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。

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
<ol><li>用高表达质粒 pCaExp 转染 白色念珠菌 菌株。<ol><li>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转染白色念珠菌获得EGFP高表达荧光菌株22（质粒图如图1所示），并将其用于后续实验。</li>
	</ol></li>
	<li>培养转染的 白色念珠菌 菌株。<ol><li>从酵母提取物蛋白胨葡萄糖培养基（YPD）板中选择 白色念珠菌 荧光菌株的单克隆菌落，并在5mL YPD液体培养基（YPD + 50μg/ mL羧苄青霉素）中培养过夜（30°C和220rpm）。</li>
		<li>在室温下以400×g离心5分钟后，将白色念珠菌重悬于生理盐水中。</li>
		<li>通过将浊度与0.5McFarland标准品进行比较，将 白色念珠菌 悬浮液的浓度调整为1×108 个细胞/ mL。</li>
	</ol></li>
	<li>准备手术器械。<ol><li>确保将所有手术器械（剪刀、镊子、止血钳、针架、缝合针）在 121°C 下高压灭菌 30 分钟。使用无菌聚乙烯导管（内径:0.28 mm;外径:0.63 mm）。 注意:本研究中使用的导管用环氧乙烷气体灭菌，并在暴露于紫外线的超洁净台中打开包装超过 30 分钟。在植入小鼠之前，将导管浸入75%乙醇中以防止污染。</li>
	</ol></li>
</ol>2. 小鼠CRI模型的建立
注意:手术过程如 图 2 所示。
<ol><li>在无特定病原体（SPF）条件下使30只BALB / c小鼠（12-16g，雄性）适应，自由获得水和食物，12小时-12小时交替光暗循环。</li>
	<li>将30只BALB / c小鼠随机分为三组（n = 10只小鼠/组）:（A）正常对照组;（B）导管组（植入的导管没有 白色念珠菌）;（C） 模型组（植入 白色念珠菌的导管）。</li>
	<li>用1-4%异氟醚麻醉小鼠，并将小鼠俯卧位放在手术台上。矫正反射丧失和对脚趾刺激无反应证实了麻醉成功。去除毛发，用三轮交替的碘伏或氯己定和酒精磨砂膏对手术部位进行消毒。</li>
	<li>将小鼠留在正常对照组中，无需任何治疗，并免费提供食物和水。</li>
	<li>对于导管组和模型组中的小鼠，将麻醉维持在3%异氟醚。通过对脚趾捏合没有反应来确认足够的麻醉深度，并根据需要调整异氟烷浓度。</li>
	<li>对于导管组中的小鼠，皮内将1mL无菌注射器针头插入脱毛区域以打孔。取出注射器针头后，将导管（长约 1 厘米）插入孔中。</li>
	<li>对于模型组中的小鼠，将20μL 白色念珠 菌悬浮液到背部脱毛区域，以模拟皮肤上的共生 白色念珠菌 。</li>
	<li>溶液被皮肤吸收后，按照步骤2.5中描述的相同程序将导管插入脱毛区域。</li>
	<li>将另外 20 μL 白色念珠 菌沿导管悬浮液到组织中，以模拟外部环境中的 白色念珠 菌。</li>
	<li>用胶带和纱布固定导管，并将小鼠放回笼子中喂食。治疗结束时，连续三天皮下注射美洛昔康（4mg / kg）作为镇痛剂。 注意:手术后，用水和食物仔细喂食小鼠。每天监测小鼠两次。如果小鼠出现喂养困难、体重显着减轻 （10-20%） 和体温过低，则通过 IACUC 批准的方法对小鼠实施安乐死。</li>
</ol>3. CRI模型的评估
<ol><li>3天后，用3%异氟醚麻醉小鼠，并通过宫颈脱位处死它们。从小鼠背部收集导管和皮肤组织样本。</li>
	<li>通过扫描电子显微镜观察导管上的白色念珠菌 和生物膜。<ol><li>将导管浸入4°C的2.5%戊二醛溶液中48小时。用无菌PBS冲洗导管三次。</li>
		<li>用1%锇酸固定导管3小时，并用无菌PBS冲洗三次。</li>
		<li>在梯度乙醇溶液中以递增浓度（50%、70%、80%、90%和100%，15分钟/梯度）将导管上的细胞脱水。</li>
		<li>将导管浸入叔丁醇中三次（每次30分钟）。</li>
		<li>在液氮中快速冷冻导管，并按照制造商的说明在冷冻干燥机中冷冻干燥样品。</li>
		<li>通过离子束沉积用 10 nm 金溅射涂覆导管样品。</li>
		<li>在扫描电子显微镜下（在高真空，1.5kV条件下）观察每组导管表面存在 白色念珠菌 及其生物膜，并记录每组的图像。</li>
	</ol></li>
	<li>通过荧光显微镜观察导管上的 白色念珠菌 。<ol><li>将导管浸入4%多聚甲醛溶液中，在4°C下固定48小时。</li>
		<li>在484nm激发下，用荧光显微镜观察每组导管表面存在 白色念珠菌 及其生物膜，并记录每组的图像。 注意:放大倍率为 400 倍。 白色念珠菌 的荧光可以在 490 nm 激发和 510 nm 发射时观察到。</li>
	</ol></li>
	<li>观察居住在小鼠皮肤中的 白色念珠菌 。<ol><li>将小鼠的背侧皮肤组织浸入4%多聚甲醛溶液中，在4°C固定48小时。</li>
		<li>在浓度递增（50%、70%、80%、90%和100%，15分钟/梯度）的梯度乙醇溶液中脱水背部皮肤组织。</li>
		<li>将脱水的背侧皮肤组织在55-60°C的石蜡中包埋。 注意温度，避免组织变脆。要去除尽可能多的杂质，请重复此步骤三次（每次 30 分钟）。</li>
		<li>用切片机切片背侧皮肤组织（厚度= 5μm）。</li>
		<li>通过将载玻片浸入二甲苯中两次20分钟来对石蜡切片进行脱蜡。</li>
		<li>通过梯度乙醇（无水乙醇，90%乙醇，75%乙醇，水）洗脱切片，每次5分钟。</li>
		<li>将切片浸入高碘酸溶液中15分钟，然后用自来水洗涤一次，蒸馏水洗涤两次，用高碘酸染色切片。</li>
		<li>用人字形染色溶液（根据制造商的说明）在黑暗中染色切片30分钟，然后在流水下冲洗切片5分钟。</li>
		<li>将切片浸入苏木精溶液中3-5分钟，然后分别用流水（2-3分钟），差异化溶液（5-10分钟）和流水洗涤。</li>
		<li>将切片浸入乙醇中三次（每次5分钟）和二甲苯两次（每次5分钟），然后用中性胶密封切片。</li>
		<li>用显微镜观察标本的图像，并分析小鼠皮肤中的 白色念珠菌 残留物。 注意: 目镜的放大倍率为 10 倍，物镜的放大倍率为 4 倍或 10 倍。</li>
	</ol></li>
	<li>观察背部皮肤组织的组织病理学变化。<ol><li>将背侧皮肤组织浸入4%多聚甲醛溶液中，在4°C固定48小时。在梯度乙醇溶液中以递增浓度（50%、70%、80%、90%和100%，15分钟/梯度）对背侧皮肤组织进行脱水。</li>
		<li>如步骤3.4.3所述，将脱水的背侧皮肤组织嵌入石蜡中。</li>
		<li>用切片机切片背侧皮肤组织（厚度= 5mm）。</li>
		<li>通过将载玻片浸入二甲苯中两次20分钟来对石蜡切片进行脱蜡。</li>
		<li>通过梯度乙醇（无水乙醇，90%乙醇，75%乙醇，水）洗脱切片，每次5分钟。</li>
		<li>用苏木精染色切片 4 分钟，然后用自来水冲洗以去除浮色。</li>
		<li>在用流水冲洗载玻片之前，用1%盐酸 - 乙醇溶液区分标本。</li>
		<li>将标本浸入85%和95%乙醇中5分钟，并用曙红溶液染色3分钟。</li>
		<li>将样品浸入梯度乙醇（70%、90%、95%和100%）和二甲苯中各2分钟，使试样脱水。</li>
		<li>用中性树脂密封试样。</li>
		<li>用显微镜观察标本的图像并分析病理变化。</li>
	</ol></li>
</ol>

建立 白色念珠菌相关导管相关感染的小鼠模型

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Phytomedicine. 71, 153223 (2020).</li><li>Cant&#243;n-Bulnes, M. L., Garnacho-Montero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+approach+to+the+management+of+catheter-related+bloodstream+infection.">Practical approach to the management of catheter-related bloodstream infection.</a> Revista Espanola de Quimioterapia. 32 Suppl 2 (Suppl 2), 38-41 (2019).</li><li>B&#246;hlke, M., Uliano, G., Barcellos, F. 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R., Abdollahi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+patterns+of+microorganisms+isolated+from+nephrology+and+kidney+transplant+wards+of+a+referral+academic+hospital.">Antibiotic resistance patterns of microorganisms isolated from nephrology and kidney transplant wards of a referral academic hospital.</a> Journal of Research in Pharmacy Practice. 5 (1), 43-51 (2016).</li><li>Huang, G., Huang, Q., Wei, Y., Wang, Y., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+and+diverse+regulation+of+the+Ras/cAMP/protein+kinase+A+pathway+in+Candida+albicans.">Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans.</a> Molecular Microbiology. 111 (1), 6-16 (2019).</li><li>Garlito-D&#237;az, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+antifungal-loaded+sol-gel+can+prevent+candida+albicans+prosthetic+joint+infection.">A new antifungal-loaded sol-gel can prevent candida albicans prosthetic joint infection.</a> Antibiotics (Basel). 10 (6), 711 (2021).</li></ol>

作者声明，他们没有已知的相互竞争的经济利益或个人关系，这些利益或关系可能会影响本文所报告的工作。

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，以支持相关CRI的治疗和预防。
为了构建CRI模型，在小鼠的背侧皮肤中加入少量 白色念珠 菌，模拟了通过常规灭菌无法在皮肤深层组织和附肢中完全根除部分 白色念 珠菌的临床情况。 插入导管后，重新接种 白色念珠 菌，以模拟手术过程中外部环境中白色 念珠菌 的存在。
本研究选择3 d时间点进行模型构建，由于生物膜形成困难，该时间点低于传统的白色念珠菌生物膜相关动物模型18,27。感染后，在该模型中，白色念珠菌粘附和生物膜形成在导管表面可见，SEM和荧光显微镜结果证明了这一点（图3和图4）。这可能是由于本研究中白色念珠菌的浓度为1×108 CFU/mL，远高于其他动物模型18,27。此外，导管周围的皮肤与外部环境不断接触。为了模拟CRI可能遇到的极端环境，在手术后再次接种白色念珠菌。
感染的复发通常是由残留在周围组织中的病原体引起的23,28,29。因此，组织中病原体的存在与否对CRI很重要。本文采用PAS染色法研究皮肤组织中白色念珠菌的残留。该方法也可用于评估新的治疗药物或方法对CRI的清除效果。
总之，使用带有eGFP的白色念珠菌菌株构建小鼠CRI模型，以促进在导管上直观观察白色念珠菌定植。该菌株还可用于评估白色念珠菌与宿主细胞之间的相互作用，例如，白色念珠菌对宿主的侵袭和粘附、治疗药物的抗白色念珠菌作用以及免疫反应。此外，采用两步接种法模拟来自外部环境和身体的病原体。值得注意的是，感染后未进行后续微生物培养。生物膜的存在是培养物敏感性低的重要因素30,31,32。既往报道表明，感染后的微生物培养具有较低的敏感性、特异性和准确性 30,31,32,33,34。相反，植入物上生物膜的存在是一个更可靠的指标。因此，本研究使用扫描电镜和荧光显微镜来观察和识别形成生物膜的白色念珠菌。
然而，该模型没有模拟患者免疫力减弱与在诊所观察到的白色念珠菌感染之间的相互作用。如果该模型可以在白色念珠菌接种之前考虑免疫功能低下的治疗（例如连续注射糖皮质激素）35，则有可能更好地模拟临床情况下发生的感染。

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。

<table><tbody><tr><td>0.5 Mactutrius turbidibris</td><td>Shanghai Lujing Technology Co., Ltd</td><td>5106063</td><td></td></tr><tr><td>2.5% glutaraldehyde fixative solution</td><td>Xingzhi Biotechnology Co., Ltd</td><td>DF015</td><td></td></tr><tr><td>4 &amp;deg;C refrigerator</td><td>Electrolux (China) Electric Co., Ltd</td><td>ESE6539TA</td><td></td></tr><tr><td>Agar</td><td>Beijing Aoboxing Bio-tech Co., Ltd</td><td>01-023</td><td></td></tr><tr><td>Analytical balances</td><td>Shimadzu</td><td>ATX124</td><td></td></tr><tr><td>Autoclaves Sterilizer</td><td>SANYO</td><td>MLS-3750</td><td></td></tr><tr><td>Butanol</td><td>Tianjin Chemio Reagent Co., Ltd</td><td>200-889-7</td><td></td></tr><tr><td>Carbenicillin</td><td>Amresco</td><td>C0885</td><td></td></tr><tr><td>Eclipse Ci Nikon upright optical microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse Ts2-FL</td><td></td></tr><tr><td>Glucose</td><td>Macklin&amp;nbsp;</td><td>D823520</td><td></td></tr><tr><td>Inoculation ring</td><td>Thermo Scientific</td><td>251586</td><td></td></tr><tr><td>Isoflurane</td><td>RWD</td><td>20210103</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Beyotime Biotechnology</td><td>P0099</td><td></td></tr><tr><td>PAS dye kit</td><td>Servicebio</td><td>G1285</td><td></td></tr><tr><td>Peptone</td><td>Beijing Aoboxing Bio-tech Co., Ltd</td><td>01-001</td><td></td></tr><tr><td>Polyethylene catheter</td><td>Shining Plastic Mall</td><td>PE100</td><td></td></tr><tr><td>RWD R550 multi-channel small animal anesthesia machine&amp;nbsp;</td><td>RWD</td><td>R550</td><td></td></tr><tr><td>SEM</td><td>Hitachi</td><td>TM-1000</td><td></td></tr><tr><td>Temperature incubator</td><td>Shanghai Zhichu Instrument Co., Ltd</td><td>ZQTY-50N</td><td></td></tr><tr><td>Ultrapure water water generator</td><td>Heal Force</td><td>NW20VF</td><td></td></tr><tr><td>Ultrasound machine</td><td>Do-Chrom</td><td>DS10260D</td><td></td></tr><tr><td>Xylene</td><td>Sinopharm&amp;nbsp; Chemical Reagent Co., Ltd</td><td>10023428</td><td></td></tr><tr><td>Yeast extract</td><td>Thermo Scientific&amp;nbsp;Oxoid</td><td>LP0021B</td><td></td></tr></tbody></table>

a catheter-related candida albicans infection model in mouse

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。

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（图4）22。导管组聚乙烯导管表面无明显荧光。然而，在模型组的导管表面上可以看到贴壁 白色念珠菌 细胞发出的强荧光。这表明大量的 白色念珠菌 细胞粘附在导管表面，这表明在小鼠中成功构建 了白色念 珠菌生物膜相关的CRI模型。
为了更直观地验证小鼠皮肤组织的感染情况，进行了Sheff Periodate染色分析。它检测真菌细胞的碳水化合物，这在临床研究中常用（图5）22。正常对照组和导管组的皮肤组织用高碘酸-希夫（PAS）染色呈阴性，表明组织中不存在 白色念珠菌 细胞。在模型组中观察到少量PAS染色的白色 念珠菌 阳性细胞，进一步验证了 对白色念珠菌相关侵袭和粘附的成功模拟。
接下来，通过组织病理学分析评估白色 念珠菌 诱导的小鼠皮肤组织的病理变化。如 图622所示，在模型组中，表皮层明显增厚并延伸到皮肤内部。炎症浸润也可见，表明 白色念珠菌 的感染在小鼠皮肤组织中引起明显的病理变化。导管组表皮层、真皮层、皮脂腺、毛囊等结构清晰完整。未观察到水肿和炎症浸润，与正常对照组相似。这些结果表明，单独插入导管不会引起皮肤组织的明显变化。模型组组织的病理变化是由 白色念珠菌引起的感染引起的。综上所述，研究结果验证了 与白色念珠菌 生物膜相关的CRI小鼠模型的成功建立。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65307/65307fig01.jpg"> 图 1:pCaExp 质粒图谱。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65307/65307fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65307/65307fig02.jpg"> 图2:白色 念珠菌相关CRI小鼠模型的示意图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65307/65307fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65307/65307fig03.jpg"> 图 3:每组导管表面的 SEM。 （A）导管组;（B） 模型组（1000x，比例尺 = 50 μm;5000x，比例尺 = 10 μm）。该图经 Mo et al.22 许可修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65307/65307fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65307/65307fig04.jpg"> 图4:每组导管表面荧光显微镜。 （A）导管组;（B） 模型组（比例尺 = 100 μm）。该图经 Mo et al.22 许可修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65307/65307fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65307/65307fig05.jpg"> 图5:每组小鼠背部皮肤的H&E染色。 （A）导管组;（二）示范组;（C）对照组（40x，比例尺= 400μm;100x，比例尺= 200μm）。该图经 Mo et al.22 许可修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65307/65307fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65307/65307fig06.jpg"> 图6:每组小鼠背部皮肤的PAS染色。 （A）导管组;（二）示范组;（C）对照组（40x，比例尺= 400μm;100x，比例尺= 200μm）。在模型组（红色矩形）中可以看到表皮层向皮肤内部的显着增厚和延伸。该图经 Mo et al.22 许可修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65307/65307fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging at JoVE.com

作者聚焦:使用荧光蛋白标记增强导管感染中的白色念珠菌检测 

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。

小鼠导管相关 白色念珠菌 感染模型

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

我们的研究旨在建立白色念珠菌相关导管相关感染的小鼠模型，其中在导管上形成生物膜。该模型有助于与临床CRI以及白色念珠菌与宿主之间的相互作用相关联。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、念珠菌病引起的侵袭性全身感染模型等，都不能很好地与临床CRI相关。为了改善导管感染中的白色念珠菌检测，我们的方案采用了具有增强绿色荧光素蛋白的染色剂。这使得白色念珠菌菌落和小鼠导管上的生物膜清晰可见，并及时检测。

a-catheter-related-candida-albicans-infection-model-in-mouse

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

A Catheter-Related Candida albicans Infection Model in Mouse

1.4K 次观看.  Xi’an Jiaotong University. 我们的研究旨在建立白色念珠菌相关导管相关感染的小鼠模型，其中在导管上形成生物膜。该模型有助于与临床CRI以及白色念珠菌与宿主之间的相互作用相关联。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、念珠菌病引起的侵袭性全身感染模型等，都不能很好地与临床CRI相关。为了改善导管感染中的白色念珠菌检测，我们的方案采用了具有增?...

视频: 作者聚焦:使用荧光蛋白标记增强导管感染中的白色念珠菌检测 

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

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.

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

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

医学

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.

<meta charset="utf8"></head><body>皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的体内功能

Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.

体内 皮肤树突状细胞差异亚群在皮下白色 念珠菌 感染中诱导 Th17 免疫的功能

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting ...

使用姜黄素类化合物治疗小鼠口腔念珠菌病的有效 aPDT

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

作者聚焦:探索姜黄素在口腔念珠菌病小鼠模型中的光动力疗法

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

小鼠导管相关白色念珠菌感染模型

副本

摘要

探索更多视频

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

研究微生物群落

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

建立和UTI和CAUTI表征在小鼠模型

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

小鼠导管相关 白色念珠菌 感染模型

副本

摘要

探索更多视频

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

研究微生物群落

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

建立和UTI和CAUTI表征在小鼠模型

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

体内 皮肤树突状细胞差异亚群在皮下白色 念珠菌 感染中诱导 Th17 免疫的功能

体内 皮肤树突状细胞差异亚群在皮下白色 念珠菌 感染中诱导 Th17 免疫的功能

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用

小鼠导管相关白色念珠菌感染模型

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能

体内皮肤树突状细胞差异亚群在皮下白色念珠菌感染中诱导 Th17 免疫的功能