Name: methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors
Uploaded: 2016-10-09T14:00:00.000+00:00
Duration: 10 min 17 s
Description: Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

Electron microscope imaging was conducted at the Electron Microscopy Unit of the Weizmann Institute of Science, supported in part by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. This research was also supported by the ISF I-CORE grant 152/1, Mr. and Mrs. Dan Kane, Ms. Lois Rosen, by a Yeda-Sela research grant, by the Larson Charitable Foundation, by Ruth and Herman Albert Scholars Program for New Scientists, by the Ilse Katz Institute for Materials Sciences and Magnetic Resonance Research grant, by the Ministry of Health grant for alternative research methods, and by the France-Israel Cooperation - Maimonide-Israel Research Program. IKG is a recipient of the Rowland and Sylvia Career Development Chair.

The authors have nothing to disclose.

Subtilis Bacillus צורות חזקה מובנה מאוד biofilms הוא נוזל (pellicles) ועל בינוני מוצק (מושבות). לפיכך, היא משמשת אורגניזם מודל אידיאלי לאפיין את מצב הפעולה של מעכבי biofilm ספציפיים. על תקשורת מוצקה, תאי טופס מבנים רבים-תאי עם מאפייני בולטים שאינם ניכרים pellicles, כמו קמטים הקורנים מהמרכ?...

קהילות חיידקים רבות תאי לשחק תפקידים משמעותיים הסביבה טבעית אנתרופוגניים, והוא יכול להיות מועיל או מזיק ביותר. מושבות רבות התאיות אלה ידועים כמו biofilms, שבה התאים הבודדים המוטבעים מטריצת חומרים פולימריים תאיים בהפקה עצמית (EPS). EPS מאוד לדבוק התאים אל פני השטח הוא ליישב. הם לשמש כמגן נגד כוחות מכניים וכימיים וליצור קשר הדוק בין תאים שכנים, בהקלת התקשורת הסלולרית 1. Biofilm ניתן לצפות כקהילת בדיל, שבו משתמשי תאי פיקוח הדוק, מתוזמר תהליכים כדי לתאם את פעילותם בתוך הקהילה, כמו גם על פני מיני 2-5. המעבר ממצב planktonic ונטול חיים של צמיחה למצב ביופילם קשור לעתים קרובות עם תהליכים התפתחותיים. דוגמה טובה לכך היא subtilis Bacillus חיידק אדמה חיובי-גראם, ולפיכך undomeזן sticated משמש כאורגניזם מודל חזק כדי ללמוד את שלבי ההתפתחות מובילים להיווצרות ביופילם. חיידק זה, תאי ניע להתארגן מבנים רבים-תאיים בולטים מבצעות משימות מיוחדות 4. קבוצה אחת של תאים, המטריצה מפיק להפריש exopolysaccharides 6, חלבון עמילואיד טס 7,8, ואת חלבון הידרופוביות משטח BslA 9,10; שכולן להשתתף באסיפה של EPS 11-13. בהתחשב השפע של biofilms בנישות טבעיות אנתרופוגניים והניזק הקטלני המשוערת הם יכולים לגרום, יש צורך דוחק למצוא דרכים למנוע היווצרותם. מעכבי מולקולה קטנים יכולים לסייע לגילוי מסלולי רגולטוריות חדשים, אנזימים וחלבונים מבניים המעורבים ביצירת ביופילם, ובכך לקדם תובנות התהליכים המורכבים של הרכבת קהילה רבה-תאית. כב &#39; subtilis הוא מודל למד היטב עבור ביוהיווצרות סרט 14,15, זה יכול לשמש כדי להעריך את ההשפעות של מעכבי ביופילם שונים. מחקר זה מתמודד עם ארבע שיטות יסוד שהן המפתח להערכת התגובה של biofilms למעכבי מולקולה קטנה. ראשית, על מנת להבטיח כי מעכבים אלה יש מטרת biofilm הספציפי, הפרדת ההשפעה על צמיחת planktonic מהשפעת על היווצרות ביופילם היא קריטית. רוב סוכני אנטיבקטריאלי למקד תאים בשלב הצמיחה planktonic שלהם, אבל מולקולות המשפיעות על אורח החיים ביופילם הם נדירים. בנוסף, כמו מולקולות שאינו משפיעות צמיחת planktonic אינן רעילות, הם יכולים להפחית את לחץ הסלקטיבי העדפת מוטציות אנטיביוטיות עמידות 16. לדוגמה, כאשר biofilms מטופלים עם חומצות אמינו D-או מולקולות מקיר מפריעים תאים אחרים מסוימים, הם גם מוטרדים או מפורקים, אבל מעכבי אלה רק במתינות להשפיע 12,17 צמיחה planktonic. לעומת זאת, אנטיביוטיקה רבה לפגוע בצמיחת planktonic דרמטית, עם little או אין השפעה על היווצרות ביופילם 17. שנית, הקמת מסגרת ניסיונית עקבית ויציב כדי לחקור את ההשפעה של המולקולות הקטנות חיונית. צפינו כי טווח הריכוז הפעיל של מעכבי מולקולה קטנים היה רגיש לתנאים מראש התרבות להגדרות הניסוי נעשו שימוש כדי לחקור את ההשפעה של מעכבי מולקולה הקטנים אלה. דיווחים שונים, במיוחד הלומדים ב subtilis, חשף וריאציות בטווח הריכוז שבו D-אמינו חומצות לדכא את היווצרות pellicles - את biofilms חיידקי צף 12,17-19. התוצאות המוצגות כאן עולות כי הגורמים הבאים להסביר את ההבדלים בטווח הריכוז הפעיל: התנאים מראש התרבות (12,17 לוגריתמים לעומת שלב מאוחר-נייח 20 צמיחה), את מדיום הגידול בשימוש המצב טרום התרבות (עשירות, undefined [מרק לוריא, LB] לעומת מוגדר [מונוסודיום גלוטמט-glycerol, MSgg]), יחס החיסון ובמיוחד הסרת המדיום מראש התרבות לפני החיסון. הטמפרטורה של צמיחה קרומית סטטי הראה תפקיד חשוב פחות בטווח הפעילות של D-לאוצין מעכב מולקולה קטנה, חומצת אמינו D-נציג השתמשו במחקר זה. לבסוף, לאחר biofilms מטופלים עם מעכבי biofilm ספציפיים, שיטות חזקות ואינפורמטיבי נדרשות לאפיין את ההשפעות של מעכבים אלה על כושר ביופילם. הנה, שתי שיטות כדי לאפיין את ההשפעה עצמאית של מעכבי מולקולה קטנים מתוארות בפירוט: (1) ההשפעה על תאים בודדים בתוך מושבה ביופילם ואת התנגדותם סוכני מיקרוביאלית. תאי biofilms הם בדרך כלל יותר עמיד לאנטיביוטיקה בהשוואת חיידקים חינם-חי 21-23. בעוד תופעה זו תלויה בכמה גורמים, היכולת של EPS כדי לצמצם את חדירת אנטיביוטיקה לעתים קרובות נחשב כהסבר מושך 24 </sup&gt;. שיטה זו מעריכה את הישרדותם של תאי biofilm שנקבע מראש לאחר חשיפה לחומרים אנטי בקטריאליים. (2) ההשפעה על אדריכלות המושבה ביופילם, מן הגדולים אל בקנה מידה קטן. מושבות Biofilm מאופיינים המבנה התלת-ממדי שלהם ואת הנוכחות של EPS. ניצול במיקרוסקופ אלקטרונים סורק, שינויים במורפולוגיה התא, מבנה המושבה ביופילם והארכיטקטורה ושפע של EPS ניתן דמיינו מן גדול (מ&quot;מ) אל בקנה מידה קטן (מיקרומטר).

<table><tbody><tr><td>Luria Broth, Lennox</td><td>Difco</td><td>240230</td><td></td></tr><tr><td>Bacto Agar</td><td>Difco</td><td>214010</td><td></td></tr><tr><td>potassium phosphate monobasic&amp;nbsp;</td><td>Sigma, 136.09 g/mol</td><td>P0662-500G</td><td></td></tr><tr><td>potassium phosphate dibasic&amp;nbsp;</td><td>Fisher Scientific, 174.18 g/mol</td><td>BP363-1</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino)propanesulfonic acid</td><td>Fisher Scientific, 209.27 g/mol</td><td>BP308-500</td><td></td></tr><tr><td>magnesium chloride hexahydrate&amp;nbsp;</td><td>Merck, 203.30 g/mol&amp;nbsp;</td><td>1.05833.0250</td><td></td></tr><tr><td>calcium chloride anhydrous</td><td>J.T. Baker, 110.98 g/mol</td><td>1311-01</td><td></td></tr><tr><td>manganese(II) chloride tetrahydrate</td><td>Sigma, 197.91 g/mol</td><td>31422-250G-R</td><td></td></tr><tr><td>iron(III) chloride hexahydrate&amp;nbsp;</td><td>Sigma, 270.30 g/mo)</td><td>F2877-500G</td><td></td></tr><tr><td>zinc chloride anhydrous&amp;nbsp;</td><td>Acros Organics, 136.29 g/mol</td><td>424592500</td><td></td></tr><tr><td>thiamine hydrochloride</td><td>Sigma, 337.27 g/mol</td><td>T1270-100G</td><td></td></tr><tr><td>L-tryptophan</td><td>Fisher Scientific, 204.1 g/mol</td><td>BP395-100</td><td></td></tr><tr><td>L-phenylalanine</td><td>Sigma, 165.19 g/mol</td><td>P5482-100G</td><td></td></tr><tr><td>L-threonine</td><td>Sigma, 119.12 g/mol</td><td>T8625-100G</td><td></td></tr><tr><td>glycerol anhydrous</td><td>Bio-Lab Itd</td><td>712022300</td><td></td></tr><tr><td>L-glutamic acid monosodium salts hydrate&amp;nbsp;</td><td>Sigma, 169.11 g/mol</td><td>G1626-1KG</td><td></td></tr><tr><td>D-leucine</td><td>Sigma, 169.11 g/mol</td><td>855448-10G</td><td></td></tr><tr><td>ethanol anhydrous</td><td>Gadot</td><td>830000054</td><td></td></tr><tr><td>razor blade</td><td>Eddison</td><td>NA</td><td></td></tr><tr><td>circular cellulose filter papers</td><td>Whatman, 90 mm</td><td>1001-090</td><td></td></tr><tr><td>glutaraldehyde</td><td>EMS (Electron Micoscopy Science), 25% in water</td><td>16220</td><td></td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>EMS, 16% in water</td><td>15710</td><td></td></tr><tr><td>sodium cacodylate</td><td>Merck, 214.05 g/mol&amp;nbsp;</td><td>8.2067</td><td></td></tr><tr><td>calcium chloride 2-hydrate</td><td>Merck, 147.02 g/mol&amp;nbsp;</td><td>1172113</td><td></td></tr><tr><td>stub-aluminium mount</td><td>EMS, sloted head</td><td>75230</td><td></td></tr><tr><td>carbon adhesive tape</td><td>EMS</td><td>77825-12</td><td></td></tr><tr><td>Shaker 37 &amp;deg;C</td><td>New Brunswick Scientific Innowa42</td><td>NA</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf table top centrifuge 5424</td><td>NA</td><td></td></tr><tr><td>Digital Sonifier, Model 250, used with Double Step Microtip</td><td>Branson</td><td>NA</td><td></td></tr><tr><td>Incubator 30 &amp;deg;C</td><td>Binder</td><td>NA</td><td></td></tr><tr><td>Incubator 23 &amp;deg;C</td><td>Binder</td><td>NA</td><td></td></tr><tr><td>Filter System, 500 ml, polystyrene</td><td>Cornig Incorporated</td><td>NA</td><td></td></tr><tr><td>Rotary Shaker - Orbitron Rotatory II</td><td>Boekel</td><td>NA</td><td></td></tr><tr><td>S150 Sputter Coater&amp;nbsp;</td><td>Edwards</td><td>NA</td><td></td></tr><tr><td>CPD 030 Critical Point Dryer</td><td>BAL-TEC</td><td>NA</td><td></td></tr><tr><td>Environmental Scanning Electron Microscope</td><td>XL30 ESEM FEG Philips (FEI)</td><td>NA</td><td></td></tr></tbody></table>

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.

את assay הקרומית היא אחת שיטות כדי לחקור את תהליכי פיקוח הדוק והדינמיים של B. subtilis רב-תאיות. מלבד זאת, assay הקרומית מתאימה לבדוק מגוון של תנאים או טרום פתיחה או ריכוזי מולקולה קטנים בצלחת multidish תא-תרבות אחת בניסוי אחד. עם זאת, ב &#39; ההיווצרות קרומית subtilis רגישה ?...

Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

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 ...

methodologies-for-studying-b-subtilis-biofilms-as-model-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

15.1K Views.  Weizmann Institute of Science. This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

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

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 ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

מתא בודד ניתוח בצילוס subtilis Biofilms באמצעות מיקרוסקופ פלואורסצנטי ו cytometry זרימה

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.&#160; In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

הקמת הריכוז החיידקי המינימלי של סוכן מיקרוביאלית לתאים פלנקטוניים (MBC-P) ותאי ביופילם (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

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.

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 ...

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 ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

שיטה חדשה לניתוח איכותי רב היקף של Biofilms חיידקית על פילמנטיות פטרייתיים המושבות Confocal שימוש במיקרוסקופ אלקטרונים

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

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 ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

גישה שיטתית לזיהוי מולקולות אנטי-מיקרוביאליות ואנטיביופילם חדשניות מתמציות ושברים של צמחים למניעת עששת שיניים

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Biology

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 ...

הערכת היווצרות ביופילם של Acinetobacter ואפיון ביופילמים

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

Author Spotlight: A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

אסטרטגיות כימות, הערכת כדאיות והדמיה עבור ביופילמים של Acinetobacter

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

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