1. תרבות חיידקים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פ גראם שלילי aeruginosa זן PAO1 גדלו במרק לוריא (LB) על 37 מעלות צלזיוס למשך 18 שעות. </li><li style=";text-align:right;direction:rtl"> התרבות של תאים הייתה אז centrifuged ב 7,500 סל&quot;ד (סיבובים לדקה) במשך 5 דקות וsupernatant הוסר. </li><li style=";text-align:right;direction:rtl"> החיידקים היו resuspended ב10% LB מחדש גדלו במשך שעה 2 אחרת כדי לאפשר התרבות להזין מחדש שלב נייח. </li><li style=";text-align:right;direction:rtl"> ההשעיה החיידקים לאחר מכן לחלק לשתי קבוצות: בקבוצה הראשונה (2 צינורות) אין אנטיביוטיקה נוספו, בקבוצה השנייה הוספנו את האנטיביוטיקה gentamycin (50 מיקרוגרם / מ&quot;ל). </li></ol> 2. נחישות של מושבה יוצרי יחידות (CFU) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לקבוע כדאיות תא 20 דגימות μl נלקחו מהניסוי בערך כל שעה 2 במסגרת הזמן של 24 שעות. דילול סידורי של הדגימות נעשו ומצופה על צלחות אגר LB וטופחו לילה בשעה 37 ° C. </li><li style=";text-align:right;direction:rtl"> עבור כל טיפול, CFUs לפלאטהדואר נקבע ונערכה השוואה בין תקופות הזמן שונים וטיפולים. הירידה ביומן CFU חושב כמתואר במשוואה. (1): התחבר הפחתה = LogU-LogC [CFU / מ&quot;ל] איפה U הוא המושבה להרכיב יחידות ערך בכל נקודת זמן; CFU הוא יחידת מושבה היוצרים בעוד שהיחידות של CFU / מ&quot;ל ​​שווה ל: CFU / מ&quot;ל ​​= (מספר הגורם לדילול מושבות x) / (נפח מחוסן) ו-C הוא CFU מצא במדגם השליטה בשלב ההתחלה. שימו לב שU מייעד מושבה להרכיב גורם ברגע מדידה. </li><li style=";text-align:right;direction:rtl"> הגורם לדילול הוא מספר דילולים ואילו בכל אחד מהם את ריכוז החיידקים צומצם בפקטור של 10. הנפח מחוסן תמיד היה 200 ליטר מיקרו וזה קשור לגודל של מבחנתנו. </li></ol> לכן, כדי לסכם את נקודת מבט של הריכוז, האנטיביוטיקה gentamycin הייתה בריכוז של 50 מיקרוגרם / מ&quot;ל. באשר לחיידקים, עד סוףהיה לנו התהליך כולל 8 דילולים. כל דילול היה בפקטור של 10 וזה נעשה בצינורות של 200 μl. נקודת המוצא הייתה 20 μl של דגימות שנוספו לתוך צינור μl 200 (ובכך הריכוז הראשוני היה 20/200C 0 = 0 0.1C0 עם C להיות הריכוז הראשוני ב20 μl של דגימות) והריכוז הסופי הופחת 8 סדרי גודל בשל דילולים 8. 3. תאורה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> CW Nd: YAG לייזר (אורך גל של 532 ננומטר וכוח אופטי הממוצע של 200 מגה ואט) פוצל לשני נתיבים אופטיים באמצעות 50% אופטיים / 50% מפצל קורה. קוטר האלומה היה על 10 מ&quot;מ. משך החשיפה היה 24 שעות. </li><li style=";text-align:right;direction:rtl"> Q-Switched פעם Nd: YAG לייזר (אורך הגל של 532 ננומטר, הספק ממוצע של 300 מגה ואט ושיא כוח אופטי של 2.5 מגה וואט) פוצל לשני נתיבים גם באמצעות 50% אופטיים / 50% מפצל קורה. קוטר הנקודה היה 6 מ&quot;מ. הרוחב פולס של לייזר ממותגת Q היה 6 NSEC ושיעור ההישנות היה 15Hz.צפיפות הספק ממוצעת הייתה 106 mW / 2 ס&quot;מ ובצפיפות הספק השיא הייתה 8.83 קילוואט / 2 מ&quot;מ. משך החשיפה היה 24 שעות. </li></ol> שים לב כי ההשעיה החיידקים עוררה במהלך הקרנה וזה היה כל הזמן תחת תנאי התרבות מתאימים להתפתחות חיידקים (בכל הצינורות היה בינונית מרק לוריא, כדי לאפשר לחיידקים לגדול).

<ol>
	<li>Feuerstein, O., Persman, N., Weiss, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxic+Effect+of+Visible+Light+on+Porphyromonas+gingivalis+and+Fusobacterium+nucleatum:+An+In+Vitro+Study.">Phototoxic Effect of Visible Light on Porphyromonas gingivalis and Fusobacterium nucleatum: An In Vitro Study.</a> Photochemistry and Photobiology. 80, 412-415 (2004).</li><li>Enwemeka, C. S., Williams, D., Enwemeka, S. K., Hollosi, S., Yens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+470-nm+light+kills+methicillin-resistant+Staphylococcus+aureus+(MRSA)+in+vitro.">Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro.</a> Photomed. Laser Surg. 27, 221-226 (2009).</li><li>Guffey, J. S., Wilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+bactericidal+effects+of+405-nm+and+470-nm.">In vitro bactericidal effects of 405-nm and 470-nm.</a> Photomed. Laser Surg. 24, 684-688 (2006).</li><li>Lipovsky, A., Nitzan, Y., Friedman, H., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Staphylococcus+aureus+strains+to+broadband+visible+light.">Sensitivity of Staphylococcus aureus strains to broadband visible light.</a> Photochem. Photobiol. 85, 255-260 (2008).</li><li>Lipovsky, A., Nitzan, Y., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+Mechanism+for+visible+light+induced+wound+healing.">A possible Mechanism for visible light induced wound healing.</a> Lasers in Surgery and Medicine. 40, 509-514 (2008).</li><li>Lipovsky, A., Nitzan, Y., Gedanken, A., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+light-induced+killing+of+bacteria+as+a+function+of+wavelength:+Implication+for+wound+healing.">Visible light-induced killing of bacteria as a function of wavelength: Implication for wound healing.</a> Lasers in Surgery and Medicine. 42, 467-472 (2010).</li><li>Feuerstein, O., Ginsburg, I., Dayan, E., Veler, D., Weiss, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Visible+Light+Phototoxicity+on+Porphyromonas+gingiwalis+and+Fusobacferium+nucleaturn.">Mechanism of Visible Light Phototoxicity on Porphyromonas gingiwalis and Fusobacferium nucleaturn.</a> Photochemistry and Photobiology. 81, 1186-1189 (2005).</li><li>Nussbaum, E. L., Lilge, L., Mazzulli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+630-,+660-,+810-,+and+905-nm+laser+irradiation+delivering+radiant+exposure+of+1-50+J/cm2+on+three+species+of+bacteria+in+vitro.">Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm2 on three species of bacteria in vitro.</a> J. Clin. Laser Med. Surg. 20, 325-333 (2002).</li><li>Dadras, S., Mohajerani, E., Eftekhar, F., Hosseini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+Photoresponses+of+Staphylococcus+aureus+and+Pseudomonas+aeruginosa+to+514,+532,+and+633+nm+Low+Level+Lasers+In+Vitro.">Different Photoresponses of Staphylococcus aureus and Pseudomonas aeruginosa to 514, 532, and 633 nm Low Level Lasers In Vitro.</a> Current Microbiology. 53, 282-286 (2006).</li><li>Stover, C. K., Pham, X. Q., Erwin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+Pseudomonas+aeruginosa+PAO1,+an+opportunistic+pathogen.">Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.</a> Nature. 406, 952-964 (2000).</li><li>Hamblin, M. R., Demidova, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+low+level+light+therapy.">Mechanisms of low level light therapy.</a> Proc. SPIE. 6140, 1-12 (2006).</li><li>Krespi, Y. P., Stoodley, P., Hall-Stoodley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+disruption+of+biofilm.">Laser disruption of biofilm.</a> Laryngoscope. 118, 1168-1173 (2008).</li><li>Reznick, Y., Banin, E., Lipovsky, A., Lubart, R., Zalevsky, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+laser+light+enhancement+of+susceptibility+of+bacteria+to+gentamycin+antibiotic.">Direct laser light enhancement of susceptibility of bacteria to gentamycin antibiotic.</a> Opt. Commun. 284, 5501-5507 (2011).</li></ol>

אין ניגודי האינטרסים הכריזו.

פוטותרפיה כבר תחום מחקר רב תחומי מתקדם בשנים האחרונות מתפתחות כגישה מבטיחה לטיפול במחלות רבות. בהקשר זה את השימוש באור בטווח הגלוי, נחקר בהרחבה. כך, למשל, נמצא כי ניתן לרפא פצעים מזוהמים בצורה יעילה יותר על ידי חשיפתם לאור הנראה אינטנסיבי למטרות עיקור. מנגנון הפעולה ל...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Lauria Broth</td>
 <td>Difco</td>
 <td>241420</td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamycin</td>
 <td>Sigma </td>
 <td>G1914</td>
 <td></td>
 </tr>
 <tr>
 <td>Bacto Agar</td>
 <td>Difco </td>
 <td>231710 </td>
 <td></td>
 </tr>
 </tbody>
</table>

the synergistic effect of visible light and gentamycin on pseudomona aeruginosa microorganisms

לאחרונה היו כמה פרסומים על השפעת bactericidal של אור הנראה, רובם בטענה כי חלק כחול של הספקטרום (400 nm-500 ננומטר) הוא אחראי להרג פתוגנים שונים 1-5. השפעת phototoxic של אור הכחול הוצעה להיות תוצאה של מיני חמצן תגובתי המושרה אור (ROS) על ידי היווצרות פוטוסנסיטייזרים חיידקים אנדוגניים אשר רובם לקלוט אור באזור הכחול 4,6,7. ישנם גם דיווחים על השפעת biocidal של אדום וליד אינפרא אדום 8, כמו גם אור ירוק 9. במחקר הנוכחי, שפתחנו שיטה שאפשרה לנו לאפיין את ההשפעה של מתח גבוה ירוק (אורך גל של 532 ננומטר) אור Q-Switched רציף (CW) ופעם (QS) בPseudomonas aeruginosa. שימוש בשיטה זו אנחנו גם בחנו את ההשפעה של אור הירוק בשילוב עם טיפול אנטיביוטי (gentamycin) על כדאיות החיידקים. פ aeruginosa הוא acהפתוגן האופורטוניסטי noscomial ommon גורם למחלות שונות. הזן הוא די עמיד לאנטיביוטיקה שונות ומכיל רבים מסוג Mex חזה AcrB / RND multidrug מערכות זרימה 10. השיטה מנוצל חיידקים נטולי חיים נייחים שלב גראם שליליים (עמ &#39;זן aeruginosa PAO1), גדלו במרק לוריא (LB) בינונית חשוף לQ-Switched ו / או CW לייזרים עם ובלי התוספת של gentamycin האנטיביוטי. כדאיות תא נקבעו בנקודות זמן שונות. התוצאות שהתקבלו הראו כי טיפול לייזר לבד לא להפחית כדאיות תא בהשוואה לשליטת מטופל וכי טיפול gentamycin לבד רק הביאה לירידה של 0.5 לוג בספירה המעשית עבור פ aeruginosa. לייזר המשולב וטיפול gentamycin, לעומת זאת, הביאו לאפקט סינרגיסטי ואת הכדאיות של פ aeruginosa הופחת ב 8 היומן של. השיטה המוצעת יכולה להיות עוד יותר impleעינה באמצעות פיתוח של צנתר כמו מכשיר שמסוגל הזרקת תמיסת אנטיביוטיקה לתוך האיבר הנגוע בזמן מאיר את האזור באור בו זמנית.

ההתקנה מבוססת לייזר מוצגת סכמטי באיור 1. תנאי הניסוי הראשון מנוצלים Nd CW: YAG לייזר בעל אורך גל של 532 ננומטר (ההרמוני שני של Nd: YAG) וכוח אופטי ממוצע של 200 מגה ואט. קרן זו פוצלה לשני נתיבים אופטיים באמצעות 50% 50% מפצל אופטי כך שכל קורה לי פיצול כוח של 100 מגה ואט אלומה /. קו...

Watch this Scientific Journal Video about The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms at JoVE.com

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

אנו מראים כי מכשיר ביו מעורב פיתחה טיפול לייזר מבוסס גלוי רציף או פעם שהוא בשילוב עם טיפול אנטיביוטי (gentamycin), גורם לאפקט סינרגיסטי מובהק סטטיסטי שמוביל לירידה בכדאיות של<em&gt; פ aeruginosa</em&gt; PAO1, על ידי 8 יומן בהשוואה לטיפול באנטיביוטיקה לבד.

האפקט סינרגיסטי של אור וgentamycin הגלויים על<em&gt; Aeruginosa Pseudomona</em&gt; מיקרואורגניזמים

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

the-synergistic-effect-visible-light-gentamycin-on-pseudomona

Research

JoVE Journal

Immunology and Infection

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

13.3K Views.  Bar-Ilan University. אנו מראים כי מכשיר ביו מעורב פיתחה טיפול לייזר מבוסס גלוי רציף או פעם שהוא בשילוב עם טיפול אנטיביוטי (gentamycin), גורם לאפקט סינרגיסטי מובהק סטטיסטי שמוביל לירידה בכדאיות של פ aeruginosa PAO1, על ידי 8 יומן בהשוואה לטיפול באנטיביוטיקה לבד.

Video: The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-מודלים של תרבות Pseudomonas aeruginosa Biofilms גדל על תאים חיים Airway האדם

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

שימוש בכימיה לחץ כדי למדוד את ההשפעה של זיהום ויראלי בסינתזת RNA Host-Cell

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

בעקבות בזמן אמת השפעת גורמי דלקת ריאות ארסיים בעכבר דלקת ריאות דגם אקוטי באמצעות חיידקי Bioluminescent

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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. ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

כולסטרול מקרופאג דלדול והשפעתו על phagocytosis של<em&gt; Neoformans קריפטוקוקוס</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

דור של Lymphocytic microparticles וזיהוי של Proapoptotic השפיעו על תאי אפיתל Airway

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

פרוטוקולים לחקירת הפצת Host-הרקמה, הילוכים-mode, ואת ההשפעה על כושר Host של Densovirus ב bollworm הכותנה

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

שכפול של הוזמן, ספריית Nonredundant של Pseudomonas aeruginosa זן PA14 Transposon ההכנסה מוטציות

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...