Assessing Biofilm Dispersal in Murine Wounds

Summary

Here, we describe ex vivo and in vivo methods for assessing bacterial dispersal from a wound infection in mice. This protocol can be utilized to test the efficacy of topical antimicrobial and anti-biofilm therapies, or to assess the dispersal capacity of different bacterial strains or species.

Abstract

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Introduction

The rise of antibiotic resistance worldwide is leading to a lack of antibiotic options to treat a variety of bacterial infections¹. In addition to antibiotic resistance, bacteria can gain antibiotic tolerance by adopting a biofilm-associated lifestyle². A biofilm is a community of microorganisms that are protected by a matrix of polysaccharides, extracellular DNA, lipids, and proteins³, collectively called the extracellular polymeric substance (EPS). As the antibiotic resistance crisis continues, new strategies that prolong the use of, or potentiate the efficacy of, antibiotics are sorely needed. Anti-biofilm agents are one promising solution⁴.

Amongst the different anti-biofilm strategies that have been proposed, the utilization of dispersal agents, which target different components of the biofilm EPS, are at the forefront of therapeutic development⁵. Glycoside hydrolases (GH) are one such class of dispersal agent. GH are a large class of enzymes that catalyze the cleavage of different bonds within the polysaccharides that provide structural integrity to the EPS. Our group, as well as others, have shown that GH can effectively degrade biofilms, induce dispersal and improve antibiotic efficacy for a number of different bacterial species, both in vitro and in vivo^{6,7,8,9,10,11}.

With a growing interest in biofilm dispersal,it is important to develop effective methods that assess dispersal efficacy. Here, we present a detailed protocol for the treatment of biofilm-associated wound infections with a dispersal agent in mice, and the assessment of dispersal efficacy, in vivo and ex vivo. The overall goal is to provide effective methods that can be used with preclinical models to measure biofilm dispersal effectively and efficiently.

A murine surgical excision infection model was used in these studies to establish a biofilm-associated infection. We have used this model for over 15 years and published our observations extensively^{7,9,12,13,14,15,16,17,18,19,20,21}. In general, this is a non-lethal infection model where bacteria remain localized to the wound bed and are biofilm-associated (bacteria seen in aggregates surrounded by EPS), setting up a chronic infection that lasts up to 3 weeks. However, if mice are immunocompromised (with Type 1 diabetes for example), they can become more susceptible to developing a fatal systemic infection in this model.

In this report, we provide protocols for assessing the dispersal of bacteria from a wound, both in vivo and ex vivo. Both protocols can be used to examine the efficacy of a dispersal agent and have their own strengths and weaknesses. For example, assessing dispersal in vivo can provide important, real-time information about the spread of bacteria to other parts of the body after dispersal, and how the host responds. On the other hand, assessing dispersal ex vivo may be more desirable for screening multiple agents, doses, or formulations, as the tissue can be divided into multiple sections that can be tested separately, thus reducing the number of mice required. When assessing multiple agents, we typically measure dispersal first in vitro as previously described ^6,9,22. We then test the most effective ex vivo and reserve in vivo testing for a limited number of very promising agents.

Protocol

This animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center (protocol number 07044). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

1. Preparing bacteria for mouse infections

NOTE: Here we describe infecting mice only with Pseudomonas aeruginosa. However, other bacterial species may be used to cause infection. Bacterial strains and materials are detailed in the Table of Materials.

1. Use Pseudomonas aeruginosa wild-type strain PAO1 carrying the luminescence plasmid pQF50-lux²³ for these experiments as previously described⁷.
2. Grow P. aeruginosa strains in baffled Erlenmeyer flasks, with shaking at 200 rpm, in Luria-Bertani (LB) broth at 37 °C for 16 to 20 h. Add a final concentration of 100 µg/mL of ampicillin to the overnight culture of PAO1(pQF50-lux) for maintenance of the plasmid.
3. Subculture P. aeruginosa by adding 100 µL of the overnight culture to an Erlenmeyer flask containing 10 mL of LB broth with a final concentration of 100 µg/mL ampicillin. Then grow for 2-2.5 h at 37 °C with shaking, to an OD_{600 nm} of 0.4, and then serially dilute (1:10) three times for an infecting dose of approximately 10⁴ cells.

2. Preparation of biofilm dispersal enzyme

NOTE: For this study we use a 10% solution of equal parts amylase and cellulase (5% of each), which will be referred to as "GH", for the dispersal treatment.

1. Use a 10% GH solution (w/v) of amylase (from Bacillus subtilis) and fungal cellulase (from Aspergillus niger) diluted in 1x phosphate buffer saline (PBS). Use PBS as the vehicle control treatment.
2. Warm all treatments to 37 °C for 30 min for enzyme activation.

3. Experimental animals and preoperative setup

1. House mice, 5 litter mates per cage, in environment-controlled conditions, with 12 h light/dark cycles and proper access to food and water.
2. Preferably, use female Swiss Webster mice, between 8 and 10 weeks old.
3. Weigh mice to determine the proper amount of anesthesia to administer.
4. Administer anesthesia by the intraperitoneal injection of 100 mg/kg ketamine 10 mg/kg xylazine.
5. Apply eye cream (e.g., Refresh P.M.) carefully onto the eye using a cotton swab to reduce dryness.
6. Monitor physiologic parameters including respiratory rate, skin coloration, and body temperature throughout the surgery.

4. Dorsal full thickness excision surgery

1. Assess surgical plane of anesthesia by toe pinch and monitoring of respiratory rate and effort. Shave the dorsal surface and apply a depilatory cream for 10 min (or as per product's instructions), after which gently remove, ensuring the skin was dry.
2. For pain management, administer 0.05 mL of lidocaine subcutaneously into the area to be excised, and inject 0.02 mL of buprenorphine subcutaneously into the scruff of the neck. Wait 10 min to ensure pain management was reached.
3. Before excision, disinfect the dorsal surface using an alcohol swab and draw a 1.5-cm diameter circle towards the posterior portion of the back. Maintain a sterile surgical field throughout the procedure and used autoclaved instruments and sterile gloves. To limit contamination, conduct the surgery by a lit Bunsen burner and use clean tools for each animal.
4. Administer a full-thickness, excisional skin wound to the level of panniculus muscle with surgical scissors.
5. After excision, immediately cover wounds with a semipermeable polyurethane dressing.
6. Inject approximately 10⁴ CFU of bacteria in 100 µL PBS under the dressing, onto the wound bed, to establish an infection.
   NOTE: Wounds can also be infected with multiple species of bacteria and/or fungi simultaneously, or by placing pre-formed biofilms¹², or even debridement tissue extracted from patients¹⁹, onto the wound bed.
7. After infection, place mice into cage with heating pads and monitor until they regained their righting reflex.
   NOTE: Ensure that mice do not get cold as this can lead to death.
8. Establish wound infections for 48 h.
9. Inspect adhesive dressings daily for tears and areas of non-adherence and replaced if needed.

5. In vivo dispersal treatment

NOTE: Here we describe administration of the dispersal agents by applying a series of 3 topical wound irrigation solutions (Figure 1). However, the protocol can be adapted for other types of delivery such as the application of gels, creams or dressings.

1. Place mice under isoflurane anesthesia (at 3% and 1 L per minute of oxygen) while administering the treatments.
2. Prepare three 1 mL syringes, with 25 G needles, containing 200 µL of treatment for each mouse.
3. Inject 200 µL of enzyme solution or PBS control onto the wound by carefully lifting and pinching the skin with forceps, slightly above the wound toward the head of the animal. Carefully pierce the syringe through the lifted skin and slowly inject the solution between the dressing and the wound bed. The dressing utilized in this study often adheres to both the wound bed and the surrounding tissue.
   1. To ensure that the entire wound bed is covered by solution, gently raise the dressing with forceps, pulling it away from the wound bed, while slowly injecting the solution.
4. After treatment, place mice back in their cages for 30 min.
5. After 30 min of exposure to the treatment, re-anesthetize the mice with isoflurane and aspirate the solution with a syringe, making sure to puncture in the same area as before.
   NOTE: This aspirated solution contains dispersed bacterial cells, which may be saved for various downstream analyses.
6. Administer the second and third irrigation treatments as the first, using a new syringe each time.

6. In vivo dispersal imaging and analysis

NOTE: If a luminescent strain of bacteria is utilized to initiate infection, an In Vivo Imaging System (IVIS) can be used to visualize dispersal from the wound bed.

1. Image mice immediately before and after treatment and at 10 and 20 h post-treatment (Figure 2 and Supplemental Figure 1).
2. Perform imaging while mice are under anesthesia by placing them individually in the IVIS and imaging each one for 5 s at a wavelength of 560 nm. Save images for further analysis.
   NOTE: Optimal wavelength and exposure time will depend on the reporter used and the IVIS instrument and imaging software.
3. For analysis, open images in the IVIS software and select the area to be analyzed in the Tool Palette.
4. To account for background, place a circle on the background of a photo and then place the same sized circle on top of the wound bed for each mouse.
5. To upload measurement values, select View -> Region of Interest (ROI) measurements and upload the table of measurements to a spreadsheet. Subtract the background circle reading from each of the wound bed measurements to calculate luminescence for each sample. Calculate percent luminescence changed by:
   ROI pre-treatment-ROI post-treatment/ ROI pre-treatment) x 100.
   ​NOTE: Control and treated mice should be analyzed simultaneously to normalize for variables that may affect image acquisition or radiance.

7. Assessing dispersal by determining CFU

1. Humanely euthanize mice by the intraperitoneal injection of 0.2 mL of pentobarbital sodium (e.g., Fatal Plus) under anesthesia with 100 mg/kg ketamine 10 mg/kg xylazine.
2. Harvest wound bed tissue by first gently removing the dressing with sterile forceps, and then cut around the perimeter of the wound bed with sterile surgical scissors. Gently lift one end of the wound bed with forceps while using scissors to cut/tease the sample away from the muscle layer.
3. Place tissue from the wound bed in pre-labeled and pre-weighed 2 mL homogenization tube containing 1 mL of PBS. Weigh the tubes again after inserting the samples, and then gently invert 3-5 times to rinse off any dispersed or loosely adhered bacteria, and remove the PBS. Add 1 mL of fresh PBS.
4. In order to harvest the spleens, place mice in a supine anatomical position and secure by pinning their extremities to a dissection surface. Soak the area to be dissected with 70% ethanol in order to disinfect the area and keep the fur from contaminating the sample.
5. Carefully make a small incision perpendicular to the midline with surgical scissors in the dermis of the lower abdomen, making sure to pull the skin up and away from the internal organs. Continue the incision interiorly, along the mid-line, until the sternum was reached. Once the peritoneal cavity was exposed and the internal organs were visible, gently separate the spleen from the connective tissue and place in a separate homogenization tube.
6. Weigh spleen and rinse with PBS, as described for the wound bed tissue.
   NOTE: Other organs of interest can be similarly harvested.
   1. When making the initial incision, take care to avoid puncturing the intestines, or other organs.
7. Homogenize samples in 2 mL pre-filled bead mill tubes using a benchtop homogenizer at 5 m/s for 60 s, twice.
8. To enumerate CFU, serially dilute samples and spot-plate on selective agar. For serial dilution, pipette 100 µL of the sample into 900 µL of PBS in a 1.5 mL tube, vortex, and repeat 5 times. Pipette 10 µL of each dilution onto an agar plate as shown in Figure 3.
   ​NOTE: If more precise CFU quantitation is required, spread 100 µL of each dilution of the sample across separate agar plates.

8. Ex vivo assessment of dispersal (Figure 4)

1. Wound mice and infect with approximately 10⁴ PAO1 as described above, and then euthanize 48 h after infection.
2. Carefully remove the dressing with forceps, using sterile technique and without disturbing the wound bed.
3. Use sterile surgical scissors to cut the perimeter of the wound bed away from the uninfected skin by using forceps to lift one end of the wound bed and scissors to excise infected wound tissue from the muscle layer underneath and surrounding uninfected tissue. Divide wound bed samples into equal parts or use whole.
4. Place wound tissue into empty, pre-weighed 2 mL bead mill homogenizing tubes. Then weigh the tubes again after adding the sample. Calculate the weight of the sample by:
   weight after adding sample - weight before adding sample.
5. Add 1 mL of PBS, and gently invert tube 3-5 times to rinse the sample and remove any planktonic cells. Remove and discard the PBS.
6. Add 1 mL of GH treatment (or PBS as a negative control) to the sample and incubate for 2 h at 37 °C with shaking at 80 rpm.
7. Remove the biofilm dispersal solution and place into a separate 1.5 mL tube. This contains the dispersed cells.
8. Add 1 mL of PBS to the remaining tissue sample and gently invert 3-5 times to rinse off any remaining dispersed cells. Remove and discard the PBS.
9. Add 1 mL of PBS to the remaining tissue and homogenize at 5 m/s for 60 s using a benchtop homogenizer.
10. Serially dilute the solution of dispersed cells and the homogenized tissue sample by placing 100 µL of sample into 900 µL of PBS in a 1.5 mL tube, and repeating five times for a total of 6 dilutions.
11. Spot-plate 10 µL of each dilution onto media selective agar (e.g., Pseudomonas Isolation Agar for P. aeruginosa) and incubate the plates at 37 °C overnight.
12. Count the CFU and calculate the 'percent dispersed' as (CFU in supernatant/ (CFU in supernatant+ CFU in biofilm tissue)) x 100.

Results

In this experiment, 8-10 week old female Swiss Webster mice were infected with 10⁴ CFU of PAO1 carrying the luminescence plasmid pQF50-lux. As described above, an infection was allowed to establish for 48 h prior to administering 3 x 30 min treatments of either PBS (vehicle control) or 10% GH (treatment) to digest the biofilm EPS. Mice were imaged pre-treatment, directly after treatment (0 h) and at 10 h and 20 h post-treatment. Figure 2A and Supplemental Figure 1

Discussion

Here we describe protocols that can be utilized to study the efficacy of biofilm dispersal agents. These protocols can be easily adapted to use with different types of dispersal agents, bacterial species or ex vivo samples, including clinical debridement samples. This protocol also provides a clinically relevant model to collect and study dispersed bacterial cells. The phenotypes of dispersed bacterial cells have been shown to be distinct from those of either planktonic or biofilm cells ⁵...

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Fisher	14823434	Use to complete serial dilutions of samples
25G 58 in needle	Fisher	14823434	Attaches to 1 mL syringe
Ampicillin Sodium Salt	Fisher	BP1760-5	Make a 50 mg/ mL stock solution and add 100 µL to 10 mL of LB broth for both overnight and subculture
Amylase	MP Biomedicals	2100447	Make a 5% w/v solution, vortex- other dispersal agents can be used
Buprenorphine SR-LAB 5 mL (1 mg/mL)	ZooPharm	RX216118	Use as pain mainagement- may use other options
Cellulase	MP Biomedicals	2150583	Add 5% w/v to the 5% w/v amylase solution, vortex, activate at 37 °C for 30 min- other dispersal agents can be used
Depilatory cream	Walmart	287746	Use a small amount to massage into the hair follicles on the back of the animal and allot 10 min to remove hair
Dressing Forceps, Serrated Tips	Fisher	12-460-536	Can use other forms of forceps
Erlenmeyer flasks baffled 125 mL	Fisher	101406	Use to grow overnights and sub-cultures of bacteria
FastPrep-24 Benchtop Homogenizer	MP Biomedicals	6VFV9	Use 5 m/s for 60 s two times to homogenize tissue
Fatal Plus	Vortech Pharmaceuticals	0298-9373-68	Inject 0.2 mL intraperitaneal for each mouse
Homogenizing tubes (Bead Tube 2 mL 2.4 mm Metal)	Fisher	15340151	Used to homogenize samples for plating
Isoflurane	Diamond Back Drugs
Ketamine hydrochloride/xylazine hydrochloride solution C-IIIN	Sigma Aldrich	K4138	Use as anasethia- other options can also be utilized to gain a surgical field of anasethia
LB broth, Miller	Fisher	BP1426-2	Add 25 g/L and autoclave
Lidocaine 2% Injectable	Diamond Back Drugs	2468	Inject 0.05 mL through the side of the marked wound bed area so it is deposited in the center of the mark. Allot 10 min prior to cutting
Meropenem	Sigma Aldrich	PHR1772-500MG	Make 5 mg/mL to add to the GH solution to apply topically and a 15 mg/mL solution to inject intraperitaneal 4 h prior and 6 h post-treatment
Non-sterile cotton gauze sponges	Fisher	13-761-52	Use to remove the depilatory cream
PAO1 pQF50-lux bacterial strain	Ref [13]	N/A	PAO1 pgF50-lux was used as the P. aeruginosa strain of interest in this paper's representative results
Petri dishes	Fisher	PHR1772-500MG
Phosphate Buffer Saline 10x	Fisher	BP3991	Dilute 10x to 1x prior to use
Polyurethane dressing	Mckesson	66024007	Cut the rounded edge off and cut the remaining square into 4 equal sections
Pseudomonas isolation agar	VWR	90004-394	Add 20 mL/L of glycerol and 45 g/mL to water, autoclave, and pour 20 mL into petri dishes
Refresh P.M.	Walmart		Use on eyes to reduce dryness during procedure.
Sterile Alcohol Prep Pads	Fisher	22-363-750	Use to clean the skin immediately prior to wounding to disinfect the area
Straight Delicate Scissors	Fisher	89515	Can also use curved scissors
Swiss Webster mice	Charles River	551NCISWWEB	Other mice strains can be used
Syring Slip Tip 1 mL	Fisher	14823434	Used to administer drugs and enzyme treatment