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. 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.
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.
Animal experiments were approved by the Institutional Animal Care and Use Committee of Michigan State University.
1. Preparation of Occlusive Dressings and Silicone Spacers
2. Experimental Animals
3. Biofilms
4. Wound Surgery
5. Postoperative Management
6. Biofilm Inoculum Preparation and Infection
7. Wound Measurement and Imaging
8. Histological Analysis
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<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).
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 7: SKH-1 diabetic mouse 6 days after wound was inoculated with the P. aeruginosa Xen 41 biofilm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 9: Total flux from wounds in SKH-1 diabetic mice infected with P. aeruginosa Xen 41 biofilms during the course of the experiment. Please click here to view a larger version of this figure.
Figure 10: Estimated CFU per wound using a standard curve of bioluminescence per CFU produced with P. aeruginosa Xen 41 biofilms. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
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.
Opsite | Smith & Nephew | Model 66000041 | Smith & Nephew Flexfix Opsite Transparent Adhesive Film Roll 4" x 11yards |
SKH-1 mice Crl:SKH1-Hrhr | Charles River Breeding Laboratories | SKH1 | Hairless mice, 8 weeks old |
Streptozotocin (STZ) | Sigma Aldrich | S0130-1G | Streptozocin powder, 1g |
AccuChek glucometer | Accu-Chek Roche | Art No. 05046025001 | ACCU-CHEK CompactPlus Diabetes Monitoring Care Kit |
Pseudomonas aeruginosa Xen 41 | Perkin Elmer | 119229 | Bioluminescent Pseudomonas aeruginosa |
Polycarbonate membrane filters | Sigma Aldrich | P9199 | Millipore polycarbonate membrane filters with 0.2 μm pore size |
Dulbelcco phosphate buffer saline (DPBS) | Sigma Aldrich | D8537 | PBS |
Tryptic soy agar | Sigma Aldrich | 22091 | Culture agar |
Meloxicam | Henry Schein Animal Health | 49755 | Eloxiject (Meloxicam) 5mg/mL, solution for injection |
10% povidone-iodine (Betadine) | Purdue Products LP | 301879-OA | Swabstick, Betadine Solution. Antiseptic. Individ. Wrapped, 200/case |
4% paraformaldehyde | Fisher Scientific | AAJ61899AK | Alfa Aesar Paraformaldehyde, 4% in PBS |
Capillary glass tube | Fisher Scientific | 22-362-566 | Heparinized Micro-Hematocrit Capillary Tubes |
Silicone to make splints | Invitrogen Life Technologies Corp | P-18178 | Press-to-Seal Silicone Sheet, 13cm x 18cm, 0.5mm thick, set of 5 sheets |
Tryptic soy broth | Sigma Aldrich | 22092 | Culture broth |
IVIS Spectrum | Perkin Elmer | 124262 | In vivo imaging system |
IVIS Spectrum Isolation chamber | Perkin Elmer | 123997 | XIC-3 animal isolation chamber |
HEPA filter | Teleflex | 28022 | Gibeck ISO-Gard HEPA Light number 28022 |
Biopsy punches | VWR International Inc | 21909-142 | Disposable Biopsy Punch, 5mm, Sterile, pack of 50. |
Biopsy punches | VWR International Inc | 21909-140 | Disposable Biopsy Punch, 4mm, Sterile, pack of 50. |
Glucose | J.T.Baker | 1916-01 | Dextrose, Anhydrous, Powder |
Citric acid | Sigma Aldrich | C2404-100G | Citric Acid |
Mastisol | Eloquest Healthcare | HRI 0496-0523-48 | Mastisol Medical Liquid Adhesive 2/3 mL vial, box of 48 |
Corning 96-well black plates | Fisher Scientific | 07-200-567 | 96-well clear bottom black polysterene microplates |
25 gauge 5/8 inch needle | BD | 305122 | Regular bevel needle |
Bransonic M Ultrasonic Cleaning Bath | Branson Ultrasonics | N/A | Ultrasonic Cleaner |