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 PAO1:lux strain of P. aeruginosa is available from our lab by request. Streak PAO1, stored as frozen glycerol stock, on Lysogeny Broth (LB) agar. For the luminescent PAO1:lux strain, LB agar should contain selective antibiotics (100 &#181;g/mL carbenicillin and 12.5 &#181;g/mL kanamycin). Grow at 37 &#176;C overnight in a bacterial incubator.</li>
	<li>Pick an isolated colony and grow overnight at 37 &#176;C in 3 ml LB medium, pH 7.4. For luminescent strains, broth should contain 100 &#181;g/mL carbenicillin. Grow under shaking, aerobic conditions.</li>
</ol>
2. Procedure preparation
<ol>
	<li>Have all personnel performing surgery wear a clean gown/Lab coat, face mask, hair net, and gloves.</li>
	<li>Autoclave all surgical tools, including scissors and forceps. Use aseptic technique to sterilize tools between animals.</li>
	<li>Clean the surgical table with ethanol and prepare a clean surgical field.</li>
</ol>
3. Hair removal
<ol>
	<li>Anesthetize 8&#8211;12 week old C57BL/6J mice using 1%&#8211;3% isoflurane. Investigators should follow their institution&#39;s veterinary staff guidelines for anesthesia when using isoflurane.
	<ol>
		<li>Start anesthesia by delivering 1%&#8211;3% isoflurane and adjust oxygen flow rate to 1.5 L/min. Place the mouse in the induction chamber.</li>
		<li>Pinch the mouse&#39;s toe to assess depth of anesthesia. When the mouse no longer responds to stimulation, remove it from the induction chamber and place it on the surgical bench with its nose in the isoflurane nose cone.</li>
		<li>Apply ocular lubricant to both eyes.</li>
	</ol>
	</li>
	<li>Weigh the mouse to obtain a baseline pre-procedure weight.</li>
	<li>Place the mouse in prone position. Inject the mouse subcutaneously with pre-warmed sterile 0.9% sodium chloride, 250 &#181;L at each flank for a total of 500 &#181;L.</li>
	<li>Shave the dorsal area of the mouse using an electric shaver. Shaving should occur at a different location than the surgical station to prevent hair contamination of the wound.</li>
	<li>Apply a thin layer of hair removal lotion. Let the lotion sit for 20&#8211;60 s. Remove the hair and excess lotion with gauze moistened in warm water. Following hair removal, proceed to the excisional wounding procedure.</li>
</ol>
4. Full thickness excisional wound surgery
<ol>
	<li>Inject sustained release buprenorphine 0.6&#8211;1 mg/kg subcutaneously using a 25 G needle at the mid-dorsal area of the mouse. Slow release buprenorphine provides pain relief over 48&#8211;72 h.</li>
	<li>Disinfect the surgical site. Wipe the dorsal surface with a sterile betadine swab. Wipe excess betadine with a sterile alcohol swab. This should be performed 3 times (alternating between betadine and alcohol), swabbing by moving from the center in a circular manner to the edge. Allow the area to air dry.</li>
	<li>Create a drape surrounding the surgical site using sterile gauze or plastic cling wrap.</li>
	<li>Stretch skin taut caudally. Use a sterile 6-mm diameter skin biopsy punch to make an initial incision through the left dorsal epidermis. Repeat on the right dorsal epidermis.</li>
	<li>Use forceps to tent the skin from the center of the left outlined wound area. Excise the epidermal and dermal layers using scissors. Repeat on the right outlined wound area to create symmetrical excisional wounds.</li>
	<li>Wash wounds with 50 &#181;L of sterile saline. Allow the surgical site and surrounding skin to air dry. Then, cover the wounds and dorsum with a transparent film dressing.</li>
	<li>Place the mouse back in a clean cage. House 1 animal per cage.</li>
	<li>Place the cage on a heating pad and monitor until the mouse wakes up.</li>
	<li>When performing the above surgery on multiple animals, use a hot bead sterilizer to clean all surgical instruments between animals.</li>
	<li>Allow 24 h for the mice to recover from the surgical procedure and for formation of a provisional wound matrix over the wounds prior to proceeding to inoculation with bacteria.</li>
</ol>
5. Inoculation with P. aeruginosa
<ol>
	<li>Dilute overnight PAO1:lux culture to OD600 = 0.05 in 75 mL of LB media containing 100 &#181;g/mL carbenicillin and grow the bacteria until the culture is in early exponential phase (OD600 &#8776; 0.3). This should take approximately 2&#8211;3 h.</li>
	<li>Dilute PAO1:lux in PBS to a concentration of (7.5 &#177; 2.5) x 102 CFU/mL. Be sure to prepare excess inoculum to ensure sufficient volume and to allow for plating after the experiment. If transporting between facilities (i.e. from the lab to the vivarium), use double containment in a leak proof box clearly marked Biohazard.</li>
	<li>Perform all work with P. aeruginosa and mice using approved personal protective equipment in an Animal Biosafety Level 2 (ABSL-2) approved biological safety cabinet (BSC). Reusable equipment such as the weighing scale should be covered with cling wrap to prevent contamination.</li>
	<li>Anesthetize using 3% isoflurane as described above. Weigh mouse and record the weight. Inject the mouse subcutaneously with pre-warmed sterile 0.9% sodium chloride, 250 &#181;L at each flank for a total of 500 &#181;L.</li>
	<li>If the mouse&#39;s transparent film dressing has come off overnight, remove any resulting scab carefully and put on a new dressing.</li>
	<li>Use a 500 &#181;L tuberculin 27 G safety cap syringe to inject 40 &#181;L of the PAO1:lux suspension through the transparent film dressing into each wound. Different mice should be used for non-inoculated/PBS wound controls in order to prevent cross-contamination from the contralateral side.</li>
	<li>Place the mouse back in its cage on a heating pad and monitor until it wakes up. All mice should be housed individually in separate cages to prevent cross-contamination.</li>
	<li>Provide high calorie nutritional supplement paste sandwiched between food pellets on the floor of the cage.</li>
	<li>Use the remaining inoculum to streak an LB agar plate. Count colonies to confirm the number of bacteria administered.</li>
</ol>
6. In vivo imaging of infected wounds
<ol>
	<li>Follow BSL-2 containment protocols for transport of mice to and from the imaging instrument, including use of a secondary container. Be careful not to transfer or drop any animal bedding during the transfer of the mouse to the induction chamber or imaging instrument.</li>
	<li>Induce anesthesia of the mouse with inhaled 1%&#8211;3% isoflurane in an induction chamber as described in step 3.1.</li>
	<li>Once the mouse is anesthetized, place it in prone position in the imaging chamber of an optical imaging system with the nose in the isoflurane nose cone.</li>
	<li>Open the software program.</li>
	<li>The acquisition parameters will vary based on the number of animals imaged simultaneously and intensity of bioluminescence. The basic parameters to set include exposure time, binning, f/stop, and field of view (FOV). Our default starting settings are exposure time 30 seconds, binning low (2), f/stop 1.2, and FOV 25. Adjust these settings as needed depending on the researcher&#39;s needs.</li>
	<li>Analyze luminescence data using an imaging program (see Table of Materials). Luminescence will be represented as a pseudocolor image overlaid on a color photograph of the mice.
	<ol>
		<li>Create a region of interest (ROI) at the wound site and measure the average flux (photons/second) detected. Note that data can also be reported as radiance (photons/second/cm&#178;/steradian), but as long as the distance of the imaging platform from the camera remains constant between imaging, flux is sufficient.</li>
		<li>Measure background by creating a ROI at a random area on the imaging platform. Subtract the background number of photons/second.</li>
		<li>Export the data to a spreadsheet for further analysis.</li>
	</ol>
	</li>
	<li>Perform imaging as described above as often as daily to track infection progression.</li>
</ol>
7. Postoperative management
<ol>
	<li>Monitor mice according to guidelines set by the researcher&#39;s IACUC protocol. We monitor all mice daily for the first 4 days, then every other day until the end of the experiment. Weigh the mice once per day for the first 4 days post-surgery in a BSC. Inject 250 &#181;L of 0.9% sodium chloride subcutaneously on post-infection days 1 and 2.</li>
	<li>Check for signs of pain/distress in the mice, including hunched posture, scruffy coat, lethargy, difficulty breathing, facial grimace, and weight loss.</li>
	<li>If animals display signs of deteriorating health, consult with a veterinarian. Any mouse that appears to show worsening signs of pain/distress and a weight loss of 20% or greater should be euthanized.</li>
</ol>
8. Wound excision
<ol>
	<li>At the end of the experiment, sacrifice the mice using CO2 inhalation, followed by cervical dislocation. Dispose of the animal carcass according to the institution&#39;s ABSL-2 protocols.</li>
	<li>Excise wound beds using sterile scissors and forceps in a BSC. Place each wound bed in 1 mL sterile PBS in a 1.5 mL polypropylene tube. Mince the wound tissue with scissors. All wounds should be treated as ASBL-2, even if they are not considered infected.</li>
	<li>Incubate on a shaker at 300 rpm for 2 h at 4 &#176;C. Vortex each tube for 10 s and serially dilute the bacterial effluent in PBS. Plate the diluted bacterial effluent on LB agar to enumerate the bacterial burden.</li>
	<li>Consider wounds infected if luminescent signal in the wound is above background luminescence and more bacteria are detected in the wound effluent than in wounds inoculated with PBS as control.</li>
</ol>

<ol>
	<li>Sen, C. K., 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=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair and Regeneration. 17 (6), 763-771 (2009).</li><li>Serra, R., 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=Chronic+wound+infections:+the+role+of+Pseudomonas+aeruginosa+and+Staphylococcus+aureus.">Chronic wound infections: the role of Pseudomonas aeruginosa and Staphylococcus aureus.</a> Expert Review of Anti-infective Therapy. 13 (5), 605-613 (2015).</li><li>Obritsch, M. D., Fish, D. N., MacLaren, R., Jung, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+infections+due+to+multidrug-resistant+Pseudomonas+aeruginosa:+epidemiology+and+treatment+options.">Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options.</a> Pharmacotherapy. 25 (10), 1353-1364 (2005).</li><li>Secor, P. R., 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=Filamentous+Bacteriophage+Produced+by+Pseudomonas+aeruginosa+Alters+the+Inflammatory+Response+and+Promotes+Noninvasive+Infection+In+Vivo.">Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.</a> Infection and Immunity. 85 (1), (2017).</li><li>Rice, S. A., 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=The+biofilm+life+cycle+and+virulence+of+Pseudomonas+aeruginosa+are+dependent+on+a+filamentous+prophage.">The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage.</a> The ISME Journal. 3 (3), 271-282 (2009).</li><li>Bayes, H. K., Ritchie, N., Irvine, S., Evans, T. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophosphamide-induced+neutropenia:+effect+on+postischemic+skin-flap+survival.">Cyclophosphamide-induced neutropenia: effect on postischemic skin-flap survival.</a> Plastic and Reconstructive Surgery. 89 (6), 1092-1097 (1992).</li><li>Sweere, J. 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=The+immune+response+to+Chronic+Pseudomonas+aeruginosa+wound+infection+in+immunocompetent+mice.">The immune response to Chronic Pseudomonas aeruginosa wound infection in immunocompetent mice.</a> Advances in Wound Care. , (2019).</li><li>Sweere, J. 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=Bacteriophage+trigger+antiviral+immunity+and+prevent+clearance+of+bacterial+infection.">Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.</a> Science. 363 (6434), (2019).</li></ol>

The authors have no competing financial interests to disclose.

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 ...

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Immunology and Infection

8.3K Views.  Stanford University. We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Video: A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

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.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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

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

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

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

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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.

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. ...

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 ...

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.

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

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...