This work was supported by National Institutes of Health Grants R01 AI129302 (to S.I.S.) and the Training Program in Pharmacology: From Bench to Bedside at UC Davis (NIH T32 GM099608 to L.S.A). The Molecular and Genomic Imaging (CMGI) at the University of California Davis provided superb technological support.

All mouse studies were reviewed and approved by the Institutional Animal Care and Use Committee at UC Davis and were performed according to the guidelines of the Animal Welfare Act and the Health Research Extension Act. Be sure to use sterile gloves when working with animals.
1. Mouse Source and Housing
<ol>
	<li>Derive LysM-eGFP mice on a C57BL/6J genetic background as described previously12. Derive LysM-EGFP&#215;MyD88-/- mice by crossing LysM-EGFP mice with MyD88-/- mice on a C57BL/6J background.</li>
	<li>House mice in a vivarium. For these studies, animals were housed at the University of California, Davis in groups prior to surgery and single housed following surgery. Use mice between the ages of 10-16 weeks.</li>
</ol>
2. Bacterial Preparation and Quantification
<ol>
	<li>Remove the bioluminescent S. aureus strain of interest from -80 &#176;C storage to thaw on ice. Streak on a 5% bovine blood agar plate. Incubate the streaked plate in a humidified incubator at 37 &#176;C overnight (16 h). 
	NOTE: In this protocol, the ALC2906 SH1000 strain was used. This strain contains the shuttle plasmid pSK236 with the penicillin-binding protein 2 promoter fused to the luxABCDE reporter cassette from Photohabdus luminescens18.</li>
	<li>Prepare tryptic soy broth (TSB) by mixing 0.03 g of TSB powder per mL of pure water, and autoclave TSB to sterilize. When cooled, add any necessary antibiotics using sterile technique. In this protocol, add 10 &#181;g/mL of chloramphenicol18 to the TSB to select for expression of the pSK236 shuttle plasmid, which contains the bioluminescence luxABCDE cassette.</li>
	<li>Pick 3-4 separate colonies from the S. aureus plate into TSB with 10 &#181;g/mL chloramphenicol to start an overnight culture. Incubate bacteria on an incubating shaker at 37 &#176;C overnight (16 h).</li>
	<li>Start a new bacterial culture from the overnight culture by diluting a sample 1:50 into TSB with 10 &#181;g/mL chloramphenicol. Culture in an incubating shaker at 200 rpm and 37 &#176;C.</li>
	<li>Two hours after splitting the S. aureus, monitor the optical density at 600 nm (OD600) on a spectrophotometer. Observe the OD600 vs. time to find mid-logarithmic phase growth. For the ALC2906 SH1000 strain, an OD600 of 0.5 is mid-logarithmic and corresponds to a concentration of 1 x 108 CFU/mL (Figure 1).</li>
	<li>When OD600 is 0.5, wash bacteria 1:1 with ice-cold DPBS. Centrifuge the bacteria for 10 min at 3,000 x g and 4 &#176;C. Carefully decant the supernatant and add additional chilled DPBS and vortex thoroughly. Centrifuge once more for 10 min at 3,000 x g and 4 &#176;C.</li>
	<li>Carefully decant the supernatant. Resuspend the bacterial pellet at a desired concentration. For these studies, collect 3 mL of ALC2906 SH1000 and resuspend in 1.5 mL of PBS, correlating to a bacteria concentration of about 2 x&#160;108 CFU/mL. Keep bacteria on ice until use.</li>
	<li>To verify bacteria concentration, dilute 100 &#181;L of the bacterial sample 1:10,000 and 1:100,000 in PBS. Plate 20 &#181;L aliquots on an agar plate. Incubate at 37 &#176;C in a humidified incubator for 16 h. Count CFUs by gross examination and calculate a bacterial concentration the following day.</li>
</ol>
3. Excisional Skin Wounding and Inoculation with S. aureus
<ol>
	<li>Administer 100 &#181;L of 0.03 mg/mL buprenorphine hydrochloride (~0.2 mg/kg) to each mouse via intraperitoneal injection.&#160;</li>
	<li>Twenty minutes post-injection, place 2-4 mice in a chamber with 2-3 LPM oxygen with 2-4% isoflurane. Once mice are fully anesthetized, transfer the mice one at a time to a nose cone connected to 2-3 LPM oxygen with 2-4% isoflurane. Verify mice are fully anesthetized by firmly pinching each rear paw between a thumb and forefinger. Proceed to the next step if the animal does not respond to the pinch.</li>
	<li>Shave a 1 inch by 2 inch section on the back of the mouse with electric clippers and clear the area of fur clippings using a clean wipe or gauze. Avoid using depilatory lotion because it may cause excess inflammation.</li>
	<li>Clean the back of the mouse first with 10% povidone-iodine soaked gauze and then with a 70% ethanol soaked gauze. Clean the area in a spiral pattern, moving outward from the center of the surgical area. Wait approximately 1 min for the surgical area to dry prior to surgery.</li>
	<li>Hold the shaved back of the mouse loosely between two fingers and firmly press a sterile 6 mm punch biopsy at the center of the prepared surgical area. Do not pull the skin taut.
	<ol>
		<li>Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin in at least one section of the outline. Be careful not to cut into the underlying fascia or tissue.</li>
		<li>Use sterile scissors and forceps to cut through the epidermis and dermis following the circular pattern imprinted by the punch biopsy.</li>
	</ol>
	</li>
</ol>
4. S. aureus Inoculation
<ol>
	<li>Fill a 28 G insulin syringe with the desired bioluminescent bacterial inoculant. In this study, administer a concentration of 1 x&#160;108 CFU/mL (50 &#181;L). Do not administer more than 100 &#181;L of volume.</li>
	<li>Inject 50 &#181;L of inoculant between the fascia and tissue in the center of the wound on the back of the mouse. Ensure that the inoculant forms a bubble at the center of the wound with minimal leakage or dispersion.
	<ol>
		<li>Pull the dermis to the side, hold the syringe nearly parallel to the tissue, and slowly push the syringe into the tissue until a sudden decrease in resistance is felt, which indicates piercing of the fascia. Carefully lead the syringe into the center of the wound and dispense the inoculant slowly. Remove the syringe slowly from the animal.</li>
	</ol>
	</li>
	<li>Inject the same volume of sterile PBS into the wounds of uninfected animals as described above.</li>
	<li>Return the animal to its cage. Place the cage under a heat lamp or on top of a heating pad, and monitor the animal until recovery from anesthesia.</li>
</ol>
5. In Vivo BLI and FLI
<ol>
	<li>Initialize the whole animal imager through the instrument software. Anesthetize mice in a chamber receiving 2-3 LPM oxygen with 2-4% isoflurane. Deliver anesthesia to the nosecones inside the imager.</li>
	<li>Place the wounded and infected mouse into the imager. Position the mouse such that the wound is as flat as possible. Use the following sequence set-up to image the mice.
	<ol>
		<li>Select Luminescence and Photograph as the imaging mode. The exposure time is 1 min at small binning and F/stop 1 (luminescence) and F/stop 8 (photograph). The emission filter is Open. Click the Acquire button to record the image.</li>
		<li>Select Fluorescence and Photograph as the imaging mode. The exposure time is 1 s at small binning and F/stop 1 (Fluorescence) and F/stop 8 (photograph) with an excitation wavelength of 465/30 nm and an emission wavelength 520/20 nm with a high lamp intensity. Click the Acquire button to record the image.</li>
	</ol>
	</li>
	<li>Return the animal to its cage and monitor until recovery from anesthesia.</li>
	<li>Image mice daily as described above.</li>
</ol>
6. Image Analysis
<ol>
	<li>Open images to be quantified.</li>
	<li>Place a large circular region of interest (ROI) over the entire wound area including surrounding skin for each mouse in the image. The neutrophil in vivo FLI EGFP signals and in vivo BLI S. aureus signals extend beyond the wound edge after several days and were&#160;included in these studies (Figure 2A and 2B). Click Measure ROI and record values for mean flux for each mouse. Plot the mean flux of each signal (p/s) versus time.</li>
	<li>If absolute numbers of neutrophils or S. aureus in the wound are desired, perform the following experiments.
	<ol>
		<li>To correlate neutrophil numbers to the in vivo FLI EGFP signals, wound C57BL/6J mice as described above, and transfer a range of bone marrow-derived neutrophils (5 x 105 to 1 x 107) from LysM-EGFP or LysM-EGFPxMyD88-/- donors directly on top of different wounds. Image as described above and correlate the in vivo FLI EGFP signals to the known quantity of neutrophils.</li>
		<li>To correlate S. aureus CFU to the in vivo BLI signals, wound and infect mice as described above. On day 1 post-infection record in vivo BLI signals from the mice and immediately euthanize and chill carcasses. Excise the wound, homogenize the tissue, and plate bacterial dilutions on agar for overnight incubation. The next day, count colonies to determine CFU per wound.</li>
	</ol>
	</li>
	<li>To measure wound healing fit a circular ROI over the wound edge and measure the area of the wound (cm2) and plot the fractional change from baseline vs.&#160;time (Figure 2C).</li>
</ol>
7. Statistics
NOTE: All data are presented as mean &#177;SEM. p &#60; 0.05 were considered statistically significant
<ol>
	<li>Determine differences between two groups on a single day using the Holm-Sidak method, with alpha = 0.05, and analyze each time point individually, without assuming a consistent SD.</li>
	<li>Compare differences between multiple groups on the same day by one-way ANOVA with the Tukey multiple-comparisons posttest. Survival between experimental groups was analyzed by the Mantel-Cox method.</li>
</ol>

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Lloyd S. Miller has received grant support from MedImmune, Pfizer, Regeneron, and the Chan Soon-Shiong Nanthealth Foundation and consulting fees from Noveome Biotherapeutics and the Chan Soon-Shiong Nanthealth Foundation that are unrelated to the work reported in this paper. The other authors have nothing to disclose.

S. aureus infection models that utilize bioluminescent S. aureus infection in a fluorescent neutrophil reporter mouse in conjunction with advanced techniques of whole animal in vivo optical imaging have advanced our knowledge of the innate immune response to infection. Previous studies using the LysM-EGFP mouse have shown that up to 1 x 107 neutrophils recruit to an S. aureus infected wound over the first 24 h of infection14, and wound-recruited neutrophils ex...

Staphylococcus aureus (S. aureus) accounts for the majority of skin and soft tissue infections (SSTIs) in the United States1. The incidence of methicillin-resistant S. aureus (MRSA) infections has increased steadily over the past two decades2, motivating the study of the mechanisms of persistence and the discovery of new treatment strategies. The standard of care for MRSA infections is systemic antibiotic therapy, but MRSA has become increasingly resistant to antibiotics over time3 and these drugs can diminish the host&#39;s beneficial microbiome, causing negative health effects, especially in children4. Preclinical studies have examined alternative strategies to treat MRSA infections5, but translating these approaches to the clinic has proved challenging due to emergence of virulence factors that thwart host immune responses6. To dissect the host-pathogen dynamics that drive S. aureus SSTIs, we combine noninvasive and longitudinal readouts of the number of neutrophils recruited to the wound bed with kinetic measures of bacterial abundance and wound area.
Neutrophils are the most abundant circulating leukocyte in humans and the first responders to a bacterial infection7. Neutrophils are a necessary component for an effective host response against S. aureus infections due to their bactericidal mechanisms, including production of reactive oxygen species, proteases, antimicrobial peptides and functional responses including phagocytosis and neutrophil extracellular trap production8,9. Human patients with genetic defects in neutrophil function, such as chronic granulomatous disease and Chediak-Higashi syndrome, show an increased susceptibility to S. aureus infection. In addition, patients with genetic (such as congenital neutropenia) and acquired (such as neutropenia seen in chemotherapy patients) defects in neutrophil numbers are also highly susceptible to S. aureus infection10. Given the importance of neutrophils in clearing S. aureus infections, enhancing their immune capacity or tuning their numbers within a S. aureus lesion may prove an effective strategy in resolving infection.
Over the past decade, transgenic mice with fluorescence neutrophil reporters have been developed to study their trafficking11,12. Combining neutrophil reporter mice with whole animal imaging techniques permits spatiotemporal analysis of neutrophils in tissues and organs. When combined with bioluminescent strains of S. aureus, it is possible to track the accumulation of neutrophils in response to S. aureus abundance and persistence in the context of bacterial virulence factors that directly and indirectly perturb neutrophil numbers in affected tissue13,14,15,16.
Mice are less susceptible to S. aureus virulence and immune evasion mechanisms than humans. As such, wild-type mice may not be an ideal animal model to investigate the efficacy of a given therapeutic to treat chronic S. aureus infection. MyD88-deficient mice (i.e., MyD88-/- mice), an immunocompromised mouse strain that lacks functional interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling, show greater susceptibility to S. aureus infection compared to wild-type mice17 and an impairment in neutrophil trafficking to a site of S. aureus infection in the skin18. Development of a mouse strain that possesses a fluorescent neutrophil reporter in MyD88-/- mice has provided an alternative model for investigating the efficacy of therapies to treat S. aureus infection compared to current neutrophil reporter mice.
In this protocol, we characterize S. aureus infection in the immunocompromised LysM-EGFP&#215;MyD88-/- mice, and compare the time course and resolution of infection with the LysM-EGFP mice. LysM-EGFP&#215;MyD88-/- mice develop a chronic infection that does not resolve, and 75% succumb to infection after 8 days. A significant defect in initial neutrophil recruitment occurs over 72 h of the inflammatory phase of infection, and 50% fewer neutrophils recruit during the latter stage of infection. The increased susceptibility of the LysM-EGFP&#215;MyD88-/- mice makes this particular strain a rigorous preclinical model to evaluate the efficacy of new therapeutic techniques targeting S. aureus infection compared to current models that utilize wild-type mice, especially techniques aiming to boost the innate immune response against infection.

<table><tbody><tr><td>14 mL Polypropylene Round-Bottom Tube</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>6 mm Disposable Biopsy Punch</td><td>Integra Miltex</td><td>33-36</td><td></td></tr><tr><td>Bioluminescent &lt;em&gt;S. aureus&lt;/em&gt;</td><td>Lloyd Miller, Johns Hopkins&amp;nbsp;</td><td>ALC 2906 SH1000</td><td></td></tr><tr><td>Bovine Blood Agar, 5%, Hardy Diagnostics</td><td>VWR</td><td>10118-938</td><td></td></tr><tr><td>Buprenoprhine hydrochloride injectable</td><td>Western Medical Supply</td><td>7292</td><td>0.3 mg/mL</td></tr><tr><td>C57BL/6J Mice</td><td>Jackson Labratory</td><td>000664</td><td></td></tr><tr><td>Chloramphenicol (crystalline powder)</td><td>Fisher BioReagents</td><td>BP904-100</td><td></td></tr><tr><td>DPBS (1x)</td><td>Gibco&amp;nbsp;</td><td>14190-144</td><td></td></tr><tr><td>Insulin Syringes</td><td>Becton, Dickson and Company</td><td>329461</td><td>0.35 mm (28 G) x 12.7 mm (1/2&#039;&#039;)</td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>Perkin Elmer</td><td>124262</td><td></td></tr><tr><td>Living Image Software &amp;ndash; IVIS Spectrum Series</td><td>Perkin Elmer</td><td>128113</td><td></td></tr><tr><td>LysM-eGFP Mice</td><td>Thomas Graff Albert Einstein College of New York&amp;nbsp;</td><td>NA</td><td></td></tr><tr><td>Microvolume Spectrophotometer</td><td>ThermoFisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>MyD88 KO Mice</td><td>Jackson Labratory</td><td>009088</td><td></td></tr><tr><td>Non-woven sponges</td><td>AMD- Ritmed Inc</td><td>A2101-CH</td><td>5 cm x 5 cm</td></tr><tr><td>Povidone Iodine 10% Solution</td><td>Aplicare</td><td>697731</td><td></td></tr><tr><td>Prism 7.0</td><td>GraphPad Software</td><td>License&amp;nbsp;</td><td></td></tr><tr><td>Tryptic Soy Broth</td><td>Becton, Dickson and Company</td><td>211825</td><td></td></tr></tbody></table>

a mouse model to assess innate immune response to staphylococcus aureus infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

LysM-EGFP&#215;MyD88-/- mice are more susceptible to S. aureus infection than LysM-EGFP mice
The strain of S. aureus used in this study, ALC290618, was constructed with a plasmid that contains the lux construct that produces bioluminescent signals from live and actively metabolizing bacteria. When inoculated into mice, in vivo bioluminescence imaging (BLI) techn...

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A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

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Research

JoVE Journal

Immunology and Infection

11.5K Views.  University of California Davis. Technieken die de bacterielast en een neutrofiele tegelijkertijd monitoren, kunnen inzicht geven in de mechanismen van Staph aureus persistentie en de werkzaamheid van behandelingsstrategieën. Het belangrijkste voordeel van deze techniek is dat meerdere parameters in de lengterichting kunnen worden gemeten in een live host gedurende de duur van de infectie. Het is van cruciaal belang om te laten zien waar de wond op een muis dorsum moet worden gezet, hoe de huid moet worden verwijderd en hoe...

Video: A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

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

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

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.

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

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

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

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

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

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

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Optimized In Vivo Models for Intracellular S. aureus Infection Studies

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

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