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. קריסטל ויולט Quantitation של ההיווצרות biofilm בא baumannii S1 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול תרבות 5 מיליליטר של א &#39; baumannii S1 במרק Lysogeny (LB) (tryptone 10 גרם / L, 5 גר &#39;/ L תמצית שמרים) על 30 מעלות צלזיוס בחממה רועדת (220 סל&quot;ד) במשך 16 שעות. </li><li style=";text-align:right;direction:rtl"> התאם את התרבות של א &#39; baumannii S1 לOD רצוי 600 של 0.8. שימוש בצלחת 96-היטב, לחסן תרבות חיידקים (1: 100 דילול) לLB טרי המכילה 10 μl של אנזים GKL מטוהר (40 מ&quot;ג / מיליליטר); הנפח הסופי של התרבות החדשה הוא 100 μl. </li><li style=";text-align:right;direction:rtl"> הכן תרבות שליטה, כמו גם; זה לא יכיל כל אנזים. חזור על תנאים דומים ללהניב את המספר הרצוי של משכפל. </li><li style=";text-align:right;direction:rtl"> מכסה את הצלחת עם מכסה ומניח אותו לתוך מיכל פלסטיק 10 ליטר אטום. דגירה את הצלחת על 30 מעלות צלזיוס במשך 3 שעות לפני בעדינות הסרת התקשורת. </li><li style=";text-align:right;direction:rtl"> להוסיף בינוני LB הטרי עוד 100 μl להיטב דגירה הצלחת במשך 21 שעות על 30 מעלות צלזיוס. </li&gt; <li style=";text-align:right;direction:rtl"> לאחר התקופה השנייה של דגירה, בעדינות להסיר את כל אמצעי התקשורת. שטוף את תא חיידקי פלנקטון עם מים סטריליים 200 μl. ודא שיש הפרעה מינימאלית בלבד לתאים במהלך כביסה. </li><li style=";text-align:right;direction:rtl"> להוסיף 1% פתרון סגול גביש 100 μl לכל היטב דגירה במשך 15 דקות ב RT. הסר את הפתרון סגול גביש על ידי שטיפה היטב עם מים סטריליים 200 μl. חזור על לשטוף לעוד פעמיים. </li><li style=";text-align:right;direction:rtl"> להוסיף 33% חומצה אצטית 100 μl לכל היטב דגירה למשך 15 דקות עם רעד עדין; זה יהיה לפזר את הצבע. </li><li style=";text-align:right;direction:rtl"> לכמת את כמות biofilm נוצרה על ידי מדידת הספיגה של סגול קריסטל ב 600 ננומטר. כמות סגולה גביש היא פרופורציונלית לסכום של biofilm נוצר. </li></ol> 2. Confocal סריקת לייזר מיקרוסקופית של א &#39; baumannii S1 Biofilm <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול תרבות 5 מיליליטר של א &#39; baumannii S1 בLB על 30 מעלות צלזיוס בחממה רועדת (220 סל&quot;ד) במשך 16 שעות. </li><li style=";text-align:right;direction:rtl"> מוֹדָעָהרק התרבות של א &#39; baumannii S1 לOD רצוי 600 של 0.8. באמצעות μ-צלחת עם תחתית זכוכית 35 מ&quot;מ, לחסן תרבות חיידקים (1: 100 דילול) לLB טרי המכילה 30 μl של אנזים GKL מטוהר (40 מ&quot;ג / מיליליטר); הנפח הסופי של התרבות החדשה הוא 1 מיליליטר. </li><li style=";text-align:right;direction:rtl"> מכסה את μ-הצלחת עם מכסה ומניח אותו במכל פלסטיק אטום 10 ליטר. דגירה μ-הצלחת על 30 מעלות צלזיוס במשך 3 שעות לפני בעדינות הסרת התקשורת. הוסף 30 μl של אנזים GKL מטוהר ובינוני טריים, הבאתו להיקף כולל של 1 מיליליטר. דגירה עבור שעות 21 נוספות על 30 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> חזור על שלב 2.3 ודגירת μ-התבשיל לשעה עוד 24 על 30 מעלות צלזיוס. לאחר מכן, להסיר את המדיה בעדינות. </li><li style=";text-align:right;direction:rtl"> הוסף 500 μl של 5 מיקרוגרם / מיליליטר 488 מצומדות- agglutinin אלכס Fluo נבט חיטה (WGA) מומס בתמיסת מלח המאוזן של האנק (HBSS) לμ-הצלחת ולדגור על 37 מעלות צלזיוס למשך 30 דקות; זה יהיה כתם biofilm נוצר. הסר את הפתרון מכתים ולשטוף tהוא μ-צלחת עם 2 מיליליטר של HBSS. חזור על השלב לשטוף עוד פעם אחת. </li><li style=";text-align:right;direction:rtl"> הוסף 500 μl של פורמלדהיד 3.7% מומסים בHBSS ולדגור על 37 מעלות צלזיוס למשך 30 דקות; זה יהיה לתקן את biofilm על μ-הצלחת. לשטוף את μ-צלחת פעם אחת עם 2 מיליליטר של HBSS ולאחר מכן להסיר את הפתרון לחלוטין. קבוע עם biofilm μ-הצלחת ניתן לאחסן בחושך על 4 מעלות צלזיוס לפני ההדמיה CLSM. </li><li style=";text-align:right;direction:rtl"> הדמיה CLSM וניתוח, להשתמש 63 הגדלת פעמים כדי ליצור 97 ערימות לכל תמונה עם מרווח של 0.21 מיקרומטר לערימה. </li></ol>

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The authors declare that they have no competing financial interests.

בשני הסטים של ניסוי, א baumannii S1 היה בתרבית בתקשורת LB ללא NaCl כריכוז מלח גבוה יכול להפחית את כמות biofilm נוצרה על ידי החיידקים 15. נוכחותו של חפץ כזה יכול לזלזל בסכום של biofilm נוצר, כמו גם את ההשפעות של אנזימי מרווה-מניין פני תנאי טיפול שונים. השימוש באנזים catalytically פעי...

אפשרויות טיפול במחלות זיהומיות כבר מסובכות על ידי הגידול המהיר בחיידקים עמיד multidrug כי הם חסינים מפני מגוון רחב של תרופות אנטיביוטיות 1. עם תחלואה גבוהה ושיעורי תמותה מזיהומים בתיווך חיידקים עמידים, יש צורך להסלים תהליכי פיתוח תרופות ו / או לחקור חלופות אנטי בקטריאלי טובים יותר כדי לשפר את האפשרויות טיפוליות. בזמן האחרון, הגישה האנטי-האלימות היא צוברת ריבית נתון את הפוטנציאל שלה במניעת אלימות באמצעות שיטות לא-bactericidal, ומכאן מקלים הסיכונים של מנגנוני התנגדות 2. מניין החישה היא &quot;מתג ראשי&quot; בארסיות ושיבוש תופעת איתות זה חיידקים הוא שיטה אנטי-אלימות נגד מבטיחה פתוגנזה 3. תחילתה של אלימות דורשת ההצטברות של מולקולות מניין בסביבה תאית לאחר שהגיעה לצפיפות אוכלוסיית חיידקים קריטית. כקווהמולקולות רום מפוזרות בחזרה לתוך המטריצה ​​תאית, מחייב עם קולטנים המקור שלהם מובילה להפעלה של גורמים ארסי כמו גם גנים הקשורים לעמידות לאנטיביוטיקה והיווצרות biofilm 4. בשיבוש כללי, מניין חישה כרוך עיכוב מולקולת מניין ואינטראקציה קולט מבלי להשפיע על מסלולי מטבוליים עיקריים. מכאן, שאין לה שום השלכה ישירה על צמיחה סלולרית. מאז כושר לא נפגעים, יש לחץ בחירה מינימאלי לחיידקים להתפתח ולהשיג התנגדות נגד טיפולים כגון 5. בנוסף, הפרעה מניין חישה יכולה להפריע למנגנוני הגנת חיידקים טבועים, כמו במקרה של היווצרות biofilm, המספקת הגנה מפני חומרים אנטי-בקטריאלי ויארח את תגובה חיסונית. הערכה היא כי 99% מחיידקים על פני כדור הארץ קיימים במטריצות כמו biofilm-מורכבות, המקנות יתרונות הישרדות חיוניות למיקרואורגניזמים שחיים בתוך המבני ESE 6. יותר מכך, היווצרות של תחומים נייחים אלה היא הסיבה לזיהומים נרכשים בבית חולים מתמשכים והכרוניים ביותר 7. Acinetobacter baumannii היא אחד מגורמי המחלה האנושיים הגדולים שמזוהה עם זיהומים נרכשים בבית חולים העולמיים והארסיות שלה מיוחסת במידה רבה למניין חישה היווצרות biofilm תיווך 8. אנזימים-מרווה מניין שימשו בהצלחה בשיבוש העברת אותות בתיווך מניין על ידי מיקוד קבוצה של תרכובות המכונית lactones -acyl N homoserine (AHLs) המיוצרים על ידי חיידקים גראם-שלילי 9. גם מספר מחקרים הרחיבו את השימוש באנזימים אלה כדי לחסום פתוגנזה של חיידקים באמצעות הפחתת מספרי ביטוי גורם ארסיות ותא בbiofilms 10,11. למרבה הצער, יש עדיין חוסר ההפגנה מוחשית של השימוש היעיל של אנזימי מרווה-מניין נגד היווצרות biofilm ידי חיידקים פתוגנים.מחדש נעשו ניסיונות להשתמש מעכבי מניין (אנלוגים AHL), במקום אנזימי מרווה-מניין, לשבש א היווצרות biofilm baumannii 12. למרות ששיטה זו של שימוש במעכבי מולקולות קטנות היא גישה חוקית, שמירה על הזמינות הביולוגית שלה בשימושים translational יכולה להיות אתגר. להיפך, השימוש באנזימי מרווה-מניין קטליטי יכול לעקוף את הבעיה הזמינות הביולוגית כאנזימים הם נוחים יותר לחוסר תנועה על משטחים של מכשירים ביו-רפואיים להשפעות טיפוליות. כאן אנו מתארים הערכה של ההשפעות של lactonases מרווה-מניין מהונדס מkaustophilus Geobacillus (GKL) 13 על היווצרות biofilm חיידקים, באמצעות מכתים סגול גביש ומיקרוסקופיה confocal סריקת לייזר (CLSM). מחקר זה הוא ההפגנה המוצלחת הראשונה של שיבוש biofilm בא רלוונטי קליני זן baumannii S1 באמצעות אנזימי מרווה-מניין. השיטות שתוארובמחקר זה הם שימושיים להערכת יעילותם של אנזימי מרווה-מניין אחרים במאמצי פיתוח טיפוליים שלאחר מכן נגד חיידקים גראם שלילית פתוגניים.

<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.

בניסוי quantitation הסגול גביש, שני אנזימי מרווה-מניין שמשו כדי להדגים היתכנות בשיבוש היווצרות biofilm: פרא-הסוג GKL ומוטציה משופר GKL כפולה (E101G / R230C). שני האנזימים הוכחו להפגין פעילות lactonase נגד 3-הידרוקסי-decanoyl-L-homoserine לקטון (3-OH-C 10 -HSL), מולקולת המניין המרכזית ...

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.

שיבוש אנטי-ארסי של Biofilms פתוגניים באמצעות Lactonases תכנון הנדסי Quorum-מרווה

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

11.2K Views.  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.

Video: 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.

Biofilm microtiter תבשיל גיבוש 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 ...

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.

הנדסת חיידקי חסיד ידי יצירת Curli אופרון סינטטי יחיד

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

Bioengineering

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.

מיקוד Associated Biofilm<em&gt; Staphylococcus aureus</em&gt; Assay רגישות בסמים בהתבסס Resazurin שימוש

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.

מתודולוגיות לימוד<em&gt; B. subtilis</em&gt; Biofilms כמודל לאפיון מולקולה קטנה Biofilm מעכבי

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.

פלטפורמה של מבחנים אנטי biofilm המתאים לחקירת ספריות תרכובת טבעיות

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.

השימוש בטכניקת הכיסוי הרך-אגר למסך תרכובות מעכבות הופקו bacterially

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.

כימות ההשפעות של אנטי-מיקרוביאלים על ארכיטקטורת ביופילם במבחנה באמצעות תוכנת COMSTAT

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.

ייצור ביופילם ביומסה חיידקית גדולה יותר באמצעות מכשירי מיקרופלאט עמוק באר עמוקה צלחת PCR

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.

הערכת יעילותן של חומצות פרוקסי אורגניות למיגור ביופילמים חלביים בגישה המשלבת שיטות סטטיות ודינמיות

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 ...

Biology

<meta charset="utf8"></head><body>הערכה ביולוגית של Thiophenesulfonamides בעיכוב חישת מניין

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>מחבר זרקור: קידום טיפולים לטיפול בויבריוזיס בבני אדם ובאורגניזמים ימיים

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.

סינתזה ובדיקה של מעכבי חישת מניין Vibrio

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

שיבוש אנטי-ארסי של Biofilms פתוגניים באמצעות Lactonases תכנון הנדסי Quorum-מרווה

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