Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Summary

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.

Abstract

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.

Introduction

Biofilms are complex communities of microorganisms embedded in a matrix of polymeric substances that have been highlighted as a contributing factor for the poor resolution of chronic wounds¹. The study of these highly organized, persistent microbial populations is particularly important for diabetic patients where poor circulation on the limbs and altered peripheral sensory mechanisms lead to undetected lesions². In the United States, it is estimated that 15% of diabetic patients will develop at least one ulcer during the course of their lives. This translates to an economic expenditure of around 28 billion dollars in treatment^3,4, not to mention the immensurable emotional and social burden. Understanding the factors that allow microbial communities to persist in the wound bed and the impact these biofilms have in the healing events is imperative to drive better care for affected patients and propel the development of new treatment approaches. Therefore, the establishment of reproducible and translatable in vivo models for exploring bacterial-host interactions is paramount.

Murine models have been successfully developed to study the impact of biofilms in chronic wounds. These models, however, often utilize haired species and evaluate biofilm clearance by plate counts for viable bacterial cells of excised tissue from sacrificed animals, making them time consuming and costly.

A biophotonic alternative to the end point sampling of animals in evaluating infection was first proposed by Contag et al. (1995)^5, who developed a method to capture luminescence from constitutively bioluminescent Salmonella typhimurium to measure antibiotic treatment efficacy. Other studies taking advantage of bioluminescence-emitting bacteria followed. For example, Rochetta et al. (2001)⁶ validated an infection model to study Escherichia coli thigh infections in mice by measuring luminescence using an intensified charge-coupled device and later, Kadurugamuwa et al. (2003)⁷ took advantage of the photon emitting properties of an engineered strain of Staphylococcus aureus to investigate the efficacy of several antibiotics in a catheter wound model in mice.

The method characterized here presents a straightforward protocol to induce diabetes in hairless mice, produce and inoculate wounds with pre-formed bioluminescent biofilms of P. aeruginosa, and conduct biophotonic monitoring of the infection using an in vivo imaging system. It offers a direct, rapid, in situ, non-invasive and quantitative process to evaluate biofilms in chronic wounds and in addition, allows for additional analysis such as microscopic imaging of the healing wounds, intermittent blood collection for cytokine measurements, and terminal tissue collection for histology.

Protocol

Animal experiments were approved by the Institutional Animal Care and Use Committee of Michigan State University.

1. Preparation of Occlusive Dressings and Silicone Spacers

1. Cut the transparent occlusive dressing to make squares approximately 1 cm x 1 cm with scissors.
2. Cut 10 mm circles on a 0.5 mm thick silicone sheet using a 10 mm biopsy punch. Center a 5 mm biopsy punch in the middle of the 10 mm circle and press firmly to create a hole to form a "donut"-like disc that will be used as a splint. 

2. Experimental Animals

1. Use 8 week old (22-26 g) male SKH-1 mice from a commercial breeder. Keep mice in standard conditions of 21 °C and a 12 h light-dark cycle with free access to food and water.
2. To induce diabetes, inject mice intraperitonially with a 13 mg/ml streptozotocin (STZ) solutionand 25% Glucose (250 µl per/ mouse) on 5 consecutive days.
   1. Make the STZ solution by diluting 65 µg of STZ in 5 µL of 100 mM Citric Acid, pH 4.5.
   2. Adjust the injected volume for each mouse to a final mass of 65 mg STZ per 1 kg of mouse body weight. Inject control mice with 100 mM Citric Acid solution pH 4.5 on the same days.
3. Confirm hyperglycemia by blood glucose monitoring with a glucometer 14 days after the last STZ injection. Diabetic mice may have polyuria and so their bedding may need to be changed more frequently to eliminate wetness and their weights should be monitored 3 times a week.

3. Biofilms

1. Biofilm preparation
   1. Grow colony biofilms⁸ to inoculate the wounds. Two days prior to surgery start an overnight culture of bioluminescent P. aeruginosa Xen 41 in tryptic soy broth (TSB) incubated at 37 °C and shaking at 200 rpm and sterilize polycarbonate membrane filters with 0.2 µm pore size by exposure to UV light in a biological safety hood for 15 min per side.
   2. One day prior to surgery centrifuge overnight culture at 20,000 x g for 2 min and wash 3 times with 1 mL of Dulbelcco's phosphate buffered saline (DPBS) by pipetting up and down.
   3. Dilute suspension in DPBS to an absorbance of 0.05 at 600 nm.
   4. Pipette 10 µL of the diluted culture on each membrane resting on a tryptic soy agar (TSA) plate. After allowing to dry, incubate the membranes at 35 °C for 72 h to grow biofilms transferring to fresh TSA plates every 24 h.
2. Standard curve
   1. Prior to the start of the experiment make a standard curve to correlate bioluminescence and bacterial counts.
   2. Prepare biofilms as described in 3.1.
   3. Make serial dilutions of biofilms ranging from ½ to 1/24 by mixing biofilm with DPBS and vortexing until a visually homogeneous solution is produced. Pipette 200µL of the diluted solutions in a black 96-well plate and image with the in vivo imaging system.
   4. Spread plate dilutions onto TSA plates and incubate at 35 °C for 24 h.
   5. Count colony forming units (CFU) on plates and create a standard curve to correlate bioluminescence and bacterial counts.

4. Wound Surgery

1. Induce general anesthesia using isoflurane in 95% oxygen/5% CO₂ (to prevent death from ketoacidosis) at a flow rate of 1 L/min and maintain anesthesia using 1-3% isoflurane. Maintain animals on heat mats during surgery.
2. Ensure the deep pedal reflexes of the mouse are suppressed by pinching the foot with tweezers and place the mouse in the prone position.
3. Administer meloxicam (0.2 mg/kg) via sub-cutaneous injection (30 μl) for pain management.
4. Wipe the skin of the back with 10% povidone-iodine three times and an isopropanol pad.
5. Use a sterile 4 mm biopsy punch to outline one circular pattern for the wound on one side of the mouse's midline at the level of the shoulders. Outline the pattern with a permanent marker.
6. Use serrated forceps to lift the skin in the middle of the outline and iris scissors to create a full-thickness wound that extends through the subcutaneous tissue including the panniculus carnosus and excise the circular piece of tissue.
7. Apply a medical waterproof skin adhesive glue to the skin of the mice and position the silicone splint applying mild pressure. Cover the wound with a transparent occlusive dressing. After surgery, individually cage the animals.

5. Postoperative Management

1. Administer meloxicam (0.2 mg/kg) once daily via sub-cutaneous injection for post-operative pain relief for the next 2 days.
2. Monitor animals daily for manifestations of pain and weight loss. Diabetic animals need insulin injections when they have lost 15% or more of the body weight.

6. Biofilm Inoculum Preparation and Infection

1. Inoculate mice 48 h after surgery as described in the following steps.
2. Scrape 72 h biofilms from the membranes using a sterile spatula, place it in a microcentrifuge tube and dilute 1:2 in DPBS. Mix by briefly pipetting up and down.
3. Spread plate inoculum onto TSA plates to calculate total CFU. To ensure that counts are accurate, break down the biofilm inoculum further by a series of two 1 min vortexing steps intercalated by a 2 min sonication step at 40 kHz in an ultrasonic cleaner.
4. Remove dressing covering the wound and silicone splint and take a micrograph of the wound with a microscope with attached camera using a ruler for reference.
5. Cut the tips of 200 µL pipette tips and pipette 10 µL of the biofilm inoculum onto each wound.
6. Image mouse using the in vivo imaging system using auto settings: exposure time 5-300 s, with medium binning, 1 f/stop and open filter, and field of view C (12.9 cm x 12.9 cm).
7. Cover wound with fresh dressing.

7. Wound Measurement and Imaging

1. Evaluate the clinical signs of animals daily⁹.
2. Provide food, water and change cages as needed.
3. Check integrity of dressings daily. When the dressing is present, only bioluminescence can be measured due to occlusion of the wound. At day 8, dressings are removed and not replaced allowing measurement of wound closure.
4. Weigh animals every other day.
5. For all days, place mice individually in an isolation chamber equipped with a HEPA filter and image using the in vivo imaging system daily or every other day until luminescence values fall below background level.
6. After day 8, induce general anesthesia and take micrographs of the wound with a microscope with attached camera using a ruler for reference every other day until wounds are healed.

8. Histological Analysis

1. Euthanize the mice with a flow of 2 l/min of CO₂ in an euthanasia chamber after luminescence falls below background levels and the wounds are completely healed. Confirm death by cervical dislocation as a second method of euthanasia.
2. Use iris scissors to create a wide, full excision around and under the wound area (around 1 cm in diameter) and preserve the tissue in 4% paraformaldehyde for histological analysis.
3. Other analysis: Cytokine detection
   1. Collect blood retro-orbitally from animals under anesthesia using a capillary glass tube and transfer it to EDTA-treated tubes.
   2. Centrifuge blood at 2,000 rpm for 20 min at 4 °C and freeze plasma for cytokine detection.

Results

In developing this new model, we observed many advantages in utilizing hairless SKH-1 over C57BL/6J mice, which we have used in the past. Animals subjected to STZ injections normally experience gradual weight loss with the onset of diabetes; however, in wound healing experiments previously conducted by our laboratories reproducing the model presented by Dunn et al. (2012)⁹ using C57BL/6J, drastic weight loss was observed (Figure 1

Discussion

Here we describe a new mouse model for the study of biofilms in diabetic chronic wounds that has many advantages to create a reproducible, translatable, and flexible model.

The first innovation is the use of hairless mice. Other mouse models have been developed to study diabetic chronic wound healing^10,11, but all have relied on the use of haired mice requiring the removal of fur by processes that involve either waxing or hair clipping...

Materials

Name	Company	Catalog Number	Comments
Opsite	Smith & Nephew	Model 66000041	Smith & Nephew Flexfix Opsite Transparent Adhesive Film Roll 4" x 11yards
SKH-1 mice Crl:SKH1-Hrhr	Charles River Breeding Laboratories	SKH1	Hairless mice, 8 weeks old
Streptozotocin (STZ)	Sigma Aldrich	S0130-1G	Streptozocin powder, 1g
AccuChek glucometer	Accu-Chek Roche	Art No. 05046025001	ACCU-CHEK CompactPlus Diabetes Monitoring Care Kit
Pseudomonas aeruginosa Xen 41	Perkin Elmer	119229	Bioluminescent Pseudomonas aeruginosa
Polycarbonate membrane filters	Sigma Aldrich	P9199	Millipore polycarbonate membrane filters with 0.2 μm pore size
Dulbelcco phosphate buffer saline (DPBS)	Sigma Aldrich	D8537	PBS
Tryptic soy agar	Sigma Aldrich	22091	Culture agar
Meloxicam	Henry Schein Animal Health	49755	Eloxiject (Meloxicam) 5mg/mL, solution for injection
10% povidone-iodine (Betadine)	Purdue Products LP	301879-OA	Swabstick, Betadine Solution. Antiseptic. Individ. Wrapped, 200/case
4% paraformaldehyde	Fisher Scientific	AAJ61899AK	Alfa Aesar Paraformaldehyde, 4% in PBS
Capillary glass tube	Fisher Scientific	22-362-566	Heparinized Micro-Hematocrit Capillary Tubes
Silicone to make splints	Invitrogen Life Technologies Corp	P-18178	Press-to-Seal Silicone Sheet, 13cm x 18cm, 0.5mm thick, set of 5 sheets
Tryptic soy broth	Sigma Aldrich	22092	Culture broth
IVIS Spectrum	Perkin Elmer	124262	In vivo imaging system
IVIS Spectrum Isolation chamber	Perkin Elmer	123997	XIC-3 animal isolation chamber
HEPA filter	Teleflex	28022	Gibeck ISO-Gard HEPA Light number 28022
Biopsy punches	VWR International Inc	21909-142	Disposable Biopsy Punch, 5mm, Sterile, pack of 50.
Biopsy punches	VWR International Inc	21909-140	Disposable Biopsy Punch, 4mm, Sterile, pack of 50.
Glucose	J.T.Baker	1916-01	Dextrose, Anhydrous, Powder
Citric acid	Sigma Aldrich	C2404-100G	Citric Acid
Mastisol	Eloquest Healthcare	HRI 0496-0523-48	Mastisol Medical Liquid Adhesive 2/3 mL vial, box of 48
Corning 96-well black plates	Fisher Scientific	07-200-567	96-well clear bottom black polysterene microplates
25 gauge 5/8 inch needle	BD	305122	Regular bevel needle
Bransonic M Ultrasonic Cleaning Bath	Branson Ultrasonics	N/A	Ultrasonic Cleaner