Okurowska Katarzyna, Roy Sanhita, Thokala Praveen, Partridge Lynda, Garg Prashant, MacNeil Sheila, Monk Peter N, Karunakaran Esther
Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield; Department of Chemical and Biological Engineering, University of Sheffield.
Hyderabad Eye Research Foundation, L V Prasad Eye Institute.
J Vis Exp. 2020 May 12(159). doi: 10.3791/61156.
When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.
在开发新型抗菌药物时，动物试验的成功取决于能否准确地将体外试验中的抗菌效果外推至体内动物感染情况。现有的体外试验通常会高估抗菌效果，因为未考虑宿主组织作为扩散屏障的存在。为克服这一瓶颈，我们利用铜绿假单胞菌作为典型生物，开发了一种细菌性角膜炎的离体猪角膜模型。本文描述了猪角膜的制备方法和建立感染的方案。定制的玻璃模具可使角膜用于感染研究的设置变得简单直接。该模型模拟体内感染，因为细菌增殖取决于细菌损伤角膜组织的能力。通过活菌平板计数评估菌落形成单位数量的增加来验证感染的建立。结果表明，使用本文所述方法可在离体角膜中以高度可重复的方式建立感染。该模型未来可扩展，以模拟由铜绿假单胞菌以外的微生物引起的角膜炎。该模型的最终目的是在更能代表体内感染的情况下，研究抗菌化疗对细菌感染进程的影响。这样做，本文所述模型将减少用于试验的动物数量，提高临床试验的成功率，并最终使新型抗菌药物能够快速转化至临床应用。
