quorum-quenching+lactonases%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases.</a> Antimicrobial Agents and Chemotherapy. 58, 1802-1805 (2014).</li>
<li>Pour, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm+formation+by+Acinetobacter+baumannii+strains+isolated+from+urinary+tract+infection+and+urinary+catheters%5BTitle%5D)+AND+%22FEMS+immunology+and+medical+microbiology%22%5BJournal%5D)">Biofilm formation by Acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters.</a> FEMS immunology and medical microbiology. 62, 328-338 (2011).</li>
</ol>

The authors declare that they have no competing financial interests.

In beiden Sätzen von Experiment A. baumannii S1 wurde in LB-Medium ohne NaCl weise einer hohen Salzkonzentration kann die Menge des Biofilms von den Bakterien 15 ausgebildet reduzieren kultiviert. Das Vorhandensein solcher Artefakt könnte die Menge der Biofilm gebildet, sowie die Auswirkungen der Quorum-Abschrecken Enzyme in verschiedenen Behandlungsbedingungen unterschätzen. Die Verwendung einer katalytisch inaktiven Enzym ist wichtig, da eine negative Kontrolle, die möglichen Effekte der Enzymb...

Behandlungsmöglichkeiten für Infektionskrankheiten durch die rasche Zunahme der Multidrug-resistenten Bakterien, die immun gegen ein breites Spektrum von Antibiotika 1 sind kompliziert. Mit hoher Morbidität und Mortalität von resistenten Bakterien-vermittelte Infektionen, gibt es einen Bedarf an Arzneimittelentwicklungsprozesse zu eskalieren und / oder entdecken bessere antibakterielle Alternativen Therapiemöglichkeiten zu verbessern. In letzter Zeit wird die anti-Virulenz Ansatz gewinnen an Interesse gegeben sein Potenzial bei der Verhinderung von Virulenz über nicht bakterizide Methoden, damit Minderung der Risiken der Resistenzmechanismen 2. Quorum-Sensing ist ein &quot;Hauptschalter&quot; in bakteriellen Virulenz und Störung dieses Signal Phänomen ist eine vielversprechende anti-Virulenz Verfahren gegen Pathogenese 3. Der Beginn der Virulenz erfordert die Ansammlung von Quorum-Moleküle in der extrazellulären Umgebung, nachdem ein kritischer bakterielle Bevölkerungsdichte erreicht ist. Wie quorum Moleküle diffundieren in die intrazelluläre Matrix, die Bindung mit ihren verwandten Rezeptoren führt zur Aktivierung von Virulenzfaktoren sowie Gene mit Antibiotikaresistenz und Biofilmbildung 4 verbunden. Im Allgemeinen Quorum-Sensing-Störung beinhaltet Hemmung Quorum Molekül und Rezeptor-Wechselwirkung, ohne die primären Stoffwechselwege. Daher spielt es keine direkten Auswirkungen auf das Zellwachstum. Da Fitness nicht beeinträchtigt wird, es eine minimale Selektionsdruck für Bakterien zu entwickeln und zu erhalten Widerstand gegen solche Behandlungen 5. Zusätzlich kann ein Quorum-sensing Störung mit inhärenten Bakterienschutzmechanismen interferieren, wie im Fall der Bildung von Biofilmen, die Schutz gegen antibakterielle Mittel bereitstellt und Wirt-Immunantworten. Es wird geschätzt, dass 99% der Mikroben auf der Erde existieren in komplexen Biofilm artigen Matrizes verleih entscheidende Lebensvorteil für die lebenden Mikroorganismen innerhalb these Strukturen 6. Noch wichtiger ist, ist die Bildung dieser sessile Domains die Ursache der hartnäckigsten und chronischer Krankenhausinfektionen 7. Acinetobacter baumannii ist eine der wichtigsten menschlichen Krankheitserreger, die mit der globalen Krankenhausinfektionen verbunden ist und dessen Virulenz ist weitgehend auf die Quorum-Sensing zurückzuführen vermittelte Biofilmbildung 8. Quorum-Abschrecken Enzyme erfolgreich in stören Quorum-vermittelte Signaltransduktion durch gezielt eine Gruppe von Verbindungen, wie N-Acyl Homoserinlactone (AHLs), die von Gram-negativen Bakterien 9 sind bekannt dafür verwendet worden. Mehrere Studien haben auch von der Verwendung dieser Enzyme erweitert, um bakterielle Pathogenese durch die Reduktion von Virulenzfaktor Expression und Zellzahlen in Biofilmen 10,11 blockieren. Leider gibt es nach wie vor ein Mangel an greifbaren Beweis für die effektive Nutzung von Quorum-Abschrecken Enzyme gegen die Biofilmbildung durch bakterielle Erreger. dasre es Versuche, Quorum-Inhibitoren (AHL-Analoga), anstelle von Quorum-Abschrecken Enzyme verwenden, um A. zu stören baumannii Biofilmbildung 12. Obwohl diese Methode der Verwendung von kleinen Molekülen Inhibitoren ist ein gültiger Ansatz können erhalt seine Bioverfügbarkeit in der translationalen Verwendungen eine Herausforderung sein. Im Gegenteil könnte die Verwendung von katalytischen quorum Abschrecken Enzyme die Bioverfügbarkeit Problem zu umgehen, wie Enzyme sind empfänglicher gegen Immobilisierung an Oberflächen von biomedizinischen Vorrichtungen für therapeutische Wirkungen. Hier beschreiben wir eine Bewertung der Auswirkungen von gentechnisch Quorum lösch Lactonasen von Geobacillus kaustophilus (GKL) 13 auf bakterielle Biofilmbildung, mit Kristallviolett-Färbung und konfokaler Laser Scanning Mikroskopie (CLSM). Diese Studie ist die erste erfolgreiche Demonstration von Biofilm Störung in einem klinisch relevanten A. baumannii S1-Stamm unter Verwendung Quorum lösch Enzyme. Beschriebenen Verfahrenin dieser Studie sind nützlich für die Beurteilung der Wirksamkeit anderer quorum Abschrecken Enzyme in nachfolgenden therapeutischen Entwicklungsbemühungen gegen pathogene gramnegative Bakterien.

<table><tbody><tr><td>Tryptone&amp;nbsp;</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>96-well plate</td><td>Costar</td><td>3596</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma-Aldrich</td><td>C6158</td><td></td></tr><tr><td>Acetic Acid</td><td>Lab-Scan</td><td>PLA00654X</td><td>Caution: Flammable</td></tr><tr><td>&amp;mu;-Dish</td><td>Ibidi</td><td>80136</td><td></td></tr><tr><td>Alex Fluo 488-conjugated WGA</td><td>Invitrogen</td><td>W11261</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Invitrogen</td><td>141475095</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Caution: Corrosive&amp;nbsp;</td></tr><tr><td>Synergy HT Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>1X-81 Inverted Fluorescence Microscope</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

In der Kristallviolett Quantifizierung Experiment wurden zwei Quorum lösch Enzyme verwendet, um Machbarkeit stören Biofilmbildung zu demonstrieren: Wildtyp-GKL und eine verbesserte GKL Doppelmutante (E101G / R230C). Beide Enzyme wurde gezeigt, Lactonase-Aktivität gegen demonstrieren 3-Hydroxy-decanoyl-L-homoserinlacton (3-OH-C 10 -HSL), der Haupt Quorum Molekül von A. verwendet baumannii S1 14. Für gültige Beurteilung der Biofilm Unterbrechun...

Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

Anti-virulenten Störung der Pathogene Biofilme mit Engineered Quorum lösch Lactonasen

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

anti-virulent-disruption-pathogenic-biofilms-using-engineered-quorum

Research

JoVE Journal

Immunology and Infection

11.5K Aufrufe.  Yong Loo Lin School of Medicine. Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

Serie: Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Mikrotiterschale Biofilmbildung Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Forschung

Immunologie und Infektion

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Engineering adhärenten Bakterien durch Schaffung eines einheitlichen Synthetic Curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Biotechnik

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

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

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

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

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

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

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Methodologien für das Studium<em&gt; B. subtilis</em&gt; Biofilme als Modell für kleine Molekül-Inhibitoren Biofilm Charakterisieren

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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 ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

Die Nutzung der Soft-Agar-Overlay-Technik zur Leinwand für bakteriell Hemmstoffe Produziert

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Quantifizierung der Auswirkungen von Antibiotika auf die In-vitro-Biofilmarchitektur mit der COMSTAT-Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

Erzeugung größerer bakterieller Biofilm-Biomasse mit PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

Bewertung der Wirksamkeit organischer Peroxysäuren zur Elimination von Milchbiofilmen unter Verwendung eines Ansatzes, der statische und dynamische Methoden kombiniert

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...

Biologie

<meta charset="utf8"></head><body>Biologische Bewertung von Thiophenesulfonamiden bei der Hemmung des Quorum Sensing

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Weiterentwicklung von Therapeutika zur Behandlung von Vibriose bei Menschen und Wasserorganismen

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Synthese und Assay von Vibrio Quorum Sensing Inhibitoren

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

Anti-virulenten Störung der Pathogene Biofilme mit Engineered Quorum lösch Lactonasen

Zusammenfassung

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