Name: a novel high-throughput ex vivo ovine skin wound model for testing emerging antibiotics
Uploaded: 2022-09-16T13:20:00
Description: Watch this Scientific Journal Video about A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics at JoVE.com

The authors would like to thank EPSRC (EP/R513313/1) for funding. The authors would like to also thank R.B Elliot and Son Abattoir in Calow, Chesterfield, for providing lambs&#39; heads and for being so accommodating in the early stages of the project, Kasia Emery for her support throughout the development of this protocol, and Fiona Wright from the Department of Infection, Immunity and Cardiovascular Disease at the University of Sheffield for processing the histology samples and being so incredibly helpful throughout this project.

Lambs&#39; heads from the R.B Elliott and Son Abattoir were used as the source of skin samples in this project. All lambs were slaughtered for consumption as food. Instead of discarding the heads, these were repurposed for research. Ethics approval was not required as the tissue was sourced from waste discarded from abattoirs.
1. Sterilization
<ol>
	<li>Disinfect forceps prior to collection of the heads by taking clean forceps and performing dry heat sterilization in an oven...

<ol>
	<li>Claeys, K. C., 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=Novel+application+of+published+risk+factors+for+methicillin-resistant+S.+aureus+in+acute+bacterial+skin+and+skin+structure+infections.">Novel application of published risk factors for methicillin-resistant S. aureus in acute bacterial skin and skin structure infections.</a> International Journal of Antimicrobial Agents. 51 (1), 43-46 (2018).</li><li>Rahim, K., 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=Bacterial+contribution+in+chronicity+of+wounds.">Bacterial contribution in chronicity of wounds.</a> Microbial Ecology. 73 (3), 710-721 (2017).</li><li>Guest, J. F., Fuller, G. W., Vowden, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costs+and+outcomes+in+evaluating+management+of+unhealed+surgical+wounds+in+the+community+in+clinical+practice+in+the+UK:+A+cohort+study.">Costs and outcomes in evaluating management of unhealed surgical wounds in the community in clinical practice in the UK: A cohort study.</a> BMJ Open. 8 (12), 022591 (2018).</li><li>Sen, C. K., 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=Human+skin+wounds:+A+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: A major and snowballing threat to public health and the economy.</a> Wound Repair and Regeneration. 17 (6), 763-771 (2009).</li><li>Wilcox, M. H., Dryden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+epidemiology+of+healthcare-acquired+bacterial+infections:+Focus+on+complicated+skin+and+skin+structure+infections.">Update on the epidemiology of healthcare-acquired bacterial infections: Focus on complicated skin and skin structure infections.</a> Journal of Antimicrobial Chemotherapy. 76, (2021).</li><li>Han, G., Ceilley, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+wound+healing:+A+review+of+current+management+and+treatments.">Chronic wound healing: A review of current management and treatments.</a> Advances in Therapy. 34 (3), 599-610 (2017).</li><li>Percival, S. L., Hill, K. E., Malic, S., Thomas, D. W., Williams, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+tolerance+and+the+significance+of+persister+cells+in+recalcitrant+chronic+wound+biofilms.">Antimicrobial tolerance and the significance of persister cells in recalcitrant chronic wound biofilms.</a> Wound Repair and Regeneration. 19 (1), 1-9 (2011).</li><li>Dheman, N., 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=An+analysis+of+antibacterial+drug+development+trends+in+the+United+States,+1980-2019.">An analysis of antibacterial drug development trends in the United States, 1980-2019.</a> Clinical Infectious Diseases. 73 (11), 4444-4450 (2021).</li><li>MacNeil, S., Shepherd, J., Smith, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+tissue-engineered+skin+and+oral+mucosa+for+clinical+and+experimental+use.">Production of tissue-engineered skin and oral mucosa for clinical and experimental use.</a> Methods in Molecular Biology. 695, 129-153 (2011).</li><li>Yang, Q., 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=Development+of+a+novel+ex+vivo+porcine+skin+explant+model+for+the+assessment+of+mature+bacterial+biofilms.">Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilms.</a> Wound Repair and Regeneration. 21 (5), 704-714 (2013).</li><li>Malachowa, N., Kobayashi, S. D., Lovaglio, J., Deleo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+Staphylococcus+aureus+skin+infection.">Mouse model of Staphylococcus aureus skin infection.</a> Methods in Molecular Biology. 1031, 109-116 (2013).</li><li>Brandenburg, K. S., Calderon, D. F., Kierski, P. R., Czuprynski, C. J., Mcanulty, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+murine+model+for+delayed+wound+healing+using+a+biological+wound+dressing+with+Pseudomonas+aeruginosa+biofilms.">Novel murine model for delayed wound healing using a biological wound dressing with Pseudomonas aeruginosa biofilms.</a> Microbial Pathogenesis. 122, 30-38 (2018).</li><li>Bledsoe, M. J., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+human+tissues+for+research:+What+investigators+need+to+know.">The use of human tissues for research: What investigators need to know.</a> Alternatives to Laboratory Animals. , (2022).</li><li>Danso, M. O., Berkers, T., Mieremet, A., Hausil, F., Bouwstra, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+human+skin+model+for+studying+skin+barrier+repair.">An ex vivo human skin model for studying skin barrier repair.</a> Experimental Dermatology. 24 (1), 48-54 (2015).</li><li>Torres, J. P., 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=Ex+vivo+murine+skin+model+for+B.+burgdorferi+biofilm.">Ex vivo murine skin model for B. burgdorferi biofilm.</a> Antibiotics. 9 (9), 1-18 (2020).</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 and Regeneration. 18 (5), 467-477 (2010).</li><li>Schierle, C. F., Dela Garza, M., Mustoe, T. A., Galiano, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+biofilms+impair+wound+healing+by+delaying+reepithelialization+in+a+murine+cutaneous+wound+model.">Staphylococcal biofilms impair wound healing by delaying reepithelialization in a murine cutaneous wound model.</a> Wound Repair and Regeneration. 17 (3), 354-359 (2009).</li><li>Tr&#248;strup, 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=Pseudomonas+aeruginosa+biofilm+aggravates+skin+inflammatory+response+in+BALB/c+mice+in+a+novel+chronic+wound+model.">Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model.</a> Wound Repair and Regeneration. 21 (2), 292-299 (2013).</li><li>Thompson, M. 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=Evaluation+of+gallium+citrate+formulations+against+a+multidrug-resistant+strain+of+Klebsiella+pneumoniae+in+a+murine+wound+model+of+infection.">Evaluation of gallium citrate formulations against a multidrug-resistant strain of Klebsiella pneumoniae in a murine wound model of infection.</a> Antimicrobial Agents and Chemotherapy. 59 (10), 6484-6493 (2015).</li><li>Maboni, 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=A+novel+3D+skin+explant+model+to+study+anaerobic+bacterial+infection.">A novel 3D skin explant model to study anaerobic bacterial infection.</a> Frontiers in Cellular and Infection Microbiology. 7, 404 (2017).</li><li>Macneil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Theuretzbacher, U., Outterson, K., Engel, A., Karl&#233;n, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+preclinical+antibacterial+pipeline.">The global preclinical antibacterial pipeline.</a> Nature Reviews Microbiology. 18 (5), 275-285 (2019).</li><li>Miethke, M., 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=Towards+the+sustainable+discovery+and+development+of+new+antibiotics.">Towards the sustainable discovery and development of new antibiotics.</a> Nature Reviews Chemistry. 5 (10), 726-749 (2021).</li><li>Guedes, G. M. M., 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=Ex+situ+model+of+biofilm-associated+wounds:+Providing+a+host-like+environment+for+the+study+of+Staphylococcus+aureus+and+Pseudomonas+aeruginosa+biofilms.">Ex situ model of biofilm-associated wounds: Providing a host-like environment for the study of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.</a> Journal of Applied Microbiology. 131 (3), 1487-1497 (2021).</li><li>Johnson, C. J., 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=Augmenting+the+activity+of+chlorhexidine+for+decolonization+of+Candida+auris+from+porcine+skin.">Augmenting the activity of chlorhexidine for decolonization of Candida auris from porcine skin.</a> Journal of Fungi. 7 (10), 804 (2021).</li><li>Horton, M. V., 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=Candida+auris+Forms+High-Burden+Biofilms+in+Skin+Niche+Conditions+and+on+Porcine+Skin.">Candida auris Forms High-Burden Biofilms in Skin Niche Conditions and on Porcine Skin.</a> mSphere. 5 (1), 00910-00919 (2020).</li><li>Ashrafi, M., 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+biofilm+formation+on+human+skin+wound+models+and+demonstration+of+clinically+translatable+bacteria-specific+volatile+signatures.">Validation of biofilm formation on human skin wound models and demonstration of clinically translatable bacteria-specific volatile signatures.</a> Scientific Reports. 8, 1-16 (2018).</li><li>Brackman, G., Coenye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+biofilm+wound+models+and+their+application.">In vitro and in vivo biofilm wound models and their application.</a> Advances in Experimental Medicine and Biology. 897, 15-32 (2016).</li><li>Rumbaugh, K. P., Carty, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Models+of+Biofilm+Infection.">In Vivo Models of Biofilm Infection.</a> Biofilm Infections. , 267-290 (2011).</li><li>Boase, S., Valentine, R., Singhal, D., Tan, L. W., Wormald, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sheep+model+to+investigate+the+role+of+fungal+biofilms+in+sinusitis:+Fungal+and+bacterial+synergy.">A sheep model to investigate the role of fungal biofilms in sinusitis: Fungal and bacterial synergy.</a> International Forum of Allergy &amp; Rhinology. 1 (5), 340-347 (2011).</li><li>Williams, D. 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=Experimental+model+of+biofilm+implant-related+osteomyelitis+to+test+combination+biomaterials+using+biofilms+as+initial+inocula.">Experimental model of biofilm implant-related osteomyelitis to test combination biomaterials using biofilms as initial inocula.</a> Journal of Biomedical Materials Research. Part A. 100 (7), 1888-1900 (2012).</li><li>Scheerlinck, J. P. Y., Snibson, K. J., Bowles, V. M., Sutton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+sheep+models:+From+asthma+to+vaccines.">Biomedical applications of sheep models: From asthma to vaccines.</a> Trends in Biotechnology. 26 (5), 259-266 (2008).</li><li>Metcalfe, A. D., Ferguson, M. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+replacement+skin:+The+crossroads+of+biomaterials,+wound+healing,+embryonic+development,+stem+cells+and+regeneration.">Tissue engineering of replacement skin: The crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration.</a> Journal of the Royal Society Interface. 4 (14), 413-417 (2007).</li><li>Kazemi-Darabadi, S., Sarrafzadeh-Rezaei, F., Farshid, A. A., Dalir-Naghadeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogenous+skin+fibroblast+transplantation+enhances+excisional+wound+healing+following+alloxan+diabetes+in+sheep,+a+randomized+controlled+trial.">Allogenous skin fibroblast transplantation enhances excisional wound healing following alloxan diabetes in sheep, a randomized controlled trial.</a> International Journal of Surgery. 12 (8), 751-756 (2014).</li><li>Martinello, T., 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=Allogeneic+mesenchymal+stem+cells+improve+the+wound+healing+process+of+sheep+skin.">Allogeneic mesenchymal stem cells improve the wound healing process of sheep skin.</a> BMC Veterinary Research. 14 (1), 1-9 (2018).</li><li>Roberts, C. D., Windsor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+pain+management+solutions+in+animals+may+provide+improved+wound+pain+reduction+during+debridement+in+humans:+An+opinion+informed+by+veterinary+literature.">Innovative pain management solutions in animals may provide improved wound pain reduction during debridement in humans: An opinion informed by veterinary literature.</a> International Wound Journal. 16 (4), 968 (2019).</li><li>Mazzone, 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=Bioengineering+and+in+utero+transplantation+of+fetal+skin+in+the+sheep+model:+A+crucial+step+towards+clinical+application+in+human+fetal+spina+bifida+repair.">Bioengineering and in utero transplantation of fetal skin in the sheep model: A crucial step towards clinical application in human fetal spina bifida repair.</a> Journal of Tissue Engineering and Regenerative Medicine. 14 (1), 58-65 (2020).</li><li>Olkowska, E., Gr&#382;ini&#263;, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+models+for+dermal+exposure+assessment+of+phthalates.">Skin models for dermal exposure assessment of phthalates.</a> Chemosphere. 295, 133909 (2022).</li><li>Couto, N., 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=Label-free+quantitative+proteomics+and+substrate-based+mass+spectrometry+imaging+of+xenobiotic+metabolizing+enzymes+in+ex+vivo+human+skin+and+a+human+living+skin+equivalent+model.">Label-free quantitative proteomics and substrate-based mass spectrometry imaging of xenobiotic metabolizing enzymes in ex vivo human skin and a human living skin equivalent model.</a> Drug Metabolism and Disposition. 49 (1), 39-52 (2021).</li></ol>

The development of antimicrobials is an important but expensive venture that is estimated to cost around $1 billion and take around 15 years to complete. Over 90% of antimicrobial drug discovery and preclinical studies of antimicrobial drug efficacy are carried out by academic researchers and small to medium companies with typically less than 50 employees22. These teams are very financially constrained, which makes the failure of lead molecules in later stages of translational research calamitous....

Skin infections are an important global issue, with large economic costs to healthcare providers around the world. The development of multidrug resistance and biofilm formation by pathogens plays a key role in the prevalence of non-healing wounds1,2,3,4. As a result of this, skin and soft tissue infections are one of the more common reasons for extended hospitalization and subsequent readmission5. Delays in wound healing are costly for both the patient and healthcare providers, with some estimates suggesting around 6.5 million patients are affected annually in the US. In the UK, skin infections and associated complications result in approximately 75,000 deaths annually2,4,6.
Staphylococcus aureus (S. aureus) is a formidable wound pathogen frequently isolated from patient wounds2,7. The rate of emergence of multidrug resistance increased drastically in the 2000s. During this time, around 60% of acute bacterial skin and skin structure infections were culture positive for methicillin-resistant S. aureus1. The increasing number of multidrug-resistant strains among Staphylococci, and indeed other pathogens, within the last 2 decades indicates an urgent need for the rapid development of antibiotics with new modes of action that can overcome resistance.
However, since the early 2000s, antibiotic discovery programs have been dominated by longer developmental times and low success rates, with only 17% of novel antibiotics entering clinical trials in the US achieving market approval8. This suggests a disparity between results from in vitro testing of emerging antibiotics and their clinical outcomes. It can be contended that this disparity is largely due to differences in bacterial physiology during infections in vivo and during conventional microbiological methods when testing the efficacy of antibiotics in the in vitro preclinical stages. Therefore, novel laboratory methods that are more representative of bacterial physiology during infection are needed to improve the success rates in antibiotic discovery programs.
Current methods for studying skin infections include studies in live animals (e.g., mice), ex vivo skin models (e.g., porcine), and 3D tissue-engineered skin models (e.g., human)9,10,11,12. Studies in live animals are strictly regulated and have relatively low throughput. In animal models, wounding and infection cause significant distress to the animals and raise ethical concerns. Human skin models, ex vivo or tissue-engineered, require ethical approval, compliance with local and global legislation (the Human Tissue Act, the Declaration of Helsinki), and there is difficulty in acquiring tissues, with some requests taking years to fulfil13,14. Both model types are labor intensive and require significant expertise to ensure experimental success. Some current ex vivo skin infection models require pre-inoculated discs and additives for the wound bed to enable infection; although these models are incredibly useful, there are limitations in the infection process as additives limit the utilization of the wound bed as a nutrient source10,15,16,17. The model described in this study uses no additives to the wound bed, which ensures that the pathology of infection and viable cell counts are a result of direct utilization of the wound bed as the only nutrient source.
Given the need for new laboratory methods, a novel high-throughput ex vivo ovine model of skin infections for use in evaluating the efficacy of emerging antibiotics has been developed. Skin infection studies face many challenges-high costs, ethical concerns, and models that do not show a full picture20,21. Ex vivo models and 3D explant models allow for better visualization of the disease process and the impact treatments can have from a more clinically relevant model. Here, the setup of a novel ovine skin model is described, which&#160;is simple, reproducible, and clinically relevant and has high throughput. Ovine skin was chosen as sheep are one of the large mammals commonly used to model responses to infections in vivo. Moreover, they are readily available from abattoirs, ensuring a steady supply of skin for research, and their carcasses are not scalded, ensuring good tissue quality. This study used S. aureus as the exemplar pathogen; however, the model works well with other microorganisms.

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	<tbody>
		<tr>
			<td>24 Well Companion Plate</td>
			<td>SLS&#160;</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>4 mm Biopsy Punch</td>
			<td>Williams Medical</td>
			<td>D7484</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes</td>
			<td>Fisher Scientific&#160;</td>
			<td>10788561</td>
			<td></td>
		</tr>
		<tr>
			<td>8 mm Biopsy Punch</td>
			<td>Williams Medical</td>
			<td>D7488</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution, sterile</td>
			<td>Sigma&#160;</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Colour Pro Style Cordless Hair Clipper</td>
			<td>Wahl</td>
			<td>9639-2117X</td>
			<td>Hair Clippers</td>
		</tr>
		<tr>
			<td>Dual Oven Incubator</td>
			<td>SLS</td>
			<td>OVe1020</td>
			<td>Sterilising oven</td>
		</tr>
		<tr>
			<td>Epidermal growth factor&#160;</td>
			<td>SLS</td>
			<td>E5036-200UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Honeywell</td>
			<td>458600-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 HAM</td>
			<td>Sigma</td>
			<td>N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum&#160;</td>
			<td>Labtech International</td>
			<td>CA-115/500</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>15307805</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Removal Cream</td>
			<td>Veet</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i</td>
			<td>Thermo Scientific</td>
			<td>15373212&#160;</td>
			<td>Tissue culture incubator</td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R</td>
			<td>VWR</td>
			<td>521-2242</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Homogenizer 220, Handheld</td>
			<td>Fisher Scientific</td>
			<td>15575809</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer 220, plastic blending cones</td>
			<td>Fisher Scientific&#160;</td>
			<td>15585819</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert Individual 24 well 0.4um membrane</td>
			<td>VWR International</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, recombinant Human</td>
			<td>SLS</td>
			<td>91077C-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199 (MK media)</td>
			<td>Sigma</td>
			<td>M0393</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, cell culture Costar 96 well</td>
			<td>Fisher Scientific</td>
			<td>10687551</td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron</td>
			<td>Infors</td>
			<td>Not applicable</td>
			<td>Bacterial incubator</td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Sigma&#160;</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SLS&#160;</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate seals</td>
			<td>Fisher Scientific</td>
			<td>ESI-B-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe 2020</td>
			<td>Fisher Scientific</td>
			<td>1284804</td>
			<td>Class II microbiology safety cabinet</td>
		</tr>
		<tr>
			<td>Scalpel blade number 15</td>
			<td>Fisher Scientific</td>
			<td>O305</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Swann Morton</td>
			<td>Fisher Scientific</td>
			<td>11849002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed Allis Tissue Forceps</td>
			<td>Rocialle</td>
			<td>RSPU500-322</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Merck Life Science UK Limited</td>
			<td>14432-500G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Merck Life Science UK Limited</td>
			<td>41298-500G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimoba Tablets</td>
			<td>Quip Labs</td>
			<td>VMTAB75BX</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel high-throughput ex vivo ovine skin wound model for testing emerging antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

The identification of a route to sterilize the skin before setting up the wound infection model was challenging. The challenge lay in sterilizing the skin without damaging the different skin layers, which may then go on to have unintended consequences in the outcome of infection. To identify an appropriate sterilization regime, different treatments were tried for varying lengths of time, as outlined in Table 1. Contamination was recorded as the development of turbidity after 48 h in the MK medium used to...

Watch this Scientific Journal Video about A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics at JoVE.com

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...

Research

JoVE Journal

Immunology and Infection

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

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Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

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The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

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