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。

这些动物是从习交通大学购买的。所有动物实验程序均经习交通大学动物伦理学委员会批准（批准号：SCXK [陕西] 2021-103）。将小鼠饲养一周，每个笼子饲养5只小鼠。他们被允许免费获得食物和水。在进行研究之前，将动物保持在室温（RT;24°C±1°C）和光/暗循环（12小时/12小时）下。
1.缓冲液和设备准备
<ol><li>白色念珠菌 细胞培养<ol><li>将酵母提取物蛋白胨葡萄糖 （YPD） 平板培养基 中的白色念珠菌 单克隆菌落 （SC5314） 接种到 5 mL YPD 液体培养基（YPD + 50 μg/mL 羧苄青霉素）中。</li>
		<li>随后在30°C下以220rpm的速度摇动 白色念珠菌 细胞过夜。</li>
		<li>在室温下以400× g 离心悬浮液5分钟，将 白色念珠菌 细胞重悬于生理盐水中，并通过目视调节浊度将细胞浓度稀释至1×106 个细胞/ mL与0.5McFarland相同。</li>
	</ol></li>
	<li>生理盐水的制备<ol><li>称取 0.9 g 氯化钠，溶于 100 mL 去离子水中，制备 0.9% 生理盐水。</li>
	</ol></li>
	<li>手术器械制备<ol><li>使用前高压灭菌（121°C，30分钟）手术器械（剪刀，镊子，止血钳，针架，缝合针）和钛镍合金丝（直径约0.5mm）。</li>
	</ol></li>
</ol>2. 小鼠PJI模型建立
<ol><li>将30只C57BL/6小鼠（雄性，15-20g）随机分为3组（10只小鼠/组），即对照组、空白植入组（钛镍丝植入无 白色念珠菌 感染）和PJI组（钛镍丝植入伴 白色念 珠菌感染）。</li>
	<li>用1-4%异氟烷吸入麻醉小鼠，然后去除左后肢上的毛发并用碘消毒。矫正反射的丧失和对脚趾刺激无反应证实了麻醉的深度。麻醉时，在双眼上涂抹眼药膏，以防止角膜干燥，并在手术和恢复期间补充热量。</li>
	<li>对于对照组中的小鼠，不提供任何治疗。为他们提供免费的水和食物。</li>
	<li>对于空白植入物组和PJI组的小鼠，用#5刀片或无菌剃须刀在每个左后肢的膝盖上做一个10毫米的纵向切口，以暴露关节。</li>
	<li>通过插入无菌注射器（26G）针头，在股骨髓内管上打一个5毫米长的孔。</li>
	<li>在用剪刀切割之前，将光滑的镍钛合金线（直径 0.5 毫米，长 5 毫米）插入孔中（图 1）。</li>
	<li>对于空白植入物组中的小鼠，在使用尼龙缝合线（直径0.15mm）逐层闭合伤口之前，沿着镍钛合金丝逐滴加入2μLYPD培养基。</li>
	<li>对于PJI组中的小鼠，将2μL白色 念珠菌 细胞（1×106个细胞 / mL）沿镍钛合金丝逐滴接种到小鼠的关节空间中，然后使用尼龙缝合线逐层闭合伤口。</li>
	<li>将小鼠饲养 14 天，免费获得水和食物。每 24 小时皮下注射 （4 mg/kg） 给药美洛昔康，最多 3 天。</li>
	<li>14天后，用3%异氟醚麻醉小鼠，然后通过颈椎脱位对小鼠实施安乐死。</li>
</ol>3. PJI模型评估
<ol><li>主要器官感染的评估<ol><li>安乐死后从小鼠身上收集肾脏、肝脏和脾脏。</li>
		<li>在每个器官中加入500μL无菌生理盐水，并在4°C的匀浆器上研磨组织。</li>
		<li>将步骤3.1.2中制备的100μL匀浆加入YPD板中，然后用弯曲的棒均匀铺开。</li>
		<li>将YPD板倒置置在37°C培养箱中48小时。</li>
		<li>目视观察并计算菌落数量。</li>
	</ol></li>
	<li>观察植入物上的 白色念珠菌 和生物膜<ol><li>在用镊子收集植入物之前，用剪刀小心地剪开小鼠关节上的皮肤。</li>
		<li>将植入物浸入2.5%戊二醛溶液中，以便在4°C下固定48小时。</li>
		<li>用无菌PBS冲洗植入物三次，然后将其浸入1%锇酸溶液中3小时。</li>
		<li>用无菌PBS冲洗植入物三次，然后将其浸入50%，70%，80%，90%和100%乙醇溶液中15分钟进行脱水。</li>
		<li>在冷冻干燥植入物之前，将植入物浸入叔丁醇中 30 分钟三次。</li>
		<li>将植入物样品固定在样品台上，用金（10 nm涂层）溅射涂覆植入物，并在高真空和1.5 kV的扫描电子显微镜（SEM）下观察它。</li>
	</ol></li>
	<li>股骨组织的病理分析<ol><li>对小鼠实施安乐死后用剪刀收集股组织。</li>
		<li>将股骨组织浸入4%多聚甲醛溶液中，在4°C下固定48小时。</li>
		<li>将股骨组织置于10%福尔马林中1周。</li>
		<li>将股组织分别浸入50%、70%、80%、90%和100%乙醇溶液中15分钟，使股组织脱水。</li>
		<li>将脱水的股组织嵌入石蜡中，然后使用切片机将组织切成4μm样品。</li>
		<li>在病理分析之前，按照标准方案用苏木精和伊红染色股骨切片21.</li>
	</ol></li>
</ol>

建立 白色念珠菌 生物膜相关 PJI 的小鼠模型

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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。

手术过程中手术器械或手术环境的污染引起的感染是大多数植入物感染的主要原因24,25,26,27。因此，本研究构建了小鼠白色念珠菌生物膜相关PJI模型。与传统的PJI模型相比，以悬浮在盐水中的无菌不锈钢颗粒为植入物，本研究使用常用的植入物材料镍钛合金丝来模拟白色念珠菌、植入物?...

白色念珠菌（白色念珠菌）共存于人体的许多部位1，这也是最常见的机会性病原体，可引起危及生命的侵袭性真菌感染，尤其是在免疫功能低下的患者中2,3。白色念珠菌可以作为多态性真菌在酵母和菌丝体状态之间转化。菌丝体状态表现出更高的毒力、更强的粘附性以及对细胞和组织的侵袭 4,5。此外，白色念珠菌可以在假牙、导管和支架等生物医学材料的表面形成生物膜1,6,7。生物膜致密的三维结构限制了抗真菌药物的浸润，表达耐药基因，下调真菌细胞的代谢以抵抗免疫系统清除6,7。因此，与生物膜相关的感染在临床中非常具有挑战性8.
金黄色葡萄球菌、凝固酶阴性葡萄球菌和肠杆菌是引起PJI9的主要病原体。虽然真菌PJI的发生率相对较低（约1%）10，但真菌PJI的治疗成本较高11，治疗周期较长11，治疗成功率低于细菌PJI10。近年来，真菌PJI的发病率逐年增加10。念珠菌PJI占真菌PJI 10,12的77%-84%，白色念珠菌是念珠菌中最常见（54%）。因此，需要研究真菌PJI。
目前，PJI 通过翻修手术进行治疗，包括 （1） 移除受感染的植入物、（2） 清创术、（3） 抗菌治疗和 （4） 再植入。彻底清创后，放置含有骨水泥的抗生素，并对患者进行全身抗生素治疗 6 周以上，以有效控制感染，然后再放置新的植入物13。然而，这种方法不能完全消除组织中的病原体，并且用长期抗菌治疗治疗的复发性感染极有可能在耐药菌株中发展14,15,16。
建立PJI的动物模型对于PJI新药或疗法的研发具有重要意义。在PJI的发展中，假体周围形成大的死腔，导致血肿的形成，血肿进一步阻塞周围组织的血液供应并损害抗生素的作用11,15。由于难以模拟假体的周围环境，传统的动物模型无法准确模拟PJI17,18的实际情况。
在本文中，通过使用临床上广泛使用的钛镍丝来模拟关节植入物，在小鼠中构建了白色念珠菌生物膜相关的PJI模型19,20。该PJI模型具有操作简单、成功率高、重复性高、临床相关性高等优点。有望成为研究白色念珠菌生物膜相关PJI防治的重要模型。

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a periprosthetic joint candida albicans infection model in mouse

假体周围关节感染 （PJI） 是由 白色念珠菌 （C. albicans） 引起的常见感染之一， 越来越受到外科医生和科学家的关注。通常，可以在感染部位形成可以保护 白色念珠菌 免受抗生素和免疫清除的生物膜。手术包括切除受感染的植入物、清创术、抗菌治疗和再植入术是治疗 PJI 的金标准。因此，建立动物PJI模型对于PJI新药或新疗法的研发具有重要意义。在这项研究中，将一根光滑的镍钛合金丝（骨科诊所广泛使用的植入物）插入 C57BL/6 小鼠的股骨关节，然后沿线将 白色念珠菌 接种到关节腔中。14天后，在扫描电子显微镜（SEM）下在植入物表面观察到成熟和厚厚的生物膜。在感染关节标本的H&E染色中发现骨小梁明显减少。综上所述，建立了具有操作简便、成功率高、重复性高、临床相关性高等优点的小鼠PJI模型。这有望成为白色 念珠菌 生物膜相关PJI预防临床研究的重要模型。

将样品转移到平板培养基上并在孵育过夜后计数菌落通常用于评估病灶附近的局部病原体载量22,23。在我们的研究中，肝脏、肾脏和脾脏样本的微生物培养呈阴性，表明本研究中的模型仅导致小鼠的局部感染而不是全身感染23。
植入物的SEM图像如 图2所示。在空白植入物组中，没有白色 念<...

Watch this Scientific Journal Video about Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections at JoVE.com

作者聚焦：推进白色念珠菌生物膜相关人工关节感染的研究 

由危险病原体引起的假体周围关节感染 （PJI） 在临床骨科中很常见。现有的动物模型无法准确模拟PJI的实际情况。在这里，我们建立了一个 白色念珠菌 生物膜相关的PJI小鼠模型，以研究和开发PJI的新疗法。

小鼠假体周围关节 白色念珠菌 感染模型

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

在这项白色念珠菌生物膜相关假体周围关节的研究中，使用通常用于模拟关节植入物的钛镍丝构建了小鼠白色念珠菌感染模型。该 PJI 模型易于操作，成功率高，并且具有临床相关性。它将有助于了解白色念珠菌生物膜相关 PJI 的预防和治疗。传统的动物模型由于过程周围的复杂环境，无法准确模拟PJI的实际情况。白色念珠菌生物膜相关小鼠PJI模型，可以弥补研究空白。PJI动物模型的建立对于PJI新药或新疗法的研究和开发具有重要意义。本文建立的白色念珠菌生物膜相关小鼠PJI模型有望成为预防和治疗白色念珠菌生物膜相关PJI的重要模型。

a-periprosthetic-joint-candida-albicans-infection-model-in-mouse

Research

JoVE Journal

Immunology and Infection

A Periprosthetic Joint Candida albicans Infection Model in Mouse

657 次观看.  Xi’an Jiaotong University. 在这项白色念珠菌生物膜相关假体周围关节的研究中，使用通常用于模拟关节植入物的钛镍丝构建了小鼠白色念珠菌感染模型。该 PJI 模型易于操作，成功率高，并且具有临床相关性。它将有助于了解白色念珠菌生物膜相关 PJI 的预防和治疗。传统的动物模型由于过程周围的复杂环境，无法准确模拟PJI的实际情况。白色念珠菌生物膜相关小鼠PJI模型，可以弥补研究空白。...

视频: 作者聚焦：推进白色念珠菌生物膜相关人工关节感染的研究 

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 ...

科研

免疫与感染

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

红光光疗法调节白色念珠菌生物膜生长

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

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 ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

小鼠假体周围关节 白色念珠菌 感染模型

小鼠假体周围关节白色念珠菌感染模型