白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像
Summary
We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.
Abstract
对生物和/或非生物表面白色念珠菌生物膜发展代表对于住院患者特定威胁。到目前为止，C。白念珠菌生物膜已主要研究了在体外 ，但存在用于在体内条件下更好地理解本动力过程的一个关键需求。我们开发了一种在体内的皮下大鼠模型来研究C.白色念珠菌生物膜的形成。在我们的模型中，多个（最多9） -感染念珠菌的设备被植入到该动物的背部的部分。这给了我们一个主要的优势中央静脉导管模型系统，因为它可以让我们学习几个独立的生物膜在一个动物。最近，我们适应这个模型来研究C.白色念珠菌生物膜发展在BALB / c小鼠。在这种模式下，成熟的C。白色念珠菌生物膜内的48小时开发和示范典型的三维生物膜结构。真菌生物膜的量化传统上分析验尸并要求主机牺牲。因为这需要使用许多动物来进行动力学研究，我们采用非侵入性的生物发光成像（BLI）纵向跟进体内成熟C.白色念珠菌生物膜发展在我们皮下的模型。C.白念珠菌细胞被工程化以表达附着到细胞壁的Gaussia princeps的荧光素酶基因（GLUC）。生物发光信号是由所加的基板腔肠素转换成光，可测得的萤光素酶生成。在BLI信号相似，从植导管获得的细胞计数。非侵入性成像的体内生物膜形成定量提供即时申请在体内条件下筛选和抗真菌药验证，以及用于基于宿主-病原体相互作用的研究，在此有助于更好地理解荷兰国际集团的导管相关感染的发病机制。
Introduction
白色念珠菌是一种共生生物，它可以在不同的地点健康个体中找到，例如在皮肤上或作为胃肠道和阴道菌群的一部分。然而，在住院治疗，尤其是免疫功能低下的患者，可能会造成大范围感染1。在这样的个人，削弱免疫系统使念珠菌细胞传播到血液中，并侵入深层组织造成危及生命的感染。此外，非生物基材如中心静脉和导尿管的存在下，人工心脏瓣膜和关节可提供一个利基念珠菌附件2。粘附于这种基材是用于进一步生物膜发展，它代表一个层嵌入在细胞外聚合物材料酵母和菌丝细胞，主要由多糖2的一个先决条件。C.白色念珠菌导管机相关性感染与高死亡率有关。生物膜的一般特征是它们减少的易感性的已知抗真菌剂，如唑类3,4。仅新类抗真菌药物，例如棘白菌素和两性霉素B的脂质体制剂被证明是有效对抗导管相关感染5-7。由于生物膜抵御抗真菌剂，治疗方法非常有限，往往导致拔除尿管及其后续替代作为唯一的解决方案。
我们大部分的电流C.理解白念珠菌生物膜发展来源于在非生物基质如聚苯乙烯，或塑料用于上述装置， 即，有机硅，聚氨酯2的制造体外研究。这些模型是相当先进的，并试图尽可能接近模拟体内的情况成为可能。然而，这些系统不涉及连续血流量和叔他宿主的免疫系统。这导致了在体内模型系统，诸如在中央静脉导管（CVC）模型8-10，口腔念珠菌11义齿口腔炎模型和导管相关念珠菌12小鼠模型的发展。此外，C。白色念珠菌生物膜发展进行了研究在体内的粘膜表面，如那些从阴道13和口腔14上。我们的实验室贡献了建立皮下的C.白念珠菌生物膜模型，这是基于对受感染的导管件上Sprague Dawley大鼠15的背面的植入物。被成功地用于在我们的实验室该模型以测试生物膜易感性氟康唑和棘白菌素药物5,16，以研究双氯芬酸和卡泊芬净17的组合治疗的效果。最近，我们采用了这种系统在BALB / c小鼠18,19使用。在与其它体内模型相比，本皮下模型的主要优点是研究每只动物植入的导管件的管腔内部开发多种生物膜的可能性。
以减少实验动物的数量，我们已经适应了这个模型来研究C的发展白色念珠菌生物膜的非侵入性地用生物发光成像（BLI）18,19。这种方法被证明是一个功能强大的技术，它可以用来通过测量在感兴趣的区域（植入导管的区域中的情况下）特定BLI信号进行量化生物膜，避免动物牺牲。相比于细菌，其可以同时表达该基因和所需的生物发光反应基板由于引入一个特定勒克斯操纵子20的，大多数真核有机体，包括C。白色念珠菌，依赖于荧光素酶基因的异源表达加上特定基底，例如D-萤光素或腔肠素21的外部施用。这可能是由于真菌细胞壁和C的存在下白色念珠菌形态发生，细胞内输送的底物萤光素酶是一种主要挑战21。为了解决这个问题，Enjalbert 等人 22工程化的菌株，其中的合成C.该基因的天然分泌的Gaussia princeps的荧光素酶（GLUC）的白色念珠菌的密码子最优化形式融合到到C.白色念珠菌PGA59基因，GPI-锚定的细胞壁蛋白。因为荧光素酶的细胞壁中存在的，可避免关于基板的胞内可用性问题。这个特殊的系统被用来研究引起C.浅表感染念珠菌 22。最近，BLI也被用来跟踪口咽念珠菌及其possibl的进展Ë治疗23。这样的研究结果支持使用BLI作为有前途的技术，研究引起自由生活的细胞，而且还与设备相关的感染的感染。
在这项研究中，我们描述了C.白念珠菌生物膜发展上的聚氨酯导管件在BALB / c小鼠和其量化使用BLI。我们期间密合性的时期，随后通过植入小鼠和随后的生物膜发展在活的动物提供体外定植聚氨酯导管的详细协议。除了 测量由C.发射的BLI信号白色念珠菌细胞，我们也决定了菌落形成单位与标准技术生物膜真菌负荷的定量比较。
Protocol
Representative Results
Discussion
使用动物模型，尤其是啮齿动物模型的，用于研究致力于微生物生物膜是非常重要的，因为宿主的免疫系统是在生物膜形成， 在体外模型不能解释的一个重要因素。在这项研究中，我们描述了一个相对简单的皮下C.白色念珠菌生物膜的小鼠模型，可以很容易地通过了一个研究实验室，也不需要很强的技术技能。最初开发这种模式来研究大鼠24 表皮葡萄球菌生物膜的形?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by the KU Leuven PF ‘IMIR’, the FWO Research community on biology and ecology of bacterial and fungal biofilms (FWO: WO.026.11N) and by the FWO project G.0804.11. SK gratefully acknowledges KU Leuven for the PDMK 11/089 fellowship and FWO for the postdoctoral fellowship. We are grateful to Nico Vangoethem for his assistance with preparation of the figures. We would like to acknowledge Celia Lobo Romero for technical assistance during in vivo experimental procedures.
Materials
| Name of Reagent | Company | Catalog Number | Comments/Description |
| Yeast extract granulated | Merck | MERC1.03753.0500 | |
| Bacteriological peptone | Oxoid | LP037B | |
| Agar granulated | Difco | 214530 | |
| D-(+)-glucose | Fluka | 49159-5KG | |
| Phosphate buffered saline | Prepared in the laboratory | for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4 | |
| RPMI1640 with L-glutamine and without sodium carbonate | Sigma | R6504-1L | Prepare according the protocol for Candida albicans drug susceptibility testing |
| 3-(N-Morpholino)propanesulfonic acid (MOPS) | Sigma | M1254 | MOPS is used to adjust the pH of RPMI medium (pH 7.0) |
| fetal bovine serum (FBS) | Sigma | F7524 | |
| Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730) | BBraun | CV-15703 | Polyurethane part cut into 1 cm pieces |
| Dexamethasone | Fagron SAS, France | 611139 | Immunosuppressant (stock solution 10 mg/ml) |
| Ampicillin | Duchefa Biochemie, The Netherlands | A0104 | Antibacterial prophylaxis |
| Ketamine 1000 | Pfizer | 804 119 | Anesthetic |
| Domitor | Pfizer | 134737-1 | Anesthetic |
| Antisedan | Pfizer | 134783-2 | Reversal of anesthesia |
| Xylocaine gel (2%) – this is Linisol | AstraZeneca | 352 1206 | Local anesthetic for the skin |
| Terramycin/ polymyxin-b ophthalmic ointment | To prevent drying and infection of eyes | ||
| Coelenterazine | Prolume (Nanolight) | NF-CTZ-FB | Light sensitive agent (must be kept in the dark) |
| Iodine isopropanol (1%) | 3M™ DuraPrep™ | Disinfectant for the skin | |
| 0.5 % chlorhexidine in 70 % alcohol. | Cedium | Disinfectant for the skin | |
| Equipment | |||
| Cell counting chamber | |||
| Insulin syringes (0.3 ml) | Terumo Myjector 29G | 324826 | For injection of coelenterazine |
| Electric razor | For small animals | ||
| Sterile surgical tools | Scissors, 2 pairs of tweezers, scalpel | ||
| Heating pad | Leica | 14042321474 | |
| Skin suture | Johnson&Johnson | K890H | Surgical thread, needle |
| Water bath sonicator | Branson 2210 | ||
| BLI camera (IVIS Spectrum) | Perkin Elmer, Alameda | IVISSPE | |
| Living Image software | Perkin Elmer, Alameda | (version 4.2) |
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