This work has received funding from the Center of Applied Nanomedicine, National Cheng Kung University from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), and Ministry of Science and Technology, Taiwan [MOST 109-2327-B-006-005] to TW Wong. J.H. Hung acknowledges funding from National Cheng Kung University Hospital, Taiwan [NCKUH-11006018], and [MOST 110-2314-B-006-086-MY3].

1. aPDT system preparation
<ol>
	<li>Cut four green light-emitting diodes (LEDs) from a LED strip (see Table of Materials) and align them with four wells of a 96-well plate (Figure 1). 
	NOTE: The LEDs were arranged into a 4 x 3 array. The back of the LED was adhered to a heat sink to disperse heat during irradiation.</li>
	<li>Measure the fluence rate11 of the LED at 540 nm with a light power meter (see Table of Materials). See Supplementary Figure 1 to ensure that the fluence rate of the LED is between 510-560 nm.</li>
	<li>Put an electric fan alongside the plate during irradiation to maintain the constant temperature (25 &#177; 1 &#176;C)11. See Supplementary Figure 2 to ensure that the constant temperature of the medium is maintained during irradiation.</li>
</ol>
2. Culturing of the yeast form of&#160;&#160;C. albicans
NOTE: A multidrug-resistant C. albicans (BCRC 21538/ATCC 10231), resistant to most antifungals, including fluconazole, is used for the experiments16.
<ol>
	<li>Determine the antimycotic drug sensitivity with a disk diffusion method following previously published report17.</li>
	<li>Grow C. albicans in yeast, hyphae, and pseudohyphae forms depending on the micro-ecological environments18. 
	&#8203;NOTE: The hyphae and pseudohyphae forms are difficult for an accurate calculation. The yeast form can be calculated precisely under a microscope or with flow cytometry. The temperature during cell growth determines its morphology. At room temperature (25 &#176;C), nearly all cells are yeast form. A 4 h short incubation of C. albicans at 30 &#176;C did not affect its yeast morphology.</li>
</ol>
3. aPDT on planktonic&#160;&#160;C. albicans
<ol>
	<li>Isolate a single colony of C. albicans from an agar plate with a sterile loop and add it to a 3 mL yeast extract peptone dextrose (YPD) medium (see Table of Materials) in a sterilized glass tube.
	<ol>
		<li>Incubate the tube at 25 &#177; 1 &#176;C overnight (14-16 h) in an incubator with a rotation speed of 155 rpm to expand C. albicans and maintain the fungus in yeast form for accurate quantification.</li>
	</ol>
	</li>
	<li>Dilute the overnight culture with medium to an OD600 value of around 0.5 at 30&#176; C and rotate at a speed of 155 rpm for 4 h to achieve a log growth phase of C. albicans.</li>
	<li>Dilute the log phase culture again with fresh YPD medium to an OD600 value of 0.65 (around 1 x 107 colony forming units, CFU/mL). Confirm the final concentration by serial dilution method on an agar plate8.</li>
	<li>Prepare a stock solution (4%) of Rose bengal (RB) by dissolving the powder in 1x PBS. Filter and sterilize it with a 0.22 &#181;m filter and store it at 4 &#176;C in the dark. The final working concentration of RB is 0.2%.</li>
	<li>Add 111 &#181;L of 2% RB to 1 mL of log-phase&#160;C. albicans in a 1.5 mL microcentrifuge tube and co-culture at different time points (0, 15, and 30 min) at room temperature to understand the absorption of RB in the cells (Figure 2).</li>
	<li>Wash the co-culture three times with 1 mL of 1x PBS with centrifugation at 16,100 x g for 2.5 min at room temperature. 
	NOTE: aPDT contains four different conditions: absolute control (no light exposure, no RB), dark control (no light but incubates with RB), light control (exposes to light without RB), aPDT (exposes to light in the presence of RB).</li>
	<li>Resuspend the C. albicans in 1 mL of 1x PBS and allocate them into three different wells in a 96 well plate for each condition. Align the wells with the LED array after washing.</li>
	<li>In light exposed groups, turn on the electric fan and light. 
	&#8203;NOTE: A different fluence (J/cm2) can be achieved by exposing the wells to varying time periods. For example, a 16.7 min light exposure comes to 10 J/cm2 with a 10 mW/cm2 LED light bulb.</li>
	<li>After irradiation, add 20 &#181;L of the co-culture solution from one well to a 1.5 mL centrifuge tube containing 180 &#181;L of 1x PBS to prepare a 10x dilution. Further, dilute ten times, subsequently following the same method.</li>
	<li>Drop three drops of 20 &#181;L of each serial dilution onto one quadrant of a YPD agar plate to achieve countable colonies the plate. Calculate the CFU/mL by multiplying the colonies with the dilution factors3.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Analyze the collected data using a graphing and statistics software (see Table of Materials).</li>
	<li>Depict data by mean &#177; standard error of the mean. Perform a two-way ANOVA analysis of variance8 to evaluate significant differences between the different test conditions.</li>
	<li>Perform Tukey&#39;s multiple comparison tests for pairwise comparisons8. For each different treatment, perform at least three independent experiments. Consider p-value &#60; 0.05 statistically significant.</li>
</ol>

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E., 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=Invasive+candidiasis+species+distribution+and+trends,+United+States,+2009-2017.">Invasive candidiasis species distribution and trends, United States, 2009-2017.</a> Journal of Infectious Diseases. 223 (7), 1295-1302 (2021).</li><li>Koehler, P., 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=Morbidity+and+mortality+of+candidaemia+in+Europe:+an+epidemiologic+meta-analysis.">Morbidity and mortality of candidaemia in Europe: an epidemiologic meta-analysis.</a> Clinical Microbiology and Infection. 25 (10), 1200-1212 (2019).</li><li>Toda, M., 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=Population-based+active+surveillance+for+culture-confirmed+candidemia+-+four+sites,+United+States,+2012-2016.">Population-based active surveillance for culture-confirmed candidemia - four sites, United States, 2012-2016.</a> Morbidity and Mortality Weekly Report Surveillance Summaries. 68 (8), 1-15 (2019).</li><li>Lee, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+CLSI+M44-A2+disk+diffusion+and+associated+breakpoint+testing+of+caspofungin+and+micafungin+using+a+well-characterized+panel+of+wild-type+and+fks+hot+spot+mutant+Candida+isolates.">Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild-type and fks hot spot mutant Candida isolates.</a> Antimicrobial Agents and Chemotherapy. 55 (5), 1891-1895 (2011).</li><li>Mukaremera, L., Lee, K. K., Mora-Montes, H. M., Gow, N. A. 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The authors declare no conflict of interest.

Encouraging results of clinical applications of RB-PDT for fungal keratitis have been reported recently19. The absorption peak of RB is at 450-650 nm. It is essential to determine the fluence rate of the light source for a successful aPDT. A high fluence (usually &#62;100 J/cm2) is required to treat cancer cells, while a lower fluence is expected to treat infected lesions6. A high fluence means a long exposure time which may not be practical in a clinical setting...

C. albicans colonizes in the gastrointestinal and genitourinary tracts of healthy individuals and can be detected as normal microbiota in about 50 percent of individuals1. If an imbalance is created between the host and the pathogen, C. albicans is capable of invading and causing disease. The infection can range from local mucous membrane infections to multiple organ failure2. In a multicenter surveillance study in the US, around half of the isolates from patients with invasive candidiasis between 2009 and 2017 is C. albicans3. Candidemia can be associated with high morbidity rates, mortality, prolonged hospital stay4. US Centers of Disease Control and Prevention reported that about 7% of all Candida blood samples tested are resistant to the antifungal drug fluconazole5. The emergence of drug-resistant Candida species raises the concern to develop an alternative or adjuvant therapy to antimycotic agents.
Antimicrobial photodynamic therapy (aPDT) involves activating a specific photosensitizer (PS) with light at the peak absorption wavelength of the PS6. After excitation, the excited PS transfers its energy or electrons to the nearby oxygen molecules and returns to the ground state. During this process, reactive oxygen species and singlet oxygen are formed and cause cell damage. aPDT has been widely used to kill microorganisms since the 1990s7. One of the benefits of aPDT is that multiple organelles are damaged in a cell by singlet oxygen and/or reactive oxygen species (ROS) during irradiation; thus, resistance to aPDT has not been found till today. Moreover, a recent study reported that the bacteria that survived after aPDT became more sensitive to antibiotics8.
The light sources used in aPDT include lasers, metal halogen lamps with filters, near-infrared light, and light-emitting diode (LED)9,10,11,12. The laser provides a high light power, usually larger than 0.5 W/cm2, that allows for the delivery of a high light dose in a very short time. It has been widely used in cases where a longer treatment time is inconvenient such as aPDT for oral infections. The drawback of a laser is that its spot size of illumination is small, ranging from a few hundred micrometers to 10 mm with a diffuser. Moreover, laser equipment is expensive and needs specific training to operate. On the other hand, the irradiation area of a metal halogen lamp with filters is relatively larger13. However, the lamp is too hefty and expensive. LED light sources have become mainstream of aPDT in the dermatologic field because it is small and less expensive. The irradiation area can be relatively large with an array arrangement of the LED light bulb. The whole face can be illuminated at the same time9. Nevertheless, most, if not all, LED light sources available today are designed for clinical use. It may not be suitable for experiments in a lab because it is space-occupying and expensive. We developed an inexpensive LED array that is very small and can be cut and assembled from a LED strip. The LEDs can be fitted into different arrangements for different experimental designs. Different conditions of aPDT can be completed in a 96-well plate or even a 384-well plate in one experiment.
Rose bengal (RB) is a colored dye widely used to enhance visualization of corneal damages in human eyes14. RB-mediated aPDT has shown killing effects on Staphylococcus aureus, Escherichia coli, and C. albicans with roughly comparable efficiency to that of Toluidine blue O15. This study demonstrates a method to validate the effect of RB-aPDT on multidrug-resistant C. albicans.

<table><tbody><tr><td>1.5 mL microfuge tube</td><td>Neptune, San Diego, USA</td><td>#3745.x</td><td></td></tr><tr><td>5 mL round-bottom tube with cell strainer cap</td><td>Falcon, USA</td><td>#352235</td><td></td></tr><tr><td>96-well plate</td><td>Alpha plus, Taoyuan Hsien, Taiwan</td><td>#16196</td><td></td></tr><tr><td>Aluminum foil</td><td>sunmei, Tainan, Taiwan</td><td></td><td></td></tr><tr><td>Aluminum heat sink</td><td>Nanyi electronics Co., Ltd., Tainan, Taiwan</td><td>BK-T220-0051-01</td><td>Disperses heat from the LED array.</td></tr><tr><td>Centrifuge</td><td>Eppendorf, UK</td><td>5415R</td><td></td></tr><tr><td>Graph pad prism software</td><td>GraphPad 8.0, San Diego, California, USA</td><td></td><td>graphing and statistics software</td></tr><tr><td>Green light emitting diode (LED) strip</td><td>Nanyi electronics Co., Ltd., Tainan, Taiwan</td><td>2835</td><td>Emission peak wavelength: 525 nm, Viewing angle: 150&amp;deg;; originated from https://www.aliva.com.tw/product.php?id=63</td></tr><tr><td>Incubator</td><td>Yihder, Taipei, Taiwan</td><td>LM-570D (R)</td><td></td></tr><tr><td>Light power meter</td><td>Ophir, Jerusalem, Israel</td><td>PD300-3W-V1-SENSOR,</td><td></td></tr><tr><td>Millex 0.22 &amp;mu;m filter</td><td>Merck, NJ, USA</td><td>SLGVR33RS</td><td></td></tr><tr><td>Multidrug-resistant Candida albicans</td><td>Bioresource Collection and Research CenterBioresource, Hsinchu, Taiwan</td><td>BCRC 21538/ATCC 10231</td><td>http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=21538</td></tr><tr><td>OD&lt;sub&gt;600&lt;/sub&gt; spectrophotometer</td><td>Biochrom, London, UK</td><td>Ultrospec 10</td><td></td></tr><tr><td>Rose Bengal</td><td>Sigma-Aldrich, MO, USA</td><td>330000</td><td>stock concentration 40 mg/mL = 4%, prepare in PBS, stored at 4 &amp;deg;C</td></tr><tr><td>Sterilized glass tube</td><td>Sunmei Co., Ltd., Tainan, Taiwan</td><td>AK45048-16100</td><td></td></tr><tr><td>Yeast Extract Peptone Dextrose Medium</td><td>HIMEDIA, India</td><td>M1363</td><td></td></tr></tbody></table>

rose bengal-mediated photodynamic therapy to inhibit candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Figure 1 shows the aPDT system being used in the present study. Since high temperatures may cause significant cell death, the LED array is cooled by an electric fan, and a heat sink is used during irradiation to maintain a constant temperature at 25 &#177; 1 &#176;C. The heat effect can be discounted. Having an even light distribution is also an important determining factor for a successful aPDT; therefore, it is critical to align the LED light bulb to the well precisely during illumination....

Watch this Scientific Journal Video about Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans at JoVE.com

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

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Immunology and Infection

3.1K Views.  National Cheng Kung University. Deze studie is een eenvoudig, veelzijdig en gemakkelijk systeem om verschillende fotodynamische effecten op verschillende micro-organismen en cellen te onderzoeken. In dit systeem kan de LED in verschillende opstellingen worden geplaatst, afhankelijk van het experimentontwerp. Verschillende condities van PDT kunnen in één experiment worden geconputeerd in een 96-well plaat of zelfs een 384-well plaat.Deze techniek kan worden gebruikt om schimmelkeratitis te behandelen, waardoor werel...

Video: Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

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.

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

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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.

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

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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.

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.

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 ...

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.

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.

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 ...

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

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.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

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.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Analyzing Petite Colony Formation and Drug Resistance in Candida glabrata

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Author Spotlight: Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug&#45;Resistant Candida for Antifungal Research

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

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