A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Summary

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

Abstract

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 7–10 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.

Introduction

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 patients^1,2. 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 sepsis^4,5, 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 gels^6,7,8. Other models use mice that are immunocompromised due to advanced age, obesity, or diabetes, or through pharmacological means such as cyclophosphamide-induced neutropenia^9,10,11,12. 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.

Protocol

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

1. Conduct all work with P. aeruginosa and animals with BSL-2 precautions per the researcher's institutional biosafety committee and animal use committee guidelines. Do all steps described here involving P. aeruginosa, including mouse inoculation, in a biosafety cabinet.
2. 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 µg/mL carbenicillin and 12.5 µg/mL kanamycin). Grow at 37 °C overnight in a bacterial incubator.
3. Pick an isolated colony and grow overnight at 37 °C in 3 ml LB medium, pH 7.4. For luminescent strains, broth should contain 100 µg/mL carbenicillin. Grow under shaking, aerobic conditions.

2. Procedure preparation

1. Have all personnel performing surgery wear a clean gown/Lab coat, face mask, hair net, and gloves.
2. Autoclave all surgical tools, including scissors and forceps. Use aseptic technique to sterilize tools between animals.
3. Clean the surgical table with ethanol and prepare a clean surgical field.

3. Hair removal

1. Anesthetize 8–12 week old C57BL/6J mice using 1%–3% isoflurane. Investigators should follow their institution's veterinary staff guidelines for anesthesia when using isoflurane.
   1. Start anesthesia by delivering 1%–3% isoflurane and adjust oxygen flow rate to 1.5 L/min. Place the mouse in the induction chamber.
   2. Pinch the mouse'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.
   3. Apply ocular lubricant to both eyes.
2. Weigh the mouse to obtain a baseline pre-procedure weight.
3. Place the mouse in prone position. Inject the mouse subcutaneously with pre-warmed sterile 0.9% sodium chloride, 250 µL at each flank for a total of 500 µL.
4. 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.
5. Apply a thin layer of hair removal lotion. Let the lotion sit for 20–60 s. Remove the hair and excess lotion with gauze moistened in warm water. Following hair removal, proceed to the excisional wounding procedure.

4. Full thickness excisional wound surgery

1. Inject sustained release buprenorphine 0.6–1 mg/kg subcutaneously using a 25 G needle at the mid-dorsal area of the mouse. Slow release buprenorphine provides pain relief over 48–72 h.
2. 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.
3. Create a drape surrounding the surgical site using sterile gauze or plastic cling wrap.
4. 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.
5. 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.
6. Wash wounds with 50 µL of sterile saline. Allow the surgical site and surrounding skin to air dry. Then, cover the wounds and dorsum with a transparent film dressing.
7. Place the mouse back in a clean cage. House 1 animal per cage.
8. Place the cage on a heating pad and monitor until the mouse wakes up.
9. When performing the above surgery on multiple animals, use a hot bead sterilizer to clean all surgical instruments between animals.
10. 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.

5. Inoculation with P. aeruginosa

1. Dilute overnight PAO1:lux culture to OD₆₀₀ = 0.05 in 75 mL of LB media containing 100 µg/mL carbenicillin and grow the bacteria until the culture is in early exponential phase (OD₆₀₀ ≈ 0.3). This should take approximately 2–3 h.
2. Dilute PAO1:lux in PBS to a concentration of (7.5 ± 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.
3. 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.
4. 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 µL at each flank for a total of 500 µL.
5. If the mouse's transparent film dressing has come off overnight, remove any resulting scab carefully and put on a new dressing.
6. Use a 500 µL tuberculin 27 G safety cap syringe to inject 40 µL 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.
7. 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.
8. Provide high calorie nutritional supplement paste sandwiched between food pellets on the floor of the cage.
9. Use the remaining inoculum to streak an LB agar plate. Count colonies to confirm the number of bacteria administered.

6. In vivo imaging of infected wounds

1. 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.
2. Induce anesthesia of the mouse with inhaled 1%–3% isoflurane in an induction chamber as described in step 3.1.
3. 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.
4. Open the software program.
5. 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's needs.
6. 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.
   1. 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/cm²/steradian), but as long as the distance of the imaging platform from the camera remains constant between imaging, flux is sufficient.
   2. Measure background by creating a ROI at a random area on the imaging platform. Subtract the background number of photons/second.
   3. Export the data to a spreadsheet for further analysis.
7. Perform imaging as described above as often as daily to track infection progression.

7. Postoperative management

1. Monitor mice according to guidelines set by the researcher'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 µL of 0.9% sodium chloride subcutaneously on post-infection days 1 and 2.
2. Check for signs of pain/distress in the mice, including hunched posture, scruffy coat, lethargy, difficulty breathing, facial grimace, and weight loss.
3. 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.

8. Wound excision

1. 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's ABSL-2 protocols.
2. 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.
3. Incubate on a shaker at 300 rpm for 2 h at 4 °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.
4. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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