The pUT-Tn5-EM7-lux-Km1 luminescent construct vector was a gracious gift from J. Hardy. Schematics were created with BioRender.com. We thank the lab of G. Gurtner for their advice on the wound infection model. We also thank T. Doyle from the Stanford Center for Innovation in In Vivo Imaging for his technical expertise. This work was supported by grants R21AI133370, R21AI133240, R01AI12492093, and grants from Stanford SPARK, the Falk Medical Research Trust and the Cystic Fibrosis Foundation (CFF) to P.L.B. C.R.D was supported by T32AI007502. A Gabilan Stanford Graduate Fellowship for Science and Engineering and a Lubert Stryer Bio-X Stanford Interdisciplinary Graduate Fellowship supported J.M.S.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University.
1. Preparation and growth of bacteria
<ol>
	<li>Conduct all work with P. aeruginosa and animals with BSL-2 precautions per the researcher&#39;s institutional biosafety committee and animal use committee guidelines. Do all steps described here involving P. aeruginosa, including mouse inoculation, in a biosafety cabinet.</li>
	<li>The luminescent PA...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+early+Pseudomonas+aeruginosa+lung+disease+with+transition+to+chronic+infection.">A murine model of early Pseudomonas aeruginosa lung disease with transition to chronic infection.</a> Scientific Reports. 6, 35838 (2016).</li><li>van Gennip, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+polymorphonuclear+leukocytes+and+Pseudomonas+aeruginosa+biofilms+on+silicone+implants+in+vivo.">Interactions between polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilms on silicone implants in vivo.</a> Infection and Immunity. 80 (8), 2601-2607 (2012).</li><li>Tr&#248;strup, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilm+aggravates+skin+inflammatory+response+in+BALB/c+mice+in+a+novel+chronic+wound+model.">Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model.</a> Wound Repair and Regeneration. 21 (2), 292-299 (2013).</li><li>Zhao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+study+of+delayed+wound+healing+in+a+biofilm-challenged+diabetic+mouse+model.">Time course study of delayed wound healing in a biofilm-challenged diabetic mouse model.</a> Wound Repair and Regeneration. 20 (3), 342-352 (2012).</li><li>Brubaker, A. L., Rendon, J. L., Ramirez, L., Choudhry, M. A., Kovacs, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+neutrophil+chemotaxis+and+infiltration+contributes+to+delayed+resolution+of+cutaneous+wound+infection+with+advanced+age.">Reduced neutrophil chemotaxis and infiltration contributes to delayed resolution of cutaneous wound infection with advanced age.</a> Journal of Immunolology. 190 (4), 1746-1757 (2013).</li><li>Watters, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilms+perturb+wound+resolution+and+antibiotic+tolerance+in+diabetic+mice.">Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.</a> Medical Microbiology and Immunology. 202 (2), 131-141 (2013).</li><li>Lee, C., Kerrigan, C. L., Picard-Ami, L. 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We have developed a novel delayed inoculation P. aeruginosa wound infection model. The strategy of delaying inoculation with bacteria until 24 h after excisional wounding enables the evaluation of wound infections over a 1-week timeframe. By using a luminescent strain of P. aeruginosa, it is possible to track infection progression throughout the infection course. The longer course of infection compared to other P. aeruginosa infection models will allow new opportunities for studying host-pathog...

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative rod-shaped bacterium with increasing relevance to human health and disease. It is responsible for extensive morbidity and mortality in nosocomial settings, particularly involving wound infections in immunocompromised patients1,2. The emergence of multidrug-resistant strains of this pathogen has provided further impetus for investigation into factors contributing to P. aeruginosa virulence, mechanisms of P. aeruginosa antibiotic resistance, and new methods for prevention and treatment of this deadly infection3. As such, the need for animal models of chronic wound infection as tools for investigating these research questions has never been greater.
Unfortunately, many animal models of P. aeruginosa infection tend to simulate acute infection with rapid resolution of infection or rapid decline due to sepsis4,5, which does not adequately simulate the oftentimes chronic nature of these infections. To address this drawback, some models utilize the implantation of foreign bodies such as agar beads, silicone implants, or alginate gels6,7,8. Other models use mice that are immunocompromised due to advanced age, obesity, or diabetes, or through pharmacological means such as cyclophosphamide-induced neutropenia9,10,11,12. However, either the use of foreign materials or immune compromised hosts likely alters the local inflammatory process, making it difficult to gain an understanding of the pathophysiology involved in chronic wound infections in hosts with otherwise normal immune systems.
We have developed a chronic model of P. aeruginosa wound infection in mice that involves delayed inoculation with bacteria after excisional wounding. Delayed inoculation allows for experiments assessing bacterial burden extending out to at least 7 days. This model opens up new opportunities for investigating both pathogenesis and new treatments of P. aeruginosa chronic infections.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride injection</td>
			<td>Hospira</td>
			<td>2484457</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G x 1 sterile needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G x 1 1/5 sterile needle</td>
			<td>BD</td>
			<td>305127</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swab</td>
			<td>BD</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Aura Imaging Software</td>
			<td>Spectral Instruments Imaging</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Frederick Company</td>
			<td>19-065534</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine SR LAB</td>
			<td>Zoopharm</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J male mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable biopsy punch, 6mm</td>
			<td>Integra</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors - Tungsten Carbide</td>
			<td>Fine Science Tools</td>
			<td>14568-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bead Dry Sterilizer</td>
			<td>Harvard Apparatus</td>
			<td>61-0183</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated Agar</td>
			<td>Fisher BioReagents</td>
			<td>BP9744</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Milliard</td>
			<td>804879481218</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe with 28 G needle</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Lago X Imaging System</td>
			<td>Spectral Instruments Imaging</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Fisher BioReagents</td>
			<td>BP1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Leur-Lok 1 mL syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Arco Animal Trimmer</td>
			<td>Wahl Professional</td>
			<td>919152</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair Hair Removal Lotion with Baby Oil</td>
			<td>Church and Dwight</td>
			<td>n/a</td>
			<td>Available at any pharmacy</td>
		</tr>
		<tr>
			<td>Octagon Forceps</td>
			<td>Fine Science Tools</td>
			<td>11041-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 1x</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Press and Seal Cling Wrap</td>
			<td>Glad</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>SafetyGlide Insulin syringe with 30 G needle</td>
			<td>BD</td>
			<td>305934</td>
			<td></td>
		</tr>
		<tr>
			<td>Safetyglide Insulin syringe, 1/2 mL, 30 G x 5/16 TW</td>
			<td>BD</td>
			<td>305934</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Ohaus Scout Pro</td>
			<td>SP202</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplical Nutritional Supplement</td>
			<td>Henry Schein Animal Health</td>
			<td>29908</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegaderm, 6 cm x 7 cm</td>
			<td>3M</td>
			<td>1624W</td>
			<td></td>
		</tr>
	</tbody>
</table>

a delayed inoculation model of chronic pseudomonas aeruginosa wound infection

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

Using a luminescent strain of PAO1 with a plasmid encoding the luxABCDE reporter system (PAO1:lux), we performed excisional wounding on mice, inoculated these wounds with planktonic P. aeruginosa 24 h later, and measured bacterial burden over time (Figure 1 and Figure 2). A representative image obtained using an imaging optical system demonstrates that this model results in detectable luminescence (F...

Watch this Scientific Journal Video about A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection at JoVE.com

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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

This protocol is significant because it describes a model for chronic Pseudomonas aeruginosa wound infection without the need for foreign material implantation or immune suppression. Delayed inoculation with Pseudomonas aeruginosa 24 hours after full thickness excisional wounding prostalls the rapid clearance and dissemination of the bacteria, establishing an infection that lasts seven to 10 days. This unique model will be a useful tool for the investigations of bacterial pathogenesis, host pathogen interactions, and the development of new therapies for chronic Pseudomonas aeruginosa wound infections.Assisting me in a demonstration of the procedure will be Dan Liu, a medical student from the Bollyky Laboratory. Before beginning the procedure, weigh an anesthetized eight to 12 week old mouse to obtain a baseline weight and place the mouse in the prone position. After confirming a lack of response to the pedal reflex, subcutaneously inject the mouse with 250 microliters of pre-warmed sterile 0.9%sodium chloride on each flank.Use an electric shaver to remove the hair from the dorsal area of the mouse and apply a thin layer of depilatory cream to the exposed skin. After 20 to 60 seconds, remove the hair and excess lotion with gauze moistened in warm water. For full thickness excisional wound surgery, first use a 25 gauge needle to subcutaneously inject 0.6 to one milligrams per kilogram of sustained release buprenorphine at the mid-dorsal area of the mouse and wipe the surgical site in a circular manner with three alternative sterile Betadine and alcohol swabs.Place a plastic cling-wrap drap over the surgical site and stretch the skin taut caudally. When the skin has dried, use a sterile six millimeter diameter skin biopsy punch to make an initial incision through the left dorsal epidermis, followed by a second incision on the right dorsal epidermis. Use forceps to tent the skin from the center of the left outlined wound area and use scissors to excise the epidermal and dermal layers.After repeating the incision on the right outlined wound area to create symmetrical excisional wounds, wash both wounds with 50 microliters of sterile saline. When the surgical site and surrounding skin have dried, cover the wounds and dorsum with a transparent film dressing and place the mouse in a clean cage on a heating pad with monitoring until full recumbency. 24 hours after the surgery, weigh the re-anesthetized mouse again and inject the animal subcutaneously with 250 microliters of pre-warmed sterile 0.9%sodium chloride in both flanks.Next, load 100 microliters of the luminescent P.aeruginosa strain suspension into a 500 microliter tuberculin safety cap syringe equipped with a 27 gauge needle and inject 40 microliters of bacteria suspension through the transparent film dressing into each wound. Ensure that the bacterial suspension is well mixed and that the transparent dressing is intact. Make sure that you puncture the transparent dressing only once with the needle bezel side up.Then return the mouse to its cage on a heating page with monitoring and provide high calorie nutritional supplement paste sandwiched between food pellets on the floor of the cage. Plate the remaining inoculum on an LB agar plate and count the colonies to confirm the number of bacteria that have been administered. For in vivo imaging of the infected wounds, follow bio safety level two containment protocols for transport of the mice to and from the imaging instrument and place the anesthetized mouse in the prone position within the imaging chamber.In the imaging software, set the exposure time, binning, f-stop, and field of view as appropriate for the experiment. Next, create a region of interest at the wound site and measure the average detected flux in photons per second. To measure the background, create a region of interest over a random area on the imaging platform and subtract the background number of photons per second.At the end of the experiment, in a bio safety cabinet, use sterile scissors and forceps to excise the wound beds and place each bed in one milliliter of sterile PBS in a 1.5 milliliter polypropylene tube. Mince the wound tissue with scissors and incubate the tissue pieces on a shaker at four degrees Celsius in 300 rotations per minute for two hours. At the end of the incubation, vortex each tube for 10 seconds before serially diluted the bacterial effluent in PBS and plating the diluted bacterial effluent on LB agar to quantify the bacterial burden.This representative image obtained using an imaging optical system demonstrates that this model results in detectable luminescence. In this experiment, the infection peaked at day three post inoculation and persisted seven days post inoculation based on both the bioluminescence and colony counts. Culture of the bacteria isolated from the wound further demonstrate that the quantified colony forming units per wound correlate with the detected luminescence.Though there is an initial rapid weight loss immediately after the injection, saline injections and supplemental nutrition can restore the mice to their initial weights. The calculated inoculation dose of which 50%of the wounds will become substantially infected has been determined to be about 7.7 times 10 to the second colony forming units per milliliter. Doses higher than one times 10 to the fourth colony forming units per milliliter result in a 100%infection rate.Various topical and systemic treatments can be applied after the wounding and inoculation procedures to investigate new potential therapies for Pseudomonas aeruginosa. All the work with Pseudomonas aeruginosa and infected mice should be conducted with BSL-2 safety precautions in accordance with the researcher's institutional bio safety and animal use committee guidelines.

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Immunology and Infection

8.3K vistas.  Stanford University. Este protocolo es significativo porque describe un modelo para la infección crónica de la herida de Pseudomonas aeruginosa sin necesidad de implantación de material extraño o supresión inmune. La inoculación tardía con Pseudomonas aeruginosa 24 horas después de la intoxicación por escisión de espesor completo prostales el rápido aclaramiento y diseminación de la bacteria, estableciendo una infección que dura de siete a 10 días. Este modelo único será una herramienta útil para...