Name: cefoperazone-treated mouse model of clinically-relevant clostridium difficile strain r20291
Uploaded: 2016-12-10T13:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291 at JoVE.com

The authors would like to thank Trevor Lawley at the Wellcome Trust Sanger Institute for C. difficile R20291 spores and James S. Guy at the North Carolina State University College of Veterinary Medicine for Vero cells, both utilized in this manuscript. Animal histopathology was performed in the LCCC Animal Histopathology Core Facility at the University of North Carolina at Chapel Hill, with special assistance from Traci Raley and Amanda Brown. The LCCC Animal Histopathology Core is supported in part by an NCI Center Core Support Grant (2P30CA016086-40) to the UNC Lineberger Comprehensive Cancer Center. We would also like to thank Vincent Young, Anna Seekatz, Jhansi Leslie, and Cassie Schumacher for helpful discussions on the Vero cell cytotoxicity assay protocol. JAW is funded by the Ruth L. Kirschstein National Research Service Award Research Training grant T32OD011130 by NIH. CMT is funded by the career development award in metabolomics grant K01GM109236 by the NIGMS of the NIH.

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The authors have nothing to disclose at this time.

This protocol characterizes the clinical course, including weight loss, bacterial colonization, toxin cytotoxicity, and histopathological changes in the gastrointestinal tract, of antibiotic-treated mice challenged with C. difficile R20291 spores. There are several critical steps within the protocol where attention to detail is essential. Accurate calculation of the C. difficile spore inoculum is critical. This calculation is based on the original C. difficile spore stock enumeration, which sho...

Clostridium difficile הינו אנאירובי, חיובי-גרם, חיידק יוצרי נבגים שגורמת מסכני חיים שלשולים 1. C. difficile זיהום (CDI) קשור לתחלואה אנושית מוגדלת ותמותה ותוצאות בלמעלה מ -4.8 מיליארדים $ בעלויות בריאות בשנה 1-4. בשנת 2013, המרכז לבקרת מחלות ומניעתן מסווג difficile ג כסיכון עמידות לאנטיביוטיקה דחוף, המציין כי זה מהווה איום דחוף בריאות הציבור 1. נכון לעכשיו, טיפול אנטיביוטי עם vancomycin ו metronidazole נחשבים רמת הטיפול CDI 5. למרבה הצער, ישנות CDI לאחר טיפול מוצלח באנטיביוטיקה הן גבוהות, המתרחשות 20 - 30% מהחולים 2,5-7. לכן, הגילוי רפוי רומן נגד הפתוגן מעיים זה הכרחי. כדי להעריך רפויים נגד C. difficile, במודל חית מתקרבות מחל אדם aclinically רלוונטי זן הוא זקוק. בתחילה, העיקרים של קוך הוקמו difficile ג בשנת 1977 תוך שימוש במודל אוגר סורי שטופלו קלינדמיצין 8. מודל זה עדיין משמש כיום כדי לחקור את ההשפעות של רעלים C. difficile על בפתוגנזה 9,10. עם זאת, CDI במודל אוגר תוצאת שיעורי תמותה גבוהים ואינו להיות קרוב ככל האפשר גילויי מחלה הפנימיים הכרוניים שניתן הנצפים באנשים לוקים 10,11 CDI. בהתבסס על הזמינות והנגישות מגיבה של פלטפורמות murine במחקר, במודל של עכברים של CDI רלוונטי. בשנת 2008, במודל של עכברים חזק של CDI הוקם על ידי טיפול בעכברים עם קוקטייל אנטיביוטי במי שתייה (kanamycin, גנטמיצין, colistin, metronidazole, ו ונקומיצין) במשך 3 ימים ואחריו זריקת intraperitoneal של קלינדמיצין 12. עכברים שניתנו זה רגיש קוליטיס CDI והחמור. לִסְמוֹךing על מינון הבידוד מנוהל, מגוון של שלטים קטלניים קליניים ניתן לצפות באמצעות מודל זה. מאז הפעם, משטרי אנטיביוטיקה שונים נחקרו המשנים את החיידקים במעיים בעכברים, הפחתת התנגדות קולוניזציה עד לנקודה שבה C. difficile יכול ליישב את מערכת העיכול (הנסקרת ב Best et al. ו Lawley אנד יאנג) 13,14. לאחרונה, צפלוספורין קשת רחבה, cefoperazone, בהתחשב במי השתייה במשך ימים 5 או 10 הופך reproducibly עכברים רגישים 15 CDI. מאז ממשל של צפלוספורינים מהדור השלישי קשורים עם סיכון מוגבר של CDI בבני אדם, השימוש במודל cefoperazone משקף באופן מדויק יותר טבעיים המתחוללים המחלה 16. Cefoperazone שטופל עכברים רגישים difficile ג כבר אתגר בשני נבגי C. difficile ותאי וגטטיבי של מגוון רחב של זנים החל קליניהרלוונטיות ארסיות 17. למרות חלק מהמחקרים המקוריים ניצול תאי צמח C. difficile כצורה זיהומיות, נבגי difficile ג נחשבים במצב העיקרי להעברת מחלה 18. בעשור האחרון, C. difficile R20291, זן NAP1 / BI / 027, התפתח, גרימת מגיפות של 19,20 CDI. אנחנו בקשנו לקבוע את המסלול הקליני של מחלה, כאשר עכברים שטופל cefoperazone הותקפו עם הזן קליני הרלוונטי וגנטי-צייתן C. difficile, R20291. פרוטוקול זה מפרט את המהלך הקליני, כולל ירידה במשקל, קולוניזציה חיידקית, cytotoxicity רעלן, ושינויים histopathological במערכת העיכול של עכברים תיגר עם נבגים R20291 C. difficile. בסך הכל, מודל העכבר הזה מוכיח להיות פלטפורמה ניסיונית ערך עבור CDI המתקרבות מחלות אנושיות. ולכן יכול להיות מנוצל במודל עכבר מאופיין זה, לאמוד את השפעתושל הרפוי רומן על השיפור של מחלה קלינית ועל השיקום של התנגדות קולוניזציה נגד difficile ג.

<table><tbody><tr><td>#62 Perisept Sporidicial Disinfectant Cleaner&amp;nbsp;</td><td>SSS Navigator</td><td>48027</td><td>This product will require dilution as recommended by the manufacturer</td></tr><tr><td>0.22 &amp;mu;m filter</td><td>Fisherbrand</td><td>09-720-3</td><td>Alternative to filter plate for indivdiual samples tested in the Vero Cell Assay</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td>Needs to be heated in water bath at 37 &amp;deg;C prior to use</td></tr><tr><td>0.4% Trypan Blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>1% Peniciilin/Streptomycin</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>10% heat inactivated FBS</td><td>Gibco</td><td>16140-071</td><td>Needs to be heated in water bath at 37 &amp;deg;C prior to use</td></tr><tr><td>1 ml plastic syringe&amp;nbsp;</td><td>BD Medical Supplies</td><td>309628</td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>2 ml Micro Centrifuge Screw Cap</td><td>Corning</td><td>430917</td><td></td></tr><tr><td>96 well cell culture flat bottom plate</td><td>Costar Corning</td><td>CL3595</td><td></td></tr><tr><td>96 well filter plate</td><td>Millipore</td><td>MSGVS2210</td><td></td></tr><tr><td>Adhesive Seal</td><td>ThermoScientific</td><td>AB-0558</td><td></td></tr><tr><td>Bacto Agar</td><td>Becton Dickinson</td><td>214010</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Bacto Proteose Peptone</td><td>Becton Dickinson</td><td>211684</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Cefoperazone</td><td>MP Bioworks</td><td>199695</td><td></td></tr><tr><td>Cefoxitine</td><td>Sigma</td><td>C47856</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Clostridium difficile Antitoxin Kit</td><td>Tech Labs</td><td>T5000</td><td>Used as control for Vero Cell Assay</td></tr><tr><td>Clostridium difficile Toxin A</td><td>List Biological Labs</td><td>152C</td><td>Positive control for Vero Cell Assay</td></tr><tr><td>D-cycloserine</td><td>Sigma</td><td>C6880</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Distilled Water</td><td>Gibco</td><td>15230</td><td></td></tr><tr><td>DMEM 1x Media</td><td>Gibco</td><td>11965-092</td><td>Needs to be heated in water bath at 37 &amp;deg;C prior to use</td></tr><tr><td>Fructose</td><td>Fisher</td><td>L95500</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Hemocytometer</td><td>Bright-Line, Sigma</td><td>Z359629</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P285-500</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Sigma</td><td>M2643</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Millex-GS 0.22 &amp;mu;m filter</td><td>Millex-GS</td><td>SLGS033SS</td><td>Filter for TCCFA plates&amp;nbsp;</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S-0876</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>NaCl</td><td>Fisher</td><td>S640-3</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Number 10 disposable scalpel blade</td><td>Miltex, Inc</td><td>4-410</td><td></td></tr><tr><td>PCR Plates</td><td>Fisherbrand</td><td>14230244</td><td></td></tr><tr><td>Plastic petri dish</td><td>Kord-Valmark Brand</td><td>2900</td><td></td></tr><tr><td>Sterile plastic L-shaped cell spreader</td><td>Fisherbrand</td><td>14-665-230</td><td></td></tr><tr><td>Syringe Stepper</td><td>Dymax Corporation</td><td>T15469</td><td></td></tr><tr><td>Taurocholate</td><td>Sigma</td><td>T4009</td><td>Part of TCCFA plates (see below)</td></tr><tr><td>Ultrapure distilled water</td><td>Invitrogen</td><td>10977-015</td><td></td></tr><tr><td>C57BL/6J Mice</td><td>The Jackson Laboratory</td><td>664</td><td>Mice should be 5 - 8 weeks of age</td></tr><tr><td>Olympus BX43F light microscope</td><td>Olympus Life Science</td><td></td><td></td></tr><tr><td>DP27 camera</td><td>Olympus Life Science</td><td></td><td></td></tr><tr><td>cellSens Dimension software&amp;nbsp;</td><td>Olympus Life Science</td><td></td><td></td></tr></tbody></table>

cefoperazone-treated mouse model of clinically-relevant clostridium difficile strain r20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

במהלך מחקר נציג, 5 שבועות בן C57BL / 6 עכברים WT טופלו קודם cefoperazone במי השתייה שלהם (0.5 מ&quot;ג / מ&quot;ל) במשך 5 ימים, מותר 2-יום לשטוף עם מי שתייה רגילים. עכברים היו תיגר עם 10 5 נבגים של C. difficile R20291 gavage דרך הפה ביום 0 (איור 1 א). עכברים נוטרו לירידה ?...

Watch this Scientific Journal Video about Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291 at JoVE.com

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

פרוטוקול זה מתאר את מודל עכבר cefoperazone של זיהום difficile Clostridium (CDI) באמצעות זן קליני רלוונטי וגנטי-צייתן, R20291. דגש על ניטור מחלה קליני, פקידת חיידקי C. difficile, cytotoxicity רעלן, ושינויי histopathological ברחבי CDI במודל של עכברים מפורטים בפרוטוקול.

Cefoperazone שטופל עכבר דגם של קליני רלוונטי<em&gt; Clostridium difficile</em&gt; R20291 הזנים

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

cefoperazone-treated-mouse-model-clinically-relevant-clostridium

Research

JoVE Journal

Immunology and Infection

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

12.2K Views.  North Carolina State University College of Veterinary Medicine.  פרוטוקול זה מתאר את מודל עכבר cefoperazone של זיהום difficile Clostridium (CDI) באמצעות זן קליני רלוונטי וגנטי-צייתן, R20291. דגש על ניטור מחלה קליני, פקידת חיידקי C. difficile, cytotoxicity רעלן, ושינויי histopathological ברחבי CDI במודל של עכברים מפורטים בפרוטוקול. 

Video: Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

<em&gt; Citrobacter rodentium</em&gt; דגם עכבר: תרומות הפתוגן ומארח לומדים קוליטיס המדבק

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

הדמיה של multimodality 4D<em&gt; Citrobacter rodentium</em&gt; זיהומים בעכברים

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

פיתוח מודל לזיהום של זחל בדג במשך שינוי קושי

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Developmental Biology

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

ההקמה ואפיון של UTI וCAUTI במודל עכבר

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Medicine

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

פרוטוקול לאפיין את השינויים המורפולוגיים של<em&gt; Clostridium difficile</em&gt; בתגובה לטיפול אנטיביוטי

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

דגם עכבר מבוקר Neonatal אלח דם Polymicrobial

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

גרימת דלקת קרום הגרון הקבוצה C בעכברים באמצעות משלוח התאיים

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

מכשיר התחממות/ריסון עכשווי להזרקות יעילות של וריד זנב במודל אלח דם פטרייתי מורין

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

זיהום חוזר בדרכי השתן של Escherichia coli שנגרם על ידי גרדנרלה וגינליס חשיפה לשלפוחית השתן בעכברים

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

הקמת מודל עכברי לזיהום הקשור לצנתר קנדידה אלביקנס

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

מחבר זרקור: שיפור זיהוי קנדידה אלביקנס בזיהומי קטטר באמצעות תיוג חלבון פלואורסצנטי 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

מודל זיהום קנדידה אלביקנס הקשור לצנתר בעכבר

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

Cefoperazone שטופל עכבר דגם של קליני רלוונטי

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הדמיה של multimodality 4D