Name: come to the light side: in vivo monitoring of pseudomonas aeruginosa biofilm infections in chronic wounds in a diabetic hairless murine model
Uploaded: 2017-10-10T13:00:00.000+00:00
Duration: 9 min 15 s
Description: Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

The authors would like to thank the American Diabetes Association for supporting this work (Grant # #7-13-BS-180), the Michigan State University Research Technology Support Facility for providing training and access to the in vivo imaging system and the Michigan State University Investigative Histopathology Lab for processing the mouse biopsies for histopathological examination.

Animal experiments were approved by the Institutional Animal Care and Use Committee of Michigan State University.
1. Preparation of Occlusive Dressings and Silicone Spacers
<ol>
	<li>Cut the transparent occlusive dressing to make squares approximately 1 cm x 1 cm with scissors.</li>
	<li>Cut 10 mm circles on a 0.5 mm thick silicone sheet using a 10 mm biopsy punch.&#160;Center a 5 mm biopsy punch in the middle of the 10 mm circle and press firmly to create a hole to form a &#34;donut&#34;-like disc that will be used as a splint.&#160;</li>
</ol>
2. Experimental Animals
<ol>
	<li>Use 8 week old (22-26 g) male SKH-1 mice from a commercial breeder. Keep mice in standard conditions of 21 &#176;C and a 12 h light-dark cycle with free access to food and water.</li>
	<li>To induce diabetes, inject mice intraperitonially with a 13 mg/ml streptozotocin (STZ) solutionand 25% Glucose (250 &#181;l per/ mouse) on 5 consecutive days.
	<ol>
		<li>Make the STZ solution by diluting 65 &#181;g of STZ in 5 &#181;L of 100 mM Citric Acid, pH 4.5.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Biofilms
<ol>
	<li>Biofilm preparation
	<ol>
		<li>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 &#176;C and shaking at 200 rpm and sterilize polycarbonate membrane filters with 0.2 &#181;m pore size by exposure to UV light in a biological safety hood for 15 min per side.</li>
		<li>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&#39;s phosphate buffered saline (DPBS) by pipetting up and down.</li>
		<li>Dilute suspension in DPBS to an absorbance of 0.05 at 600 nm.</li>
		<li>Pipette 10 &#181;L of the diluted culture on each membrane resting on a tryptic soy agar (TSA) plate. After allowing to dry, incubate the membranes at 35 &#176;C for 72 h to grow biofilms transferring to fresh TSA plates every 24 h.</li>
	</ol>
	</li>
	<li>Standard curve
	<ol>
		<li>Prior to the start of the experiment make a standard curve to correlate bioluminescence and bacterial counts.</li>
		<li>Prepare biofilms as described in 3.1.</li>
		<li>Make serial dilutions of biofilms ranging from &#189; to 1/24 by mixing biofilm with DPBS and vortexing until a visually homogeneous solution is produced. Pipette 200&#181;L of the diluted solutions in a black 96-well plate and image with the in vivo imaging system.</li>
		<li>Spread plate dilutions onto TSA plates and incubate at 35 &#176;C for 24 h.</li>
		<li>Count colony forming units (CFU) on plates and create a standard curve to correlate bioluminescence and bacterial counts.</li>
	</ol>
	</li>
</ol>
4. Wound Surgery
<ol>
	<li>Induce general anesthesia using isoflurane in 95% oxygen/5% CO2 (to prevent death from ketoacidosis) at a flow rate of 1 L/min and&#160;maintain anesthesia using 1-3% isoflurane. Maintain animals on heat mats during surgery.</li>
	<li>Ensure the deep pedal reflexes of the mouse are suppressed by pinching the foot with tweezers and place the mouse in the prone position.</li>
	<li>Administer meloxicam (0.2 mg/kg) via sub-cutaneous injection (30 &#956;l) for pain management.</li>
	<li>Wipe the skin of the back with 10% povidone-iodine three times and an isopropanol pad.</li>
	<li>Use a sterile 4 mm biopsy punch to outline one circular pattern for the wound on one side of the mouse&#39;s midline at the level of the shoulders. Outline the pattern with a permanent marker.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Postoperative Management
<ol>
	<li>Administer meloxicam (0.2 mg/kg) once daily via sub-cutaneous injection for post-operative pain relief for the next 2 days.</li>
	<li>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.</li>
</ol>
6. Biofilm Inoculum Preparation and Infection
<ol>
	<li>Inoculate mice 48 h after surgery as described in the following steps.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Cut the tips of 200 &#181;L pipette tips and pipette 10 &#181;L of the biofilm inoculum onto each wound.</li>
	<li>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).</li>
	<li>Cover wound with fresh dressing.</li>
</ol>
7. Wound Measurement and Imaging
<ol>
	<li>Evaluate the clinical signs of animals daily9.</li>
	<li>Provide food, water and change cages as needed.</li>
	<li>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.</li>
	<li>Weigh animals every other day.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
8. Histological Analysis
<ol>
	<li>Euthanize the mice with a flow of 2 l/min of CO2 in an euthanasia chamber&#160;after luminescence falls below background levels and the wounds are completely healed. Confirm death by cervical dislocation as a second method of euthanasia.</li>
	<li>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.</li>
	<li>Other analysis: Cytokine detection
	<ol>
		<li>Collect blood retro-orbitally from animals under anesthesia using a capillary glass tube and transfer it to EDTA-treated tubes.</li>
		<li>Centrifuge blood at 2,000 rpm for 20 min at 4 &#176;C and freeze plasma for cytokine detection.</li>
	</ol>
	</li>
</ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18 (4), 593-603 (1995).</li><li>Rocchetta, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+noninvasive,+real-time+imaging+technology+using+bioluminescent+Escherichia+coli+in+the+neutropenic+mouse+thigh+model+of+infection.">Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.</a> Antimicrob Agents Chemother. 45 (1), 129-137 (2001).</li><li>Kadurugamuwa, J. 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(75), e50265 (2013).</li><li>Zhao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+wound+healing+in+diabetic+(db/db)+mice+with+Pseudomonas+aeruginosa+biofilm+challenge+-+a+model+for+the+study+of+chronic+wounds.">Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge - a model for the study of chronic wounds.</a> Wound Repair Regen. 18 (5), 467-477 (2010).</li><li>Holley, A. K., Xu, Y., Noel, T., Bakthavatchalu, V., Batinic-Haberle, I., St. Clair, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+superoxide+dismutase-mediated+inside-out+signaling+in+HaCaT+human+keratinocytes+and+SKH-1+mouse+skin.">Manganese superoxide dismutase-mediated inside-out signaling in HaCaT human keratinocytes and SKH-1 mouse skin.</a> Antioxid Redox Signal. 20 (15), 2347-2360 (2014).</li><li>Abbas, S., Alam, S., Pal, A., Kumar, M., Singh, D., Ansari, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UVB+exposure+enhanced+benzanthrone-induced+inflammatory+responses+in+SKH-1+mouse+skin+by+activating+the+expression+of+COX-2+and+iNOS+through+MAP+kinases/NF-&#312;B/AP-1+signalling+pathways.">UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-&#312;B/AP-1 signalling pathways.</a> Food Chem Toxicol. 96, 183-190 (2016).</li><li>Watters, C., Everett, J. A., Haley, C., Clinton, A., Rumbaugh, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+treatment+modulates+the+host+immune+system+to+enhance+Pseudomonas+aeruginosa+wound+biofilms.">Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.</a> Infect Immun. 82 (1), 92-100 (2014).</li></ol>

The authors declare that they have no competing financial interests.

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 healing10,11, but all have relied on the use of haired mice requiring the removal of fur by processes that involve either waxing or hair clipping combined with depilatory creams. This step is not only time consuming and messy but potentially injures the skin of the animals in the area where the wound will be placed. While hairless mice have been used in a series of carcinogenesis studies12,13, these mice have not been used to evaluate biofilm persistence in chronic wounds. Another common problem solved by the use of hairless animals, especially in long term studies, is hair re-growth in the wound area, which may jeopardize evaluation of wound healing and disrupt bandaging.
SKH-1 animals also proved amenable to STZ-induced Type I diabetes and, in comparison with C57BL/6J mice, had statistically significant lower weight loss during the course of the experiment, making dosing with insulin unnecessary. This is a particularly interesting trait as treatment with insulin can potentially impact the infection outcome as evidenced by Watters et al. (2014)14 who described an increase in bacterial counts in insulin-treated diabetic animals infected with P. aeruginosa biofilms in comparison to no insulin counterparts. In addition, in our model, there was a drastic reduction in mortality rates in the hairless cohort indicating that the animals are potentially more resilient in dealing with infection.
A second feature of this model is the application of measured pre-formed biofilm inoculum slurry to infect the wounds in contrast to planktonic grown cells. By delivering an already metabolically complex and structured bacterial community to the wound, the bacterial cells are able to evade the immune system and the immediate effects of the biofilms on the lesions can be determined.
The third advantage of this new wound model is the use of a microbial strain capable of producing bioluminescence that can be measured with an in vivo imaging system to spatially localize and quantify the bacteria. This allows real-time tracking of biofilm evolution over time. The P. aeruginosa Xen 41 strain possesses a single stable copy of the P. luminescences luxCDABE operon on the bacterial chromosome that results in the constitutive emission of luminescence, which can be captured by the ultra-sensitive camera in the imaging system. This real-time, non-invasive, in situ feature allows measuring of biofilm by bioluminescence even while the splint and cover are in place. This feature drastically decreases the number of animals needed per study, as there is no need to sacrifice animals at certain time points for biofilm monitoring. However, the presence of biofilm and pus occluded measuring wound healing. In this study, we removed the dressing at day 8 to allow visualization of wound healing, but this parameter could be modified depending on the questions being addressed.
Lastly, as the imaging system is capable of detecting bioluminescence up to 2.5 cm in depth, the newly proposed model is amenable to testing of antimicrobial therapies whether in form of solution or gels or incorporated to occlusive dressings. A real-time infection monitoring model allows much greater flexibility to measure the impact of different dosing concentrations and durations as opposed to a static end-point assay. This model can contribute to the validation of potential novel treatments to eradicate biofilms in chronic wounds.

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 treatment3,4, 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.

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

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.

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). In contrast, when using this wound model with SKH-1 mice a statistically significant lower weight loss was observed (P&#60;0.0001, Mann-Whitney U test). Furthermore, no deaths occurred in the diabetic SKH-1 mice cohort infected with P. aeruginosa Xen 41 biofilms while a 40% mortality rate was observed for C57BL/6J infected mice in previous experiments (Figure 2).
Another advantage to the model presented here is that the experimental procedure for the hair removal step mandatory for C57BL/6J mice is unnecessary for SKH-1 mice. Although in our previous experiments with haired mice special attention was given to minimize irritation to the skin, some damage inevitably occurred (Figure 3). Notably, however, the greatest advantage in utilizing hairless mice in this model is the elimination of the problem of hair re-growth observed in long term wound healing studies. In our experience with C57BL/6J mice, hair re-growth varied from animal to animal but given the long-term nature of the studies, it always occurred and interfered with wound area measurements or dislocated wound splints and/or dressings used to cover infected wounds, potentially resulting in drying of the wound (Figure 4).
In the SKH-1 wound healing model, after diabetes is confirmed, surgery can easily be executed to create a circular full-thickness wound on the back of the animal. The silicone splint is kept in place by a medical waterproof skin adhesive and avoids direct contact from the occlusive dressing with the newly created wound bed (Figure 5).
P. aeruginosa Xen 41 bioluminescent biofilms grown on polycarbonate membranes (Figure 6) are easily and aseptically transferred to a syringe to be prepared for delivery to the wounds and the inoculated mice are monitored daily for clinical signs of infection (Figure 7). For this model, we implemented two distinct phases. In the first phase, after inoculation of the biofilm the wound was surrounded by a splint covered with a transparent occlusive dressing. This results in pus accumulation that occluded the wound. Biofilm-containing wounds were imaged daily with the in vivo imaging system to monitor infection development and assess biofilm evolution (Figure 8 and Figure 9). Bioluminescence, recorded as total flux (p/s), can be correlated with bacterial density using a standard curve (Figure 10).
At day 8, the splint and dressing were removed to allow visualization of wound healing. Bioluminescence subsequently drops due to the loss of the pus surrounding the wound; however, bacteria remained associated with the wound as determined by histology. This approach of removing the dressing to measure wound healing has been utilized in other chronic wounds healing studies (REFs). Wound healing progression can be determined by taking micrographs with a camera attached to a microscope (Figure 11).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55991/55991fig1.jpg" /> 
Figure 1: Comparative percentage weight loss of SKH-1 and C57BL/6J diabetic mice. Day zero corresponds to weight at the day of last (5th) STZ injection. n = 10 mice for SKH-1 and n = 12 mice for C57BL/6J. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55991/55991fig2.jpg" /> 
Figure 2: Percent survival rates of SKH-1 and C57BL/6J diabetic mice after P. aeruginosa Xen 41 biofilm application (day 1). n = 5 mice for SKH-1 and n = 10 mice for C57BL/6J. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55991/55991fig3.jpg" /> 
Figure 3: Skin lacerations in the future wounded area after shaving and using depilatory cream on C57BL/6J mice. (A): day of procedure; (B): 4 days after procedure. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55991/55991fig4.jpg" /> 
Figure 4: (A) C57BL/6J mouse with a partially removed wound splint. (B) Lifting of the splint revealed a healed wound surrounded by fully re-grown hair. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55991/55991fig5.jpg" /> 
Figure 5: Surgical procedure for wounding SKH-1 mice. (A) demarcation with biopsy punch; (B) outline of the demarcation; (C) wounding completed; (D) application of medical waterproof skin adhesive; (E) glueing splint; (F) wound covered with occlusive dressing. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55991/55991fig6.jpg" /> 
Figure 6: Preparation of the biofilm inoculum. (A) 72 h colony biofilms of Pseudomonas aeruginosa Xen 41 grown on polycarbonate membranes; (B) measurement of biofilm using a syringe. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55991/55991fig7.jpg" /> 
Figure 7: SKH-1 diabetic mouse 6 days after wound was inoculated with the P. aeruginosa Xen 41 biofilm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55991/55991fig8.jpg" /> 
Figure 8: Monitoring of biofilm infection by tracking bioluminescence evolution over time in diabetic SKH-1 mice. (A) day of biofilm application; (B) 5 days post-biofilm; (C) 8 days post-biofilm; (D) 12 days post-biofilm; (E) 16 days post-biofilm; (F) 20 days post-biofilm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55991/55991fig9.jpg" /> 
Figure 9: Total flux from wounds in SKH-1 diabetic mice infected with P. aeruginosa Xen 41 biofilms during the course of the experiment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/55991/55991fig10.jpg" /> 
Figure 10: Estimated CFU per wound using a standard curve of bioluminescence per CFU produced with P. aeruginosa Xen 41 biofilms.&#160; <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/55991/55991fig11.jpg" /> 
Figure 11: Micrograph timeline of wounds infected with P. aeruginosa Xen 41 biofilms in diabetic SKH-1 mouse showing progression of healing. The days after biofilm infection are indicated in the bottom left corner of each picture. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55991/55991fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

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.

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. ...

come-to-light-side-vivo-monitoring-pseudomonas-aeruginosa-biofilm

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.1K Views.  Michigan State University. Hier beschrijven we een roman diabetische lymfkliertest model met behulp van haarloze muizen voor real-time, niet-invasieve, bewaking van biofilm wond infecties van bioluminescente Pseudomonas aeruginosa. Deze methode kan worden aangepast aan de infectie van andere bacteriesoorten en genetisch gemodificeerde micro-organismen, met inbegrip van meerdere soorten biofilms, evalueren en testen van de werkzaamheid van antibiofilm strategieën.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

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

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 ...

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 ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

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.

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 ...

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 ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

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

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