The authors would like to acknowledge the University of Lille and the Pasteur Institute of Lille, especially Thierry Chassat and Jean-Pierre Decavel, responsible for animal housing breeding safety and husbandry. This work was supported by the "Soci&#233;t&#233; de Pathologies Infectieuses de Langue Fran&#231;aise" (SPILF).

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The authors have nothing to disclose.

מודלים של בעלי חיים, במיוחד יונקים, שימושיים להבהיר מנגנונים מורכבים של אינטראקציה מארח הפתוגן בתחומי חסינות. כמובן, את הצורך במידע זמין רק ממודלים של בעלי החיים חייבים להיות חיוני; אחרת, שימוש בבעלי החיים חייב להיות מוחלף על ידי מודלים במבחנה. מודל חיה זה ממחיש ?...

<html> מודלים של בעלי חיים, בעיקר עכברים, נעשו שימוש נרחב כדי לחקור את תגובה חיסונית נגד פתוגנים. למרות חסינות מולדת ונרכשת שונה בין מכרסמים ובני האדם 7, הקלות בגידול והפיתוח של knockouts לגנים רבים, להפוך עכברי מודל מצוין ללמוד תגובות חיסוני 8. התגובה החיסונית היא מורכבת ותוצאות מהאינטראקציה של הפתוגן, (תאי האפיתל, תאי האנדותל) צמחיית תושב חיידקים וכמה (לימפוציטים, נויטרופילים, מקרופאגים) חיסוניים ואינם חסינים סוגים סלולריים 2. במבחנה אינה מאפשרות התבוננות אינטראקציות המורכבות אלה ומתמקדים בעיקר באינטראקציות התא-פתוגן ייחודיים. בעוד מודלים של בעלי חיים יש להשתמש בזהירות ובמוגבלת לשאלות מאוד ספציפיות ורלוונטיות, מודלים עכבר מספקים תובנה טובה לתגובה החיסונית יונק in vivo ויכולים להתייחס לחלקים של שאלות קליניות חשובות 7. &quot;Jove_content&quot;&gt; בדרכי הנשימה, קהילת החיידקים היא מורכבת שיוך מספר גדול של מיקרואורגניזמים שונים 6. בעוד מה שמהווה Microbiome &quot;נורמלי&quot; בדרכי נשימה עדיין לא נקבע, קהילות תושב לעתים קרובות polymicrobial, ומקורן ממקורות אקולוגיים מגוונים. חולים עם מחלת ריאות כרונית מוגלתית (סיסטיק פיברוזיס, bronchectasis) או חולי מאוורר מכאני התערוכה צמחייה מסוימת בשל התישבות של דרכי הנשימה על ידי מיקרואורגניזמים לסביבה שנרכשו-9. אלביקנס Pseudomonas aeruginosa וקנדידה שניהם הבעיה פתוגנים 5, לעתים קרובות מבודדת יחד מדגימות tracheobronchial , ואחראי לזיהום אופורטוניסטי חמור בחולים אלה, במיוחד ביחידה לטיפול הנמרצת (ICU) 4. בידוד של מיקרואורגניזמים אלה בדלקת ריאות חריפות בתוצאות טיפול נמרץ בטיפול אנטי-מיקרוביאלי נגד פ aeruginosa בשמרי ut בדרך כלל לא נחשבים פתוגניים באתר זה 5. במבחנה אינטראקציות בין פ aeruginosa וג אלביקנס כבר דווח בהרחבה, והראה כי מיקרואורגניזמים אלה יכולים להשפיע על הצמיחה וההישרדות של אחד את השני, אבל מחקרים לא הצליחו להסיק אם נוכחותם של ג אלביקנס הוא מזיק או מועיל למארח 10. מודלים העכבר פותחו כדי לטפל בזה רלוונטי של פ aeruginosa וג אלביקנס in vivo, אבל האינטראקציה בין מיקרואורגניזמים לא הייתה נקודת המפתח. ואכן, המודל הוקם כדי להעריך את מעורבותו של ג אלביקנס בתגובה מארח חיסוני, ותוצאה. דגם קודם שנקבע על ידי רו et al כבר השתמש התישבות ראשונית עם ג אלביקנס אחרי דלקת ריאות חריפה הנגרמת על ידי פ &#39; aeruginosa. שימוש במודל שלהם, החוקרים מצאו תפקיד מזיק של prior ג אלביקנס קולוניזציה 11. עם זאת רו et al משמש עומס גבוה של ג אלביקנס במודל שלהם עם 2 x 10 6 CFU / עכבר במהלך 3 ימים רצופים. הקמנו מודל 4 ימים של ג קולוניזציה בדרכי הנשימה אלביקנס, או בהתמדה לפחות ללא פגיעת ריאות, במודל זה ג אלביקנס אוחזר עד 4 ימים לאחר החדרה יחידה של 10 5 CFU לכל עכבר (איור 2) 12,13. לאחר 4 ימים, אין עדות לגיוס דלקתי תא, ייצור ציטוקינים דלקתי ולא נזק אפיתל נצפתה. ב24 - 48 שעות, בנוכחות השיא של ג אלביקנס, למרות שתגובה חיסונית מולדת סלולרית וציטוקינים נצפתה, לא היה עדות לפגיעת ריאות. באופן מפתיע, עכברים וכך יישבו עם ג אלביקנס 48 שעות לפני intranasal החדרה של פ aeruginosa שנחלש זיהום בהשוואה לעכברים עם פ זיהום aeruginosa לבד. אניndeed, עכברים הציגו פציעת ריאות פחותה וירידה בניטל חיידקי 12,13. כמה השערות יכולות להסביר השפעה חיובית זו של התישבות מוקדמת עם ג אלביקנס על פ aeruginosa בתיווך דלקת ריאות חריפה. ראשון, שיחה בין-interspecies מעורב כל מיקרואורגניזמים מערכות חישת מניין, פ מבוסס homoserinelactone מערכת aeruginosa וג מבוסס פרנסול מערכת אלביקנס, הוערכה. שנית, ג אלביקנס פועל כמטרה &quot;דמה&quot; לפ aeruginosa הסטת הפתוגן מתאי האפיתל ריאות נחקר. שני ההשערות ביטלו (נתונים שלא פורסמו). ההשערה השלישית הייתה של &quot;תחול&quot; של מערכת החיסון המולדת על ידי ג אלביקנס האחראי לתגובה מולדת הבאה משופרת נגד פ aeruginosa. ההשערה האחרונה זו אושרה. אכן ג קולוניזציה אלביקנס הובילה ליחול חסינות מולדת throuGH IL-22, בעיקר מופרש על ידי תאי הלימפה מולדים, וכתוצאה מכך אישור חיידקים גדל ומופחת פגיעת ריאות 12. לסיכום, המארח הוא שחקן מרכזי ביחסי הגומלין בין מיקרואורגניזמים ויסות התגובה חיסונית המולדת ומעורבים סוגי תאים דלקתיים שונים. בעוד שניתן גזור אינטראקציות חיסוני המורכבות אלה במבחנה ניתן לספק השערות הראשוניות רק על ידי מתאים בדגמי vivo. הפרוטוקול הבא מספק דוגמא למחקר vivo של אינטראקציה הפתוגן בתיווך מארח שעשוי להיות מותאם למייקרו-אורגניזמים אחרים.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sevorane, Sevoflurane</td>
			<td style="width:185px;">Abott</td>
			<td style="width:161px;">05458-02</td>
			<td style="width:167px;">250 mL plastic bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescence Reader Mithras&#160; LB940</td>
			<td style="width:185px;">Berthold Technologies</td>
			<td style="width:161px;">reference in first column</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromo-cresol purple agar</td>
			<td style="width:185px;">Biomerieux</td>
			<td align="right" style="width:161px;">43021</td>
			<td>x20 per unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital sodique 5,47%</td>
			<td style="width:185px;">CEVA</td>
			<td align="right" style="width:161px;">6742145</td>
			<td>100 mL plastic bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-headed valve&#160;</td>
			<td style="width:185px;">Distrimed</td>
			<td align="right" style="width:161px;">92831</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile inoculation loop 10 &#181;L</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">10175</td>
			<td>x1000 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insuline syringes 1 mL</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">30003</td>
			<td>per 100 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 positions Culture tube 8 mL</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">64300</td>
			<td>no comment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrospec 10&#160;</td>
			<td style="width:185px;">General Electric life sciences</td>
			<td style="width:161px;">80-2116-30</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemolysis tubes 13 x 75 mm&#160;</td>
			<td style="width:185px;">Gosselin</td>
			<td style="width:161px;">W1773X</td>
			<td>per 100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS - Phosphate-Buffered Saline</td>
			<td style="width:185px;">Life technologies</td>
			<td align="right" style="width:161px;">10010023</td>
			<td>packaged in 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amikacin 1g</td>
			<td style="width:185px;">Mylan</td>
			<td align="right" style="width:161px;">62516778</td>
			<td>per 10&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin 10 000 UI in 2 mL</td>
			<td style="width:185px;">Pan pharma</td>
			<td align="right" style="width:161px;">9128701</td>
			<td>x 10 per unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RAL 555 coloration kit</td>
			<td style="width:185px;">RAL Diagnostics</td>
			<td align="right" style="width:161px;">361550</td>
			<td>3 flacons of 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,5 mL microcentrifuge tube</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:161px;">55.526.006</td>
			<td>x&#160; 1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transparent 300 &#181;L 96-well plate</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:161px;">82 1581500</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yest-peptone-Dextrose Broth</td>
			<td style="width:185px;">Sigma</td>
			<td align="right" style="width:161px;">95763</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-albumin</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:161px;">A9771</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Luria Bertani Broth</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:161px;">L3022</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25-gauge needle</td>
			<td style="width:185px;">Terumo or unisharp</td>
			<td style="width:161px;">A231</td>
			<td>x100 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytocentrifuge</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:161px;">A78300003</td>
			<td>no comment</td>
		</tr>
	</tbody>
</table>

studying microbial communities in vivo: a model of host-mediated interaction between candida albicans and pseudomonas aeruginosa in the airways

לימוד אינטראקציה מארח הפתוגן מאפשר לנו להבין את המנגנון הבסיסי של פתוגניות במהלך זיהום חיידקים. הפרוגנוזה של המארח תלוי במעורבות של תגובה חיסונית מותאמת נגד הפתוגן 1. תגובה חיסונית היא מורכבת ותוצאות מהאינטראקציה של פתוגנים וכמה סוגים חיסוניים תאיים או אי-חיסון 2. מחקרים במבחנה לא יכולים לאפיין אינטראקציות אלה ולהתמקד באינטראקציות תא-הפתוגן. יתר על כן, בדרך הנשימה 3, במיוחד בחולים עם מחלת ריאות כרונית מוגלתית או בחולי מאוורר מכאני, קהילות polymicrobial נמצאים ולסבך אינטראקציה מארח הפתוגן. אלביקנס Pseudomonas aeruginosa וקנדידה שניהם הבעיה פתוגנים 4, לעתים קרובות מבודדת מדגימות tracheobronchial, ו קשורים לזיהומים חמורים, במיוחד ביחידה לטיפול נמרץ 5. יש אינטראקציות חיידקיםדווח בין פתוגנים אלה במבחנה, אך ההשפעה הקלינית של אינטראקציות אלה נותרה 6 לא ברורים. כדי לחקור את יחסי הגומלין בין C. אלביקנס ופ aeruginosa, מודל עכברי של ג קולוניזציה אלביקנס דרכי הנשימה, ואחריו פ דלקת ריאות חריפה בתיווך aeruginosa- בוצעה.

כפי שניתן לראות בעבר בתיאור הפרוטוקול, הניסוי צריך 5 ימים כדי להשלים (איור 1: ציר זמן ניסוי). מפעיל אחד שידל במשך כל הריצה של הניסוי ויכול להתמודד עם התהליכים עד למקסימום של 10 עכברים. אם יותר בעלי חיים נדרשים, שני אנשים נדרשים במיוחד עבור אוסף מדגם כירורגית. ואכ?...

Watch this Scientific Journal Video about Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways at JoVE.com

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

לומד קהילות חיידקים<em&gt; In vivo</em&gt;: דגם של אינטראקציה בתיווך בין מארח<em&gt; קנדידה אלביקנס</em&gt; ו<em&gt; Pseudomonas aeruginosa</em&gt; באיירווייס

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

studying-microbial-communities-vivo-model-host-mediated-interaction

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

8.4K Views.  Université Lille. While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Video: Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

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 ...

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

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

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

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

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 ...

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. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

לצידו. אור: בויוו ניטור של דלקות Biofilm Pseudomonas aeruginosa לפצעים כרוניים במודל מאתר של שיער סוכרתית

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

שימוש משלים של טכניקות מיקרוסקופיים וקריאה פלואורסצנטית ללמוד קריפטוקוקוס-אמבה אינטראקציות

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

ויזואליזציה של קנדידה אלביקנס במערכת העיכול מוריין באמצעות פלורסנט היברידיזציה באתרו

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

מדבקת החיסון נגד הידבקות כרונית בנגיף הדלקת בפצע

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

הבהרת והדמיה של קנדידה ביוביאמים

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Ex vivo Assay לחקר קנדידה אלביקנים מקף מורפוגנזה במערכת העיכול

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

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.

לומד קהילות חיידקים

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