用于测试新兴抗生素的新型高通量 离体 绵羊皮肤伤口模型
Summary
该协议描述了一种逐步建立感染金黄色葡萄球菌的离体绵羊受伤皮肤模型的方法。与传统微生物学技术相比，这种高通量模型更好地模拟体内感染，并为研究人员提供了一个生理相关的平台来测试新兴抗菌剂的功效。
Abstract
抗菌药物的开发是一个昂贵的过程，成功率越来越低，这使得对抗菌发现研究的进一步投资变得不那么有吸引力。如果在先导物优化阶段可以实施快速失败和廉价失败的方法，那么抗菌药物的发现和随后的商业化可以变得更加有利可图，研究人员可以更好地控制药物设计和配方。本文介绍了一种感染金黄色葡萄球菌的离体绵羊损伤皮肤模型的建立，该模型简单、经济高效、通量高且可重复。模型中的细菌生理学模拟了感染期间细菌增殖取决于病原体破坏组织的能力。伤口感染的建立通过与接种物相比活细菌计数的增加来验证。该模型可用作在先导物优化阶段测试新兴抗菌剂功效的平台。可以说，该模型的可用性将为开发抗菌剂的研究人员提供快速失败和失败便宜的模型，这将有助于提高后续动物试验的成功率。该模型还将促进减少和改进动物用于研究，并最终能够更快，更具成本效益地将用于皮肤和软组织感染的新型抗菌剂转化为临床。
Introduction
皮肤感染是一个重要的全球性问题，给世界各地的医疗保健提供者带来了巨大的经济成本。病原体对多重耐药性和生物膜形成的发展在伤口不愈合的流行中起着关键作用1，2，3，4。因此，皮肤和软组织感染是延长住院和随后再次入院的更常见原因之一5。伤口愈合的延迟对患者和医疗保健提供者来说都是代价高昂的，据估计，美国每年约有 650 万患者受到影响。在英国，皮肤感染和相关并发症每年导致约75，000人死亡2，4，6。
金黄色葡萄球菌（S. aureus） 是一种强大的伤口病原体，经常从患者伤口中分离出来2，7。多重耐药性的出现率在2000年代急剧增加。在此期间，大约 60% 的急性细菌性皮肤和皮肤结构感染对耐甲氧西林 金黄色葡萄球菌1 呈培养阳性。在过去20年中， 葡萄球菌和其他病原体中耐多药菌株的数量不断增加，这表明迫切需要快速开发具有克服耐药性的新作用模式的抗生素。
然而，自 2000 年代初以来，抗生素发现计划一直以较长的开发时间和低成功率为主，只有 17% 的新型抗生素进入美国临床试验获得市场批准8.这表明新兴抗生素的 体外 检测结果与其临床结果之间存在差异。可以争辩说，这种差异主要是由于 体内 感染期间细菌生理学的差异以及在 体外 临床前阶段测试抗生素功效时常规微生物学方法的差异。因此，需要更能代表感染期间细菌生理学的新型实验室方法，以提高抗生素发现计划的成功率。
目前研究皮肤感染的方法包括活体动物（例如小鼠）、离体皮肤模型（例如猪）和3D组织工程皮肤模型（例如人类）的研究9，10，11，12。对活体动物的研究受到严格监管，通量相对较低。在动物模型中，伤害和感染会给动物带来巨大的痛苦，并引起伦理问题。人体皮肤模型，离体或组织工程，需要伦理批准，遵守当地和全球立法（人体组织法，赫尔辛基宣言），并且难以获得组织，有些要求需要数年才能满足13，14。这两种模型类型都是劳动密集型的，需要大量的专业知识才能确保实验成功。目前一些离体皮肤感染模型需要预先接种的椎间盘和伤口床添加剂才能使感染;尽管这些模型非常有用，但在感染过程中存在局限性，因为添加剂限制了伤口床作为营养来源的利用10，15，16，17。本研究中描述的模型不对伤口床使用添加剂，这确保了感染的病理学和活细胞计数是直接利用伤口床作为唯一营养来源的结果。
鉴于对新实验室方法的需求，已经开发出一种用于评估新兴抗生素疗效的新型高通量离 体 羊皮肤感染模型。皮肤感染研究面临许多挑战 – 高成本，伦理问题和无法显示全貌的模型20，21。 离 体模型和3D外植体模型可以更好地可视化疾病过程以及更具临床相关性的模型对治疗的影响。这里描述了一种新的绵羊皮肤模型的建立，该模型简单，可重复，临床相关且具有高通量。选择绵羊皮肤是因为绵羊是通常用于模拟 体内感染反应的大型哺乳动物之一。此外，它们很容易从屠宰场获得，确保为研究提供稳定的皮肤供应，并且它们的尸体不会被烫伤，确保良好的组织质量。这项研究使用 金黄色葡萄球菌 作为典型病原体;但是，该模型适用于其他微生物。
Protocol
来自R.B Elliott和Son屠宰场的羔羊头被用作该项目的皮肤样本来源。所有的羔羊都被宰杀作为食物食用。这些没有丢弃头部，而是被重新用于研究。不需要道德批准，因为组织来自屠宰场丢弃的废物。 1. 灭菌 在收集头部之前对镊子进行消毒，方法是取干净的镊子并在200°C的烤箱中进行干热灭菌1小时。使用前将所有玻璃器皿在121°C高压灭菌15分钟。 在…
Representative Results
在建立伤口感染模型之前确定对皮肤进行消毒的途径具有挑战性。挑战在于在不损害不同皮肤层的情况下对皮肤进行消毒，这可能会继续对感染结果产生意想不到的后果。为了确定适当的灭菌方案，如 表1所示，在不同的时间内尝试了不同的处理方法。污染记录为用于维持皮肤样品的MK培养基中48小时后浑浊的发展。通过组织学监测组织完整性，然后在处理后立即用苏木精和伊红（H&E?…
Discussion
抗菌剂的开发是一项重要但昂贵的冒险，估计耗资约10亿美元，大约需要15年才能完成。超过90%的抗菌药物发现和抗菌药物疗效的临床前研究是由学术研究人员和通常少于50名员工的中小型公司进行的22。这些团队在财务上非常拮据，这使得铅分子在转化研究后期阶段的失败是灾难性的。抗微生物药物耐药性的上升速度超过了新型抗菌药物的发展速度，这进一步加剧了负责任地管?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者要感谢EPSRC（EP/R513313/1）的资助。作者还要感谢切斯特菲尔德卡洛的R.B Elliot和Son屠宰场提供羊头并在项目的早期阶段如此包容，感谢Kasia Emery在整个协议开发过程中的支持，以及谢菲尔德大学感染，免疫和心血管疾病系的Fiona Wright处理组织学样本，并在整个项目中提供了令人难以置信的帮助。
Materials
| 24 Well Companion Plate | SLS | 353504 | |
| 4 mm Biopsy Punch | Williams Medical | D7484 | |
| 50 ml centrifuge tubes | Fisher Scientific | 10788561 | |
| 8 mm Biopsy Punch | Williams Medical | D7488 | |
| Amphotericin B solution, sterile | Sigma | A2942 | |
| Colour Pro Style Cordless Hair Clipper | Wahl | 9639-2117X | Hair Clippers |
| Dual Oven Incubator | SLS | OVe1020 | Sterilising oven |
| Epidermal growth factor | SLS | E5036-200UG | |
| Ethanol | Honeywell | 458600-2.5L | |
| F12 HAM | Sigma | N4888 | |
| Foetal bovine serum | Labtech International | CA-115/500 | |
| Forceps | Fisher Scientific | 15307805 | |
| Hair Removal Cream | Veet | Not applicable | |
| Heracell VIOS 160i | Thermo Scientific | 15373212 | Tissue culture incubator |
| Heraeus Megafuge 16R | VWR | 521-2242 | Centrifuge |
| Homogenizer 220, Handheld | Fisher Scientific | 15575809 | |
| Homogenizer 220, plastic blending cones | Fisher Scientific | 15585819 | |
| Insert Individual 24 well 0.4um membrane | VWR International | 353095 | |
| Insulin, recombinant Human | SLS | 91077C-1G | |
| Medium 199 (MK media) | Sigma | M0393 | |
| Microplate, cell culture Costar 96 well | Fisher Scientific | 10687551 | |
| Multitron | Infors | Not applicable | Bacterial incubator |
| PBS tablets | Sigma | P4417-100TAB | |
| Penicillin-Streptomycin | SLS | P0781 | |
| Plate seals | Fisher Scientific | ESI-B-100 | |
| Safe 2020 | Fisher Scientific | 1284804 | Class II microbiology safety cabinet |
| Scalpel blade number 15 | Fisher Scientific | O305 | |
| Scalpel Swann Morton | Fisher Scientific | 11849002 | |
| Sodium bicarbonate | Sigma | S5761-1KG | |
| Toothed Allis Tissue Forceps | Rocialle | RSPU500-322 | |
| Tryptic Soy Agar | Merck Life Science UK Limited | 14432-500G-F | |
| Tryptic Soy Broth | Merck Life Science UK Limited | 41298-500G-F | |
| Vimoba Tablets | Quip Labs | VMTAB75BX |
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Citar este artículo
Regan, H. C., Taylor, A. F., Karunakaran, E. A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics. J. Vis. Exp. (187), e64041, doi:10.3791/64041 (2022).