This work was supported by grants from the National Institutes of Health (R21 AI137462-01A1), the Ted Nash Long Life Foundation, the Jasper L. and Jack Denton Wilson Foundation and the Department of Defense (DoD MIDRP W0318_19_NM_PP).

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.
<ol>
	<li>Use Pseudomonas aeruginosa wild-type strain PAO1 carrying the luminescence plasmid pQF50-lux23&#160;for these experiments as previously described7.</li>
	<li>Grow P. aeruginosa strains in baffled Erlenmeyer flasks, with shaking at 200 rpm, in Luria-Bertani (LB) broth at 37 &#176;C for 16 to 20 h. Add a final concentration of 100 &#181;g/mL of ampicillin to the overnight culture of PAO1(pQF50-lux) for maintenance of the plasmid.</li>
	<li>Subculture P. aeruginosa by adding 100 &#181;L of the overnight culture to an Erlenmeyer flask containing 10 mL of LB broth with a final concentration of 100 &#181;g/mL ampicillin. Then grow for 2-2.5 h at 37 &#176;C with shaking, to an OD600 nm of 0.4, and then serially dilute (1:10) three times for an infecting dose of approximately 104 cells.</li>
</ol>
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 &#34;GH&#34;, for the dispersal treatment.
<ol>
	<li>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.</li>
	<li>Warm all treatments to 37 &#176;C for 30 min for enzyme activation.</li>
</ol>
3. Experimental animals and preoperative setup
<ol>
	<li>House mice, 5 litter mates per cage, in environment-controlled conditions, with 12 h light/dark cycles and proper access to food and water.</li>
	<li>Preferably, use female Swiss Webster mice, between 8 and 10 weeks old.</li>
	<li>Weigh mice to determine the proper amount of anesthesia to administer.</li>
	<li>Administer anesthesia by the intraperitoneal injection of 100 mg/kg ketamine 10 mg/kg xylazine.</li>
	<li>Apply eye cream (e.g., Refresh P.M.) carefully onto the eye using a cotton swab to reduce dryness.</li>
	<li>Monitor physiologic parameters including respiratory rate, skin coloration, and body temperature throughout the surgery.</li>
</ol>
4. Dorsal full thickness excision surgery
<ol>
	<li>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&#39;s instructions), after which&#160;gently remove, ensuring the skin was dry.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Administer a full-thickness, excisional skin wound to the level of panniculus muscle with surgical scissors.</li>
	<li>After excision, immediately cover wounds with a semipermeable polyurethane dressing.</li>
	<li>Inject approximately 104 CFU of bacteria in 100 &#181;L 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.</li>
	<li>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.</li>
	<li>Establish wound infections for 48 h.</li>
	<li>Inspect adhesive dressings daily for tears and areas of non-adherence and replaced if needed.</li>
</ol>
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.
<ol>
	<li>Place mice under isoflurane anesthesia (at 3% and 1 L per minute of oxygen) while administering the treatments.</li>
	<li>Prepare three 1 mL syringes, with 25 G needles, containing 200 &#181;L of treatment for each mouse.</li>
	<li>Inject 200 &#181;L 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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>After treatment, place mice back in their cages for 30 min.</li>
	<li>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.</li>
	<li>Administer the second and third irrigation treatments as the first, using a new syringe each time.</li>
</ol>
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.
<ol>
	<li>Image mice immediately before and after treatment and at 10 and 20 h post-treatment (Figure 2 and Supplemental Figure 1).</li>
	<li>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.</li>
	<li>For analysis, open images in the IVIS software and select the area to be analyzed in the Tool Palette.</li>
	<li>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.</li>
	<li>To upload measurement values, select View -&#62; 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. 
	&#8203;NOTE: Control and treated mice should be analyzed simultaneously to normalize for variables that may affect image acquisition or radiance.</li>
</ol>
7. Assessing dispersal by determining CFU
<ol>
	<li>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.</li>
	<li>Harvest wound bed tissue by first gently removing the dressing with sterile forceps, and then cut&#160;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Weigh spleen and rinse with PBS, as described for the wound bed tissue. 
	NOTE: Other organs of interest can be similarly harvested.
	<ol>
		<li>When making the initial incision, take care to avoid puncturing the intestines, or other organs.</li>
	</ol>
	</li>
	<li>Homogenize samples in 2 mL pre-filled bead mill tubes using a benchtop homogenizer at 5 m/s for 60 s, twice.</li>
	<li>To enumerate CFU, serially dilute samples and spot-plate on selective agar. For serial dilution, pipette 100 &#181;L of the sample into 900 &#181;L of PBS in a 1.5 mL tube, vortex, and repeat 5 times. Pipette 10 &#181;L of each dilution onto an agar plate as shown in Figure 3. 
	&#8203;NOTE: If more precise CFU quantitation is required, spread 100 &#181;L of each dilution of the sample across separate agar plates.</li>
</ol>
8. Ex vivo assessment of dispersal (Figure 4)
<ol>
	<li>Wound mice and infect with approximately 104 PAO1 as described above, and then euthanize 48 h after infection.</li>
	<li>Carefully remove the dressing with forceps, using sterile technique and without disturbing the wound bed.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add 1 mL of GH treatment (or PBS as a negative control) to the sample and incubate for 2 h at 37 &#176;C with shaking at 80 rpm.</li>
	<li>Remove the biofilm dispersal solution and place into a separate 1.5 mL tube. This contains the dispersed cells.</li>
	<li>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.</li>
	<li>Add 1 mL of PBS to the remaining tissue and homogenize at 5 m/s for 60 s using a benchtop homogenizer.</li>
	<li>Serially dilute the solution of dispersed cells and the homogenized tissue sample by placing 100 &#181;L of sample into 900 &#181;L of PBS in a 1.5 mL tube, and repeating five times for a total of 6 dilutions.</li>
	<li>Spot-plate 10 &#181;L of each dilution onto media selective agar (e.g., Pseudomonas Isolation Agar for P. aeruginosa) and incubate the plates at 37 &#176;C overnight.</li>
	<li>Count the CFU and calculate the &#39;percent dispersed&#39; as (CFU in supernatant/ (CFU in supernatant+ CFU in biofilm tissue)) x 100.</li>
</ol>

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The authors have nothing to disclose.

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...

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 vivo6,7,8,9,10,11.
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,&#160;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 extensively7,9,12,13,14,15,16,17,18,19,20,21. 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.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Fisher</td><td>14823434</td><td>Use to complete serial dilutions of samples</td></tr><tr><td>25G 58 in needle</td><td>Fisher</td><td>14823434</td><td>Attaches to 1 mL syringe</td></tr><tr><td>Ampicillin Sodium Salt</td><td>Fisher</td><td>BP1760-5</td><td>Make a 50 mg/ mL stock solution and add 100 &amp;micro;L to 10 mL of LB broth for both overnight and subculture</td></tr><tr><td>Amylase</td><td>MP Biomedicals</td><td>2100447</td><td>Make a 5% w/v solution, vortex- other dispersal agents can be used</td></tr><tr><td>Buprenorphine SR-LAB 5 mL (1 mg/mL)</td><td>ZooPharm</td><td>RX216118</td><td>Use as pain mainagement- may use other options</td></tr><tr><td>Cellulase</td><td>MP Biomedicals</td><td>2150583</td><td>Add 5% w/v to the 5% w/v amylase solution, vortex, activate at 37 &amp;deg;C for 30 min- other dispersal agents can be used</td></tr><tr><td>Depilatory cream</td><td>Walmart</td><td>287746</td><td>Use a small amount to massage into the hair follicles on the back of the animal and allot 10 min to remove hair</td></tr><tr><td>Dressing Forceps, Serrated Tips</td><td>Fisher</td><td>12-460-536</td><td>Can use other forms of forceps</td></tr><tr><td>Erlenmeyer flasks baffled 125 mL</td><td>Fisher</td><td>101406</td><td>Use to grow overnights and sub-cultures of bacteria</td></tr><tr><td>FastPrep-24 Benchtop Homogenizer</td><td>MP Biomedicals</td><td>6VFV9</td><td>Use 5 m/s for 60 s two times to homogenize tissue</td></tr><tr><td>Fatal Plus</td><td>Vortech Pharmaceuticals</td><td>0298-9373-68</td><td>Inject 0.2 mL intraperitaneal for each mouse</td></tr><tr><td>Homogenizing tubes (Bead Tube 2 mL 2.4 mm Metal)</td><td>Fisher</td><td>15340151</td><td>Used to homogenize samples for plating</td></tr><tr><td>Isoflurane</td><td>Diamond Back Drugs</td><td></td><td></td></tr><tr><td>Ketamine hydrochloride/xylazine hydrochloride solution C-IIIN</td><td>Sigma Aldrich</td><td>K4138</td><td>Use as anasethia- other options can also be utilized to gain a surgical field of anasethia</td></tr><tr><td>LB broth, Miller</td><td>Fisher</td><td>BP1426-2</td><td>Add 25 g/L and autoclave</td></tr><tr><td>Lidocaine 2% Injectable</td><td>Diamond Back Drugs</td><td>2468</td><td>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</td></tr><tr><td>Meropenem</td><td>Sigma Aldrich</td><td>PHR1772-500MG</td><td>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</td></tr><tr><td>Non-sterile cotton gauze sponges</td><td>Fisher</td><td>13-761-52</td><td>Use to remove the depilatory cream</td></tr><tr><td>PAO1 pQF50-lux bacterial strain</td><td>Ref [13]</td><td>N/A</td><td>PAO1 pgF50-lux was used as the P. aeruginosa strain of interest in this paper&#039;s representative results</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>PHR1772-500MG</td><td></td></tr><tr><td>Phosphate Buffer Saline 10x</td><td>Fisher</td><td>BP3991</td><td>Dilute 10x to 1x prior to use</td></tr><tr><td>Polyurethane dressing</td><td>Mckesson</td><td>66024007</td><td>Cut the rounded edge off and cut the remaining square into 4 equal sections</td></tr><tr><td>Pseudomonas isolation agar</td><td>VWR</td><td>90004-394</td><td>Add 20 mL/L of glycerol and 45 g/mL to water, autoclave, and pour 20 mL into petri dishes</td></tr><tr><td>Refresh P.M.</td><td>Walmart</td><td></td><td>Use on eyes to reduce dryness during procedure.</td></tr><tr><td>Sterile Alcohol Prep Pads</td><td>Fisher</td><td>22-363-750</td><td>Use to clean the skin immediately prior to wounding to disinfect the area</td></tr><tr><td>Straight Delicate Scissors</td><td>Fisher</td><td>89515</td><td>Can also use curved scissors</td></tr><tr><td>Swiss Webster mice</td><td>Charles River</td><td>551NCISWWEB</td><td>Other mice strains can be used</td></tr><tr><td>Syring Slip Tip 1 mL</td><td>Fisher</td><td>14823434</td><td>Used to administer drugs and enzyme treatment</td></tr></tbody></table>

assessing biofilm dispersal in murine wounds

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.

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 </strong...

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Assessing Biofilm Dispersal in Murine Wounds

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.

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

assessing-biofilm-dispersal-in-murine-wounds

Research

JoVE Journal

Immunology and Infection

4.3K Views.  Texas Tech University Health Sciences Center. Microbiologen en artsen van infectieziekten beginnen zich te realiseren hoe belangrijk biofilm-geassocieerde infecties zijn bij chronische ziekten. De NIH heeft zelfs gezegd dat meer dan 80% van de chronische infecties te wijten zijn aan bacteriële biofilms. Dit heeft dus aangedrongen op de nadruk op het aanpakken van biofilm en het ontwikkelen van therapieën die het biofilmprobleem aanpakken.En veel van die therapieën zijn gericht op het verspreiden van bacteriële cellen die zich ...

Video: Assessing Biofilm Dispersal in Murine Wounds

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

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. ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

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.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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

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

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Developing a Long-Term Swine Model for Biofilm-Infected Burn Wound Studies

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Biology

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.

Source: Anderson, L. S., et al. A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection. J. Vis. Exp. (2019).
This video ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to generate a murine model of skin wound infection. Upon infecting mice &#8212; engineered to express a fluorescent protein in neutrophils &#8212; with a genetically modified bioluminescent Staphylococcus aureus &#8212; a pathogenic bacteria. The bacterial burden at the infection site and the neutrophil recruitment dynamics can be studied using whole animal imaging.

Assessing Biofilm Dispersal in Murine Wounds

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