Name: visualization of biofilm formation in candida albicans using an automated microfluidic device
Uploaded: 2017-12-14T15:00:00.000+00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

우리 biofilm 분석에 유용한 토론 Nobile 연구소의 모든 구성원을 감사합니다. 이 연구는 건강 (NIH) 부여 R21 AI125801 (C.J.N.)의 국가 학회에 의해 지원 되었다. D.L.R.는 멕시코와 미국 (UC-MEXUS)와 Consejo 나시오날 드 많은 y Technologia (CONACYT) 캘리포니아 연구소의 대학에서 박사 과정 장학금에 의해 지원 되었다.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell-surface+genes+and+biofilm+formation+by+the+C.+albicans+transcription+factor+Bcr1p.">Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p.</a> Curr Biol. 15 (12), 1150-1155 (2005).</li><li>Baker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the bench: A laboratory navigator. 27, (2005).</li></ol>

고모 J. Nobile BioSynesis, Inc., 저 해제 및 C. albicans biofilms의 진단 개발 회사의 설립자 이다.

여기에 설명 된 사용자 지정 가능한 미세 biofilm 분석 결과 수에 biofilm 형성의 시각화에 대 한 고정된 속도 층 흐름과 일정 한 온도에 노출 되 면 단일 셀 수준에 실시간으로. 그것은 야생 형 및 돌연변이 체 긴장에 biofilms의 개발 연구에 강력한 수단을 제공 하 고 생리 조건 모방 조건 하에서 biofilms에 항균 성 에이전트 처리의 효과 임상 설정에서 관찰. 대부분 체 외에서 biofilm 분석 실험와 달?...

그러나 그것은 또한는 기회 주의 병원 체, 표면 하 고 심한 곰 팡이 감염1,2를 일으키는 능력이 candida albicans 인간의 microbiota의 공생 회원입니다. C. albicans 의 주요 독성 특성 양식 복구 하는 기능 이며, 약물 내성 biofilms, 세포의 표면에 준수 고 기질 소재1,3에. C. albicans biofilms는 고도로 구조화, 포함 하는 여러 종류의 세포 여러 레이어 (신진 효 모-양식 라운드 셀, 타원형 pseudohyphal 셀 및 관 hyphal 셀)4. C. albicans biofilm 개발 표면에,이 세포의 확산에 의해 따라 (에서 biofilm 시드), 표면에 둥근 효 모 형태의 세포의 부착으로 시작 하 고 다음 미 숙 biofilm의 성숙으로 구조를 완벽 하 게 형성 된 biofilm 기질 소재4에 의해 둘러싸여. 성숙한 biofilm biofilm4건축 안정성을 제공 밀도 연결 네트워크를 형성 하는 길쭉한 hyphal 세포 주로 구성 됩니다. Biofilm 라이프 사이클에 걸쳐 신진 효 모 라운드 세포 성숙 biofilm에서 분산 하 고 전파 감염 시키거나 다른 사이트4,5에서 새로운 biofilms 씨앗 몸의 다른 지역에 여행 수 있습니다. C. albicans 는 biotic 표면, 점 막 표면에와 같은 및 호스트 조직, 전체에 카 테 터, 심장, 틀니, 인공 관절 등의 비 생물 적인 표면에 biofilms를 형성할 수 있다. Biofilms의 고집 불통 속성으로 인해 그들은 매우 어려운 근절, 그리고 많은 경우에서 유일 하 게 효과적인 치료 전략은 감염 된 장치4의 제거. 따라서 조사 임상 설정에서 관찰 된 것과 유사한 조건 하에서 biofilm 형성에 결정적 이다.
몇 가지 중요 한 vivo에서 동물 모델이 C. albicans biofilm 형성6,,78; 공부 하는 데 사용 그러나, 이러한 연구 비용, 시간이 소요 될 수 있으며 긴장 및 한 번에 테스트할 수 있는 항균 성 대리인의 수에 의해 제한 됩니다. 생체 외에서 biofilm 분석 실험, 다른 한편으로, 항진균 성 화합물 및 돌연변이 체 긴장의 급속 한, 높은 처리량 평가 대 한 허용 하 고 훨씬 더 비용 효율적이 고 윤리적인 biofilm 분석 실시 보다는 동물에서 모델9, 10,,1112,,1314. 여기는 생체 외에서 분석 결과 우리가 개발 하 고 사용자 지정 가능한 미세 장치14,15를 사용 하 여 층 류에서 일시적으로 biofilm 형성을 관찰 하도록 최적화에 대해 설명 합니다. 분석 결과 시각화 biofilm 형성, 초기 부착 단계, 세포 증식, biofilm 성숙, 세포 분산 등의 각 단계에 대 한 수 있습니다. 분석 결과 시각화는 biofilm의 개발을 통해 세포 형태학 변화에도 유용 합니다.
일반적으로 체 외에서 biofilm 분석 실험, 높은 처리량을 동안에 대 한 활용 결정 접시 제어 흐름 조건에 대 한 허용 하지 않습니다. 전통적인 층 흐름 전지 시스템 제어 흐름 조건에서 biofilm 형성의 지속적인 평가 대 한 허용 하지만 이러한 시간이 소요를 설정 하 고 동적 범위 컨트롤 및 처리량 제한 하는 경향이 많습니다. 여기 활용 미세 장치 내장 층 류 챔버와 높은 처리량 판 (48 웰 스 포함)를 결합 하 여 이러한 한계를 극복 하 고 매우 재현 하 고 다재 다능 한, 사용자 정의.
우리가 야생-타입 C. albicans 의 biofilm 형성 평가를 상업적으로 사용할 수 있는 자동화 미세 장치 사용에 대 한 프로토콜을 설명 하는 여기, 스트레인, biofilm, biofilm의 개발에 알려진 항진균 제의 효과 두 돌연변이 체 긴장 (bcr1Δ/Δ와 efg1 Δ/Δ) 이전 biofilm를 보고 된에서 대형 생체 외에서 그리고 vivo에서16,,1718결함. Biofilm의 개발을 통해 biofilm 형성 억제에 항균 성 대리인의 효능을 테스트 하 고 돌연변이 라이브러리를 검사 하 여 정상적인 biofilm 개발에 필요한 유전자를 식별 하는 설명된 프로토콜을 사용할 수 있습니다.

<table><tbody><tr><td>BioFlux 1000z</td><td>Fluxion</td><td></td><td>Automated microfluidic device for live cell analysis</td></tr><tr><td>48-well plate 0-20 dyne</td><td>Fluxion</td><td>910-0047</td><td>Microfluidic plate</td></tr><tr><td>Montage Software</td><td>Fluxion</td><td>Version 7.8.4.0</td><td>Visualization analysis software</td></tr><tr><td>ImageJ Software</td><td>NIH</td><td>https://imagej.nih.gov/ij/</td><td></td></tr><tr><td>Yeast Extract</td><td>Criterion</td><td>C7341</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD Biosciences</td><td>211677</td><td></td></tr><tr><td>Dextrose (D-Glucose)</td><td>Fisher Scientific</td><td>D163</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R6504</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M3183</td><td></td></tr><tr><td>Nutrient Broth</td><td>Criterion</td><td>C6471</td><td></td></tr><tr><td>Difco D-Mannitol</td><td>BD Biosciences</td><td>217020</td><td></td></tr><tr><td>Agar</td><td>Criterion</td><td>C5001</td><td></td></tr><tr><td>Amphotericin B</td><td>Corning</td><td>30-003-CF</td><td></td></tr><tr><td>Sterile Inoculating Loops</td><td>VWR</td><td>30002-094</td><td></td></tr><tr><td>Petri Dishes with Clear Lid</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Disposable Cuvettes</td><td>Fisher Scientific</td><td>14-955-127</td><td></td></tr><tr><td>Lens Paper</td><td>VWR</td><td>52846-001</td><td></td></tr><tr><td>Microplate and Cuvette Spectrophotometer</td><td>BioTek</td><td>EPOCH2TC</td><td></td></tr><tr><td>Shaking Incubator</td><td>Eppendorf</td><td>M12820004</td><td></td></tr></tbody></table>

visualization of biofilm formation in candida albicans using an automated microfluidic device

Candida albicans 인간의 병원 획득 패 혈 증 경우의 약 15%를 일으키는 가장 일반적인 곰 팡이 병원 체 이다. C. albicans 의 주요 독성 특성은 그것의 양식 biofilms 능력, biotic 및 abiotic 표면에 연결 된 세포의 구조적된 커뮤니티. C. albicans biofilms 호스트 조직, 점 막 층, 같은 카 테 터, 심장, 의치, 공동 prostheses 등 의료 기기에 형성할 수 있다. 그들은 물리적, 화학적 물결에 높게 저항 하기 때문에 씨앗에 저수지 전파 감염으로 작동할 수 있다 Biofilms 중요 한 임상 도전 포즈. 분석 실험 체 외에 다양 한 C. albicans biofilm 형성, 결정 플레이트 분석 실험, 건조 중량 측정, 세포 생존 능력 분석 실험, confocal 레이저 스캐닝 현미경 검사 법 등 공부를 이용 되어 있다. 이 분석 실험의 모두 단일 끝점 분석 실험, biofilm 형성 특정 시간에 평가 됩니다. 여기, 우리 층 흐름 조건 하에서 자동된 미세 장치를 사용 하 여 실시간에 biofilm 형성 연구 프로토콜을 설명 합니다. 이 메서드는 biofilm 혈관 카 테 터에서 발생 하는 등 호스트의 모방 하는 사용자 지정 조건을 사용 하 여 시간이 지남에 개발로 biofilm 형성의 관찰에 대 한 수 있습니다. 유전 돌연변이의 biofilm 결함 뿐만 아니라 실시간으로 biofilm 개발에 항균 성 대리인의 억제 효과 평가 하기 위해이 프로토콜을 사용할 수 있습니다.

우리 수행 미세 biofilm 분석 결과 설명 여기 야생-타입을 사용 하 여 두 미디어 조건 (RPMI 1640 그리고 거미 미디어) C. albicans 스트레인, 알려진된 항진균 약물 RPMI, 암포 B (16 µ g/mL)의 야생-타입 스트레인 그리고 2 개의 돌연변이 체 긴장 이전 거미 미디어에 biofilm 형성 (bcr1Δ/Δ와 efg1 Δ/Δ)에서 결함을 보고.
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Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans 자동 미세 장치를 사용 하 여에서 Biofilm 형성의 시각화

이 프로토콜에서는 호스트 생리 적 조건 하에서 Candida albicans 에 biofilm 형성을 시각화 하는 사용자 지정 가능한 자동화 된 미세 장치를 사용 하 여를 설명 합니다.

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

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

10.5K 조회수.  University of California, Merced. 이 프로토콜에서는 호스트 생리 적 조건 하에서 Candida albicans 에 biofilm 형성을 시각화 하는 사용자 지정 가능한 자동화 된 미세 장치를 사용 하 여를 설명 합니다.

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

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.

모니터링 형성과 Antifungal 자화율 테스트를위한 96 자 Microtiter 플레이트 기반 방법 캔디다 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 ...

연구

면역학 및 감염병학

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

높은 처리량 Antifungal 약물 검사를위한 칸디다 albicans Biofilm 칩 (CA BChip)

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

생체공학

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

마이크로 기둥 미세 유체 장치에서 바이오 필름 보리 형성을위한 프로토콜

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

높은 처리량의 사용<em&gt; 체외</em&gt; 미세 유체 시스템 구강 다중 종의 바이오 필름을 개발하는

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

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.

생물 발광 영상에 이어 마우스 피하 모델의 의학 관련 외국 기관에 칸디다 알비 칸스 (Candida albicans)에 바이오 필름 ​​개발

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

의학

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

실시간 이미징 및 2 단계 반복 흐름 시스템을 사용 하 여 곰 팡이 Biofilm 개발의 정량화

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

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.

칸디다 알비칸스 바이오필름 의 명확화 및 이미징

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

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

칸디다 트로피칼리스 생물막에 대한 항체의 효과를 평가하기 위한 가용성 테트라졸륨 기반 환원 분석

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://app.jove.com/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

칸디다 알비칸스 In Vivo 마우스 모델의 생물막 형성

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

Candida albicans 자동 미세 장치를 사용 하 여에서 Biofilm 형성의 시각화

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