Mannala Gopala K, Rupp Markus, Walter Nike, Scholz Konstantin J, Simon Michaela, Riool Martijn, Alt Volker
Department of Trauma Surgery, University Hospital Regensburg, Regensburg, Germany.
Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany.
J Orthop Res. 2023 Nov;41(11):2547-2559. doi: 10.1002/jor.25572. Epub 2023 Apr 30.
Fungal implant-associated bone infections are rare but difficult to treat and often associated with a poor outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play a major role to study biofilm development and potential new treatment options; however, there are only a very few in vivo models to study fungi-associated biofilms. Furthermore, mammalian infection models are replaced more and more due to ethical restrictions with other alternative models in basic research. Recently, we developed an insect infection model with Galleria mellonella larvae to study biofilm-associated infections with bacteria. Here, we further expanded the G. mellonella model to study in vivo fungal infections using Candida albicans and Candida krusei. We established a planktonic and biofilm-implant model to test different antifungal medication with amphotericin B, fluconazole, and voriconazole against the two species and assessed the fungal biofilm-load on the implant surface. Planktonic infection with C. albicans and C. krusei showed the killing of the G. mellonella larvae at 5 × 10 colony forming units (CFU). Treatment of larvae with antifungal compounds with amphotericin B and fluconazole showed significant survival improvement against planktonic C. albicans infection, but voriconazole had no effect. Titanium and stainless steel K-wires were preincubated with C. albicans and implanted inside the larvae to induce biofilm infection on the implant surface. The survival analysis revealed significantly reduced survival of the larvae with Candida spp. infection compared to noninfected implants. The treatment with antifungal amphotericin B and fluconazole resulted in a slight and nonsignificant improvement survival of the larvae. The treatment with the antifungal compounds in the biofilm-infection model was not as effective as in the planktonic infection model, which highlights the resistance of fungal biofilms to antifungal compounds like in bacterial biofilms. Scanning electron microscopy (SEM) analysis revealed the formation of a fungal biofilm with hyphae and spores associated with larvae tissue on the implant surface. Thus, our study highlights the use of G. mellonella larvae as alternative in vivo model to study biofilm-associated implant fungal infections and that fungal biofilms exhibit high resistance profiles comparable to bacterial biofilms. The model can be used in the future to test antifungal treatment options for fungal biofilm infections.
真菌植入物相关骨感染虽罕见但难以治疗，且常导致患者预后不良。念珠菌属约占所有真菌感染的90%。体内生物膜模型在研究生物膜形成及潜在新治疗方案方面发挥着重要作用；然而，用于研究真菌相关生物膜的体内模型却非常少。此外，由于基础研究中的伦理限制，哺乳动物感染模型越来越多地被其他替代模型所取代。最近，我们开发了一种用大蜡螟幼虫的昆虫感染模型来研究细菌生物膜相关感染。在此，我们进一步扩展了大蜡螟模型，以研究白色念珠菌和克鲁斯念珠菌的体内真菌感染。我们建立了浮游菌和生物膜植入模型，用两性霉素B、氟康唑和伏立康唑测试针对这两种真菌的不同抗真菌药物，并评估植入物表面的真菌生物膜负荷。白色念珠菌和克鲁斯念珠菌的浮游菌感染在5×10菌落形成单位（CFU）时显示能杀死大蜡螟幼虫。用两性霉素B和氟康唑等抗真菌化合物处理幼虫，对浮游白色念珠菌感染显示出显著的生存改善，但伏立康唑无效。将钛和不锈钢克氏针与白色念珠菌预孵育后植入幼虫体内，以诱导植入物表面的生物膜感染。生存分析显示，与未感染植入物相比，念珠菌属感染的幼虫存活率显著降低。用抗真菌两性霉素B和氟康唑治疗，幼虫存活率略有提高，但无显著差异。生物膜感染模型中抗真菌化合物的治疗效果不如浮游菌感染模型，这突出了真菌生物膜与细菌生物膜一样对抗真菌化合物具有抗性。扫描电子显微镜（SEM）分析显示，植入物表面形成了与幼虫组织相关的带有菌丝和孢子的真菌生物膜。因此，我们的研究强调了使用大蜡螟幼虫作为替代体内模型来研究生物膜相关植入物真菌感染，以及真菌生物膜表现出与细菌生物膜相当的高抗性特征。该模型未来可用于测试真菌生物膜感染的抗真菌治疗方案。
