אנו מכירים את תמיכתם של בריטניה המכון הלאומי לחקר הבריאות, ד&quot;ר Hadwen אמון למחקר להומניות, מובילה הצדקה רפואית בבריטניה למימון מחקר בלעדי שאינם מהחי שיטות מחקר כדי להחליף ניסויים בבעלי חיים, ואת האמון Wellcome (גרנט 089215). כמו כן, אנו מכירים נוברטיס פרמצבטיקה בריטניה בע&quot;מ (בלתי מוגבל החינוך מענק).

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אין ניגוד עניינים הצהיר.

במחקר זה השתמשנו במודל חוץ גופית הרומן מבוסס על ASM לשכפל פ biofilm aeruginosa התנאים בתוך 4 ריאות CF. מודל שונה בהצלחה בדיקות בקנה מידה קטן תפוקה גבוהה, של סוכני מיקרוביאלית. השלבים הקריטיים של assay זה הם: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנה עקבית של התקשורת ASM ועקרות שמירה. אנחנו הקדישו שעות רבות כדי לייעל את האופן שבו כל רכיב נוסף כדי להשיג תוצאות לשחזור בכל פעם. סינון של ASM איטי אבל עדיף מעוקר, אשר עלול לגרום נזק מרכיב mucin. אנחנו לא ממליצים על תוספת של אנטיביוטיקה, כפי שהוצע על ידי אחרים 11, כיוון שהדבר עלול להטיל לחצים בררניים ניכרים, מוטציות מניעות, לגרום תמוגה prophage 16 ו לשנות באופן משמעותי את הביטוי של גנים חיידקים רבים. </li><li style=";text-align:right;direction:rtl"> Assay צריך להיות מותאם על פי נפח Oו ASM בשימוש. מהירויות רועדות מוגדלים עבור אמצעי אחסון קטנות יותר biofilm מחזור חיים קצר יותר הוא ציין. </li></ol> היישום הברור של דגם בקנה מידה קטן biofilm ASM היא קביעה מציאותית יותר של רגישויות biofilm מיקרוביאלית (BSMIC 90). נישות אנאירוביים ו microaerophilic נמצאים ריאות CF ויש ראיות לכך חמצן מוגבל עמוק בתוך biofilms בוגרת 2, 17. כאן אנו מראים כי 10/14 פ קליני מבודד aeruginosa מ CF המטופלת ספוטה התערוכה רב (4 - ≥ 128 לקפל) ירידה רגישות tobramycin בתנאים microaerophilic ב ASM. תוצאות המחקר הזה מצביעים על כך אנטיביוטיקה, כגון tobramycin, עשוי להיות פחות יעיל נגד פ aeruginosa דלקות ריאות CF מכפי שצוין על ידי שיטות בדיקה המקובלות הרגישות. תוצאות אלו משקפות מחקרים קודמים על הרגישות מיקרוביאלית של biofilms 10. קטןמבחני ASM בקנה מידה ובכך לספק פלטפורמה פשוטה תפוקה גבוהה ליצירת משמעות הנתונים הרגישות אנטיביוטיות כדי ליידע טוב יותר החלטות טיפוליות. Assay מוגבל באותו אופן כמו בדיקה רגילה רגישות אנטיביוטיקה כי מושבות בודדות נקטפים להקרנה כי לא יכול להיות מייצג של האוכלוסייה כולה. עם זאת, אנו מאמינים כי הגישה (א) שימוש לא לפני השטח צמיחה biofilm המצורף (ב) החל על תנאים microaerophilic, מהווה חלופה ברורה שיפור פוטנציאל השיטות הקיימות. אנו מסיקים כי assay הזה הוא מודל מתאים ללמוד פ &#39; biofilm aeruginosa אוכלוסיות. בדיקות נוספות במסגרות קליניות היה לברר אם רגישויות אנטיביוטיות על בסיס biofilm, גדל פ aeruginosa יכול להוביל לבחירות אנטיביוטיות שונות עם תוצאות מיקרוביולוגיות וקליניים שיפור פוטנציאלי. חקירות דומות תוך שימוש במודלים הקלאסיים biofilm הראו כי ערכי BSMIC להובילהמלצות שונות לטיפול אנטיביוטי 5,17. בנוסף בדיקת היעילות של אנטי זיהומיות סוכנים, מערכת ASM מייצג אלטרנטיבה זולה, פשוטה לשחזור כדי בבעלי חיים ללימודים כגון אלה להבנת פיזור פ aeruginosa אוכלוסיות. ראינו הטרוגניות רב האוכלוסיות הטבעיות של פ &#39; aeruginosa התאושש CF המטופלת ספוטה 18, 19. גיוון פנוטיפי ו genotypic דומה ניתן לראות במהלך הצמיחה ASM 4 (נתונים שלא פורסמו שלנו), מה שהופך אותו אטרקטיבי במודל חוץ גופית התנאים ריאות CF. הפשטות היחסית של המודל ASM מקל לתכנן לטווח ארוך ניסויים התאמה מכוונת, למשל, על ניטור ההשפעות של אנטיביוטיקה או לחצים אחרים על פ &#39; האוכלוסייה aeruginosa סטייה. בנוסף, חיידקים פתוגנים נוספים ניתן לגדלASM. לדוגמה, Fouhy et al. 2007 השתמשו ASM ללמוד היווצרות biofilm ש &#39; maltophillia 20.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> ה-DNA של הזרע דגים </td><td> Sigma-Aldrich </td><td> 74782 </td></tr><tr><td> Mucin מ חזירי הקיבה, סוג ב &#39; </td><td> Sigma-Aldrich </td><td> M2378 </td></tr><tr><td> L-אלאנין </td><td> Acros Organics </td><td> 102830250 </td></tr><tr><td> L-ארגינין </td><td> Sigma-Aldrich </td><td> A5006 </td></tr><tr><td> L (+)-Asparagine מונוהידראט </td><td> Acros Organics </td><td> 175271000 </td></tr><tr><td> L (+), חומצה אספרטית </td><td> Acros Organics </td><td> 105041000 </td></tr><tr><td> L-ציסטאין </td><td> Sigma-Adrich </td><td> 168149 </td></tR&gt; <tr><td> L (+), חומצה גלוטמית </td><td> Acros Organics </td><td> 156211000 </td></tr><tr><td> L-גלוטמין </td><td> Sigma-Aldrich </td><td> G3126 </td></tr><tr><td> גליצין </td><td> Acros Organics </td><td> 220911000 </td></tr><tr><td> L-היסטידין </td><td> Sigma-Adrich </td><td> H8000 </td></tr><tr><td> L-isoleucine </td><td> Sigma-Aldrich </td><td> I2752 </td></tr><tr><td> L-לאוצין </td><td> Sigma-Aldrich </td><td> L8000 </td></tr><tr><td> L (+), ליזין monohydrochloride </td><td> Acros Organics </td><td> 125222500 </td></tr><tr><td> L-מתיונין </td><td> Sigma-Aldrich </td><td> M9625 </td></tr><tr><td> L-פנילאלנין </td><td> Acros Organics </td><td> 130310250 </td></tr><tr><td> L-פרולין </td><td> Sigma-Aldrich </td><td> P0380 </td></tr><tr><td> L-סרין </td><td> Acros Organics </td><td> 132660250 </td></tr><tr><td> L-תראונין </td><td> Acros Organics </td><td> 138930250 </td></tr><tr><td> L (-)-טריפטופן </td><td> Acros Organics </td><td> 140590250 </td></tr><tr><td> L-טירוזין </td><td> Acros Organics </td><td> 140641000 </td></tr><tr><td> L-valine </td><td> Sigma-Aldrich </td><td> V0500 </td></tr><tr><td> Diethylenetriaminepentaacetic חומצה </td><td> Sigma-Aldrich </td><td> 32318 </td></tr><tr><td> NaCl </td><td> פישר סיינטיפיק </td><td> S/3160/60 </td></tr><tr><td> KCl </td><td> BDH </td><td> BDH0258 </td></tr><tr><td&gt; KOH </td><td> BDH </td><td> BDH0262 </td></tr><tr><td> חלמון ביצה תחליב </td><td> Sigma-Aldrich </td><td> 17148 </td></tr><tr><td> ME 2 ואקום משאבת דיאפרגמה </td><td> Vacuubrand </td><td> 696126 </td></tr><tr><td> Steritop מסננים (גודל הנקבוביות: 0.22 מיקרומטר, גודל צוואר: 45 מ&quot;מ) </td><td> Millipore </td><td> SCGPT10RE </td></tr><tr><td> לוריא, Bertani בינוני </td><td> סיגמא </td><td> L2897 </td></tr><tr><td> 96-microtitre גם צלחות </td><td> Sarstedt </td><td> 82.1581 </td></tr><tr><td> 24 גם רקמת התרבות שטופלו צלחות </td><td> Iwaki </td><td> 3820-024 </td></tr><tr><td> הדור CampyGen גז חבילות </td><td> Oxoid </td><td> CN0025 </td></tr><tr><td> Microaerophilic קאמרית </td><td> Oxoid </td><td> HP0011 </td></tr><tr><td> Tobramycin סולפט </td><td> Sigma-Aldrich </td><td> T1783 </td></tr><tr><td> Cellulase, החל אספרגילוס ניז&#39;ר </td><td> Sigma-Aldrich </td><td> 22178 </td></tr><tr><td> Resazurin </td><td> Sigma-Aldrich </td><td> 199303 </td></tr><tr><td> Citrate.H 2 0 </td><td> BDH </td><td> BDH0288 </td></tr><tr><td> Fluostar אומגה microplate הקורא </td><td> BMG-Labtech </td><td> SPECTROstar אומגה </td></tr></tbody></table>

use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

גוברת הדאגה לגבי הרלוונטיות של במבחנה הבדיקות הרגישות מיקרוביאלית כשמדובר מבודד של פ &#39; aeruginosa מ סיסטיק פיברוזיס (CF) חולים. שיטות קיימות להסתמך על יחיד או מבודד כמה גדל aerobically ו planktonically. מראש לחתוך-offs רגילים להגדיר האם החיידקים רגישים או עמידים בפני אנטיביוטיקה נתון 1. עם זאת, במהלך דלקות ריאה כרוניות ב CF, פ אוכלוסיות aeruginosa קיימים biofilms ויש ראיות לכך הסביבה היא microaerophilic בעיקר 2. ההבדל שטרק בתנאים בין חיידקים בריאה לבין אלה במהלך בדיקות אבחון בספק את הרלוונטיות אמינות ואפילו של בדיקות אלה 3. בינוני כיח מלאכותית (ASM) היא בינונית התרבות המכיל את מרכיבי כיח החולה CF, כולל חומצות אמינו, mucin ו-DNA חופשי. פ aeruginosa &lt;/ Em&gt; גידול בצמיחה מחקה ASM במהלך זיהומים CF, עם הקמתה של עצמי צבירה מבנים biofilm ו סטייה האוכלוסייה 4,5,6. מטרת מחקר זה הייתה לפתח assay microtitre-הצלחת ללמוד הרגישות מיקרוביאלית של פ &#39; aeruginosa מבוסס על הצמיחה ASM, שהוא החלים על שני התנאים microaerophilic ו אירובית. Assay ASM פותחה בפורמט צלחת microtitre. פ biofilms aeruginosa הורשו לפתח במשך 3 ימים לפני הדגירה עם סוכני מיקרוביאלית בריכוזים שונים עבור 24 שעות. לאחר הפרעה biofilm, כדאיות התא נמדדה על ידי מכתימה עם resazurin. Assay זה נעשה שימוש כדי לקבוע את התא המינימלי נייחים ריכוז מעכבות (SMIC) של tobramycin עבור 15 פ &#39;שונה מבודד aeruginosa בתנאים אירוביים ו microaerophilic וערכים SMIC הושוו לאלו שהושגו עם צמיחה מרק סטנדרטי. בעוד שישראיות עבור ערכים מיקרופון מוגבר מבודד שגודלו ASM בהשוואה לעמיתיהם planktonic שלהם, ההבדלים הגדולים ביותר נמצאו עם חיידקים שנבדקו בתנאים microaerophilic, אשר הראו עמידות מוגברת יותר של עד פי 128&gt;, לקראת tobramycin במערכת ASM כאשר לעומת מבחני שבוצעו בתנאים אירוביים. היעדר קשר בין בדיקות שוטפות שיטות הרגישות ואת התוצאה הקלינית הטיל ספק בתוקפו של השיטות הקיימות 3. כמה במודלים חוץ גופית שימשו בעבר כדי ללמוד פ &#39; aeruginosa biofilms 7, 8. עם זאת, שיטות אלו מבוססות על biofilms משטח המצורפת, בעוד biofilms ASM דומות לאלו שנצפו ריאות CF 9. בנוסף, ריכוז חמצן מופחת ריר הוכח לשנות את אופן הפעולה של פ &#39; aeruginosa 2 ולהשפיע על רגישות לאנטיביוטיקה 10. לכן usinגרם ASM בתנאים microaerophilic יכול לספק סביבה מציאותית יותר שבו ללמוד הרגישות מיקרוביאלית.

Watch this Scientific Journal Video about Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung at JoVE.com

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

בדיקות אבחון רגישות מיקרוביאלית הנוכחי מסתמך על צמיחה של planktonic מבודד ב מזין, עשיר תנאים אירוביים. כאן, אנו מעסיקים בינוני חלופה כיח מלאכותית ללמוד הרגישות מיקרוביאלית של Pseudomonas aeruginosa biofilms תחת שני תנאים אירוביים ו microaerophilic נציג נוסף של הריאה סיסטיק פיברוזיס.

שימוש בינוני כיח מלאכותית לבדיקת יעילות אנטיביוטי נגד<em&gt; Pseudomonas aeruginosa</em&gt; בתנאי רלוונטיים יותר ריאות סיסטיק פיברוזיס

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

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Immunology and Infection

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

32.5K Views.  University of Liverpool. בדיקות אבחון רגישות מיקרוביאלית הנוכחי מסתמך על צמיחה של planktonic מבודד ב מזין, עשיר תנאים אירוביים. כאן, אנו מעסיקים בינוני חלופה כיח מלאכותית ללמוד הרגישות מיקרוביאלית של Pseudomonas aeruginosa biofilms תחת שני תנאים אירוביים ו microaerophilic נציג נוסף של הריאה סיסטיק פיברוזיס.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa ו biofilm Saccharomyces cerevisiae בתוך תאים Flow

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

RNA בידוד Pseudomonas aeruginosa מיישבים את מערכת העיכול Murine

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

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

כרוני לטווח ארוך<em&gt; Pseudomonas aeruginosa</em&gt; זיהום דרכי הנשימה בעכברים

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

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

Pseudomonas aeruginosa Induced Lung Injury Model

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

Pseudomonas aeruginosa מושרה ריאות פגיעה דגם

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

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

מודל דלקת ריאות חזק במכרסמי immunocompetent להערכת יעילות אנטיבקטריאלי נגד<i&gt; S. pneumoniae</i&gt;,<i&gt; H. אינפלואנזה</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; או<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

דגם IL-8 עכבר transgenized חולף עבור<em&gt; בויבו</emמעקב ארוך טווח אחר תגובות דלקתיות

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

שיטה לבדיקת יעילות שטיפת הידיים להסרת הפתוגנים המזהמים

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

שיטות זיהוי ציטוטוקסיות Amyloids הבאים זיהום של אנדותל תאי הריאות על ידי Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

יישום טיפול Phage כדי לנטרל זיהום aeruginosa פסאודומונס בעוברי דגי זברה סיסטיק פיברוזיס

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.