概要

小鼠导管相关 白色念珠菌 感染模型

Published: March 22, 2024
doi:

概要

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 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?…

開示

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

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記事を引用
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).

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