小鼠导管相关 白色念珠菌 感染模型
Summary
我们建立了 白色念珠菌相关导管相关感染 （CRI） 的小鼠模型，其中在导管上形成生物膜，白色 念 珠菌与宿主之间的相互作用与临床 CRI 密切相关。该模型有助于筛选白色 念珠菌 生物膜相关CRI的治疗方法，为临床转化奠定基础。
Abstract
导管相关感染 （CRI） 是导管植入过程中由 白色念珠菌 引起的常见院内感染。通常，在导管的外表面形成生物膜并导致播散性感染，这对患者来说是致命的。诊所没有有效的预防和治疗管理。因此，迫切需要建立CRI的动物模型，以期临床前筛选其预防和治疗的新策略。在这项研究中，将一根聚乙烯导管（一种广泛使用的医用导管）插入脱毛后 BALB/c 小鼠的背部。 白假丝酵母 随后将表达增强绿色荧光蛋白的ATCC MYA-2876（SC5314）沿导管接种在皮肤表面。3天后，在荧光显微镜下在导管表面观察到强烈的荧光。通过扫描电子显微镜在导管表面发现了成熟而厚的生物膜。这些结果表明白色 念珠菌 在导管表面的粘附、定植和生物膜形成。表皮增生和皮肤标本中炎症细胞浸润表明CRI相关皮肤的组织病理学变化。综上所述，成功建立了小鼠CRI模型。该模型有望有助于白色 念珠菌 相关CRI治疗管理的研究和开发。
Introduction
近年来，随着生物医学材料的发展和应用，种植体相关感染正在成为临床难题1,2。随着医用导管在诊所的广泛应用，每年相关的感染和死亡人数都很大3,4。导管相关感染（CRI）的常见感染途径包括：（1）皮肤表面的病原体浸润到体内并粘附在导管的外表面5,6,7;（2）无菌操作不当的病原体侵入、粘附和定植在导管上;（3）血液循环中的病原体粘附在导管上并定植;（4）被病原微生物污染的药物。
念珠菌是 CRI 8,9 的第三大常见原因。在植入物表面形成生物膜后，极有可能引起血流感染和其他危及生命的侵袭性念珠菌病。预后较差，死亡率高2。据报道，在中心静脉插入后2周内在导管表面形成生物膜，几周后在导管腔内形成生物膜10,11。
在医用导管上形成的白色念珠菌（白色念珠菌）生物膜表现出由酵母、基质和菌丝体组成的双层网络12,13。 白色念珠菌生物膜的形成不仅是耐药性和免疫逃避的关键13，而且对产生播散性孢子也至关重要，这会导致进一步的血源性感染2,12，并导致更严重甚至危及生命的后果。白色念珠菌相关CRI是临床真菌血流感染的主要原因7,14，超过40%的中心静脉导管白色念珠菌感染患者会发展为菌血症15。
根据美国传染病学会的说法，念珠菌 CRI 的推荐治疗方法包括 （1） 拔除受感染的导管;（2）对患者进行14天全身抗真菌治疗8;（3） 重新植入新导管4.然而，在临床应用中，导管有时无法完全移除。一些患者只能用全身性抗生素和抗菌锁定疗法治疗，并伴有强烈的副作用16,17。
现有的白色念珠菌动物模型，如口咽念珠菌病模型、阴道念珠菌病模型和念珠菌病引起的侵袭性全身感染模型18,19 不能很好地与临床 CRI 相关。因此，在这项研究中，建立了小鼠白色念珠菌相关的CRI模型。临床常用的聚乙烯导管被用作皮下植入物20,21，并将白色念珠菌接种在皮肤表面，以模拟白色念珠菌与医用导管的粘附和生物膜的形成。
该模型已成功用于我们的实验室，以筛选不同疗法的抗生物膜作用22。此外，由于导管感染后对 白色念珠 菌的滞后检测，构建了一种含有增强绿色荧光蛋白（EGFP）的 白色念 珠菌菌株，并将其接种在小鼠体内，以利于直观观察植入导管上 白色念 珠菌的菌落和生物膜。
Protocol
实验动物，雄性BALB / c小鼠（12-16g），购自习交通大学医学中心实验动物中心。所有程序均经习交通大学机构动物伦理委员会批准，许可证号为SCXK（陕西）2021-103。 1.缓冲液和设备准备 用高表达质粒 pCaExp 转染 白色念珠菌 菌株。从ATCC购买白色念珠菌（SC5314）。通过用含有EGFP基因完整开放阅读框的高表达质粒pCaExp转染白色念珠…
Representative Results
扫描电镜可以观察到导管上的 白色念珠菌 和生物膜。如 图322所示，导管组聚乙烯导管表面光滑，未观察到粘附的病原微生物。然而，在模型组的聚乙烯导管表面可见成熟致密的 白色念 珠菌生物膜，表明在实验条件下， 白色念 珠菌能够在小鼠的导管表面成功定植并形成生物膜。此外，荧光显微镜结果进一步验证了上述结论（<strong clas…
Discussion
CRI 是临床实践中最常见的院内感染之一23.皮肤附属物中的病原体，如表皮、皮脂腺和毛囊，都是 CRI23,24 的可能原因。念珠菌是引起CRI的第三大病原体，其中白色念珠菌是最常见的生物膜感染类型25,26。因此，我们旨在建立白色念珠菌生物膜相关CRI的相关动物模型，以支?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
感谢陕西省自然科学基金（批准号：2021SF-118）和国家自然科学基金（批准号：81973409,82204631）的财政支持。
Materials
| 0.5 Mactutrius turbidibris | Shanghai Lujing Technology Co., Ltd | 5106063 | |
| 2.5% glutaraldehyde fixative solution | Xingzhi Biotechnology Co., Ltd | DF015 | |
| 4 °C refrigerator | Electrolux (China) Electric Co., Ltd | ESE6539TA | |
| Agar | Beijing Aoboxing Bio-tech Co., Ltd | 01-023 | |
| Analytical balances | Shimadzu | ATX124 | |
| Autoclaves Sterilizer | SANYO | MLS-3750 | |
| Butanol | Tianjin Chemio Reagent Co., Ltd | 200-889-7 | |
| Carbenicillin | Amresco | C0885 | |
| Eclipse Ci Nikon upright optical microscope | Nikon | Eclipse Ts2-FL | |
| Glucose | Macklin | D823520 | |
| Inoculation ring | Thermo Scientific | 251586 | |
| Isoflurane | RWD | 20210103 | |
| Paraformaldehyde | Beyotime Biotechnology | P0099 | |
| PAS dye kit | Servicebio | G1285 | |
| Peptone | Beijing Aoboxing Bio-tech Co., Ltd | 01-001 | |
| Polyethylene catheter | Shining Plastic Mall | PE100 | |
| RWD R550 multi-channel small animal anesthesia machine | RWD | R550 | |
| SEM | Hitachi | TM-1000 | |
| Temperature incubator | Shanghai Zhichu Instrument Co., Ltd | ZQTY-50N | |
| Ultrapure water water generator | Heal Force | NW20VF | |
| Ultrasound machine | Do-Chrom | DS10260D | |
| Xylene | Sinopharm Chemical Reagent Co., Ltd | 10023428 | |
| Yeast extract | Thermo Scientific Oxoid | LP0021B |
References
- Kojic, E. M., Darouiche, R. O. Candida infections of medical devices. microbiology reviews. 17 (2), 255-267 (2004).
- Giri, S., Kindo, A. J. A review of Candida species causing blood stream infection. Indian Journal of Medical Microbiology. 30 (3), 270-278 (2012).
- Weinstein, R. A., Darouiche, R. O. Device-associated infections: A macroproblem that starts with microadherence. Clinical Infectious Diseases. 33 (9), 1567-1572 (2001).
- Mermel, L. A., et al. Guidelines for the management of intravascular catheter-related infections. Clinical Infectious Diseases. 32 (9), 1249-1272 (2001).
- Seidler, M., Salvenmoser, S., Müller, F. -. M. C. In vitro effects of micafungin against Candida biofilms on polystyrene and central venous catheter sections. International Journal of Antimicrobial Agents. 28 (6), 568-573 (2006).
- Chaves, F., et al. Diagnosis and treatment of catheter-related bloodstream infection: Clinical guidelines of the Spanish Society of Infectious Diseases and Clinical Microbiology and (SEIMC) and the Spanish Society of Spanish Society of Intensive and Critical Care Medicine and Coronary Units (SEMICYUC). Medicina Intensiva. 42 (1), 5-36 (2018).
- Raad, I. I., Bodey, G. P. Infectious complications of indwelling vascular catheters. Clinical Infectious Diseases. 15 (2), 197-208 (1992).
- Paul DiMondi, V., Townsend, M. L., Johnson, M., Durkin, M. Antifungal catheter lock therapy for the management of a persistent Candida albicans bloodstream infection in an adult receiving hemodialysis. Pharmacotherapy. 34 (7), e120-e127 (2014).
- Bouza, E., Guinea, J., Guembe, M. The role of antifungals against candida biofilm in catheter-related candidemia. Antibiotics (Basel). 4 (1), 1-17 (2014).
- Raad, I., et al. Ultrastructural analysis of indwelling vascular catheters: a quantitative relationship between luminal colonization and duration of placement. The Journal of Infectious Diseases. 168 (2), 400-407 (1993).
- Yousif, A., Jamal, M. A., Raad, I. Biofilm-based central line-associated bloodstream infections. Advances in Experimental Medicine and Biology. 830, 157-179 (2015).
- Douglas, L. J. Candida biofilms and their role in infection. Trends in Microbiology. 11 (1), 30-36 (2003).
- Mack, D., et al. Biofilm formation in medical device-related infection. International Journal of Artificial Organs. 29 (4), 343-359 (2006).
- Schinabeck, M. K., et al. Rabbit model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock therapy. Antimicrobial Agents and Chemotherapy. 48 (5), 1727-1732 (2004).
- Anaissie, E. J., Rex, J. H., Uzun, O., Vartivarian, S. Predictors of adverse outcome in cancer patients with candidemia. The American Journal of Medicine. 104 (3), 238-245 (1998).
- Fujimoto, K., Takemoto, K. Efficacy of liposomal amphotericin B against four species of Candida biofilms in an experimental mouse model of intravascular catheter infection. Journal of Infection and Chemotherapy. 24 (12), 958-964 (2018).
- Shuford, J. A., Rouse, M. S., Piper, K. E., Steckelberg, J. M., Patel, R. Evaluation of caspofungin and amphotericin B deoxycholate against Candida albicans biofilms in an experimental intravascular catheter infection model. The Journal of Infectious Diseases. 194 (5), 710-713 (2006).
- Koh, A. Y., Köhler, J. R., Coggshall, K. T., Van Rooijen, N., Pier, G. B. Mucosal damage and neutropenia are required for Candida albicans dissemination. PLoS Pathogens. 4 (2), e35 (2008).
- Tucey, T. M., et al. Glucose homeostasis is important for immune cell viability during candida challenge and host survival of systemic fungal infection. Cell Metabolism. 27 (5), 988-1006 (2018).
- Lawrence, E. L., Turner, I. G. Materials for urinary catheters: a review of their history and development in the UK. Medical Engineering & Physics. 27 (6), 443-453 (2005).
- Schumm, K., Lam, T. B. Types of urethral catheters for management of short-term voiding problems in hospitalized adults: a short version Cochrane review. Neurourology and Urodynamics. 27 (8), 738-746 (2008).
- Mo, F., et al. Development and evaluation of a film forming system containing myricetin and miconazole nitrate for preventing candida albicans catheter-related infection. Microbial Drug Resistance. 28 (4), 468-483 (2022).
- Balikci, E., Yilmaz, B., Tahmasebifar, A., Baran, E. T., Kara, E. Surface modification strategies for hemodialysis catheters to prevent catheter-related infections: A review. Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 109 (3), 314-327 (2021).
- María, L. T., Alejandro, G. S., María Jesús, P. G. Central venous catheter insertion: Review of recent evidence. Best Practice & Research. Clinical Anaesthesiology. 35 (1), 135-140 (2021).
- Kojic, E. M., Darouiche, R. O. Candida infections of medical devices. Clinical Microbiology Reviews. 17 (2), 255-267 (2004).
- He, Y., et al. Retrospective analysis of microbial colonization patterns in central venous catheters, 2013-2017. Journal of Healthcare Engineering. 2019, 8632701 (2019).
- Mo, F., et al. In vitro and in vivo effects of the combination of myricetin and miconazole nitrate incorporated to thermosensitive hydrogels on C. albicans biofilms. Phytomedicine. 71, 153223 (2020).
- Cantón-Bulnes, M. L., Garnacho-Montero, J. Practical approach to the management of catheter-related bloodstream infection. Revista Espanola de Quimioterapia. 32 Suppl 2 (Suppl 2), 38-41 (2019).
- Böhlke, M., Uliano, G., Barcellos, F. C. Hemodialysis catheter-related infection: prophylaxis, diagnosis and treatment. The Journal of Vascular Access. 16 (5), 347-355 (2015).
- Fang, X., et al. Effects of different tissue specimen pretreatment methods on microbial culture results in the diagnosis of periprosthetic joint infection. Bone & Joint Research. 10 (2), 96-104 (2021).
- Naumenko, Z. S., Silanteva, T. A., Ermakov, A. M., Godovykh, N. V., Klushin, N. M. Challenging diagnostics of biofilm associated periprosthetic infection in immunocompromised patient: A clinical case. Open Access Macedonian Journal of Medical Sciences. 7 (5), 786-790 (2019).
- Cai, Y., et al. Metagenomic next generation sequencing improves diagnosis of prosthetic joint infection by detecting the presence of bacteria in periprosthetic tissues. International Journal of Infectious Diseases. 96, 573-578 (2020).
- Samanipour, A., Dashti-Khavidaki, S., Abbasi, M. R., Abdollahi, A. Antibiotic resistance patterns of microorganisms isolated from nephrology and kidney transplant wards of a referral academic hospital. Journal of Research in Pharmacy Practice. 5 (1), 43-51 (2016).
- Huang, G., Huang, Q., Wei, Y., Wang, Y., Du, H. Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans. Molecular Microbiology. 111 (1), 6-16 (2019).
- Garlito-Díaz, H., et al. A new antifungal-loaded sol-gel can prevent candida albicans prosthetic joint infection. Antibiotics (Basel). 10 (6), 711 (2021).
Cite This Article
Yang, C., Mo, F., Zhang, J., Zhang, P., Li, Q., Zhang, J. A Catheter-Related Candida albicans Infection Model in Mouse. J. Vis. Exp. (205), e65307, doi:10.3791/65307 (2024).