The authors thank&#160;the financial support from FAPESP (S&#227;o Paulo Research Foundation, process number FAPESP #2013/07276-1 (CePID CePOF) and 2008/00601-6. We also thank Dr. Ana Paula Silva for providing the information about the CUR-based water-soluble salt.&#160;

The research protocol for the use of mice was approved by the Ethics Committee for Animal Use (case numbers 05/2008 and 09/2020) at the School of Dentistry, Araraquara, UNESP. C. albicans (ATCC 90028) was used as the reference strain. Six-week-old female Swiss mice (n = 45), with a body mass range of 20-30 g, were used for the present study. The animals were provided by S&#227;o Paulo State University, UNESP, Botucatu.
1. Preparation of PS and selection of the light source for aPDT
<ol>
	<li>Prepare the PS according to the specifications for the substance to be tested. 
	NOTE: In this study, a water-soluble mixture of curcuminoids (CUR-based water-soluble salt mixture12) was used as the PS (see Table of Materials and Figure 1). Dilutions at 20 &#181;M, 40 &#181;M, and 80 &#181;M (15.6, 29.2, and 58.4 mg/L, respectively) were prepared in ultra-pure water immediately before use and kept at 5 &#176;C.</li>
	<li>Select a light equipment with an appropriate wavelength (the same as that of the PS absorption) capable of illuminating the entire photosensitized target uniformly and simultaneously without heating the tissue. 
	&#8203;NOTE: In this study, a handpiece containing a light-emitting diode (LED, see Table of Materials), that was designed at the Instituto de F&#237;sica de S&#227;o Carlos (University of S&#227;o Paulo, S&#227;o Carlos, SP, Brazil), was used. The output power delivered by the light was 89.2 mW/cm2 at a blue wavelength of approximately 455 nm.</li>
</ol>
2. Preparation of C. albicans inoculum
<ol>
	<li>Keep the reference strain C. albicans (ATCC 90028) in yeast-peptone-glucose (YEPD, see Table of Materials) with 50% glycerol at -80 &#176;C until use.</li>
	<li>Thaw the frozen strain at room temperature, spread a 100 &#181;L aliquot on a Petri dish with Sabouraud dextrose agar (SDA) with chloramphenicol (see Table of Materials), and incubate at 37 &#176;C for 48 h.</li>
	<li>Transfer five colonies into 10 mL of yeast nitrogen broth with 2% glucose (YNBg) medium and grow aerobically at 37 &#176;C for 16 h.</li>
	<li>Dilute the yeast culture in fresh YNBg (1:10) and incubate aerobically at 37 &#176;C until the optical density reaches the mid-log phase of growth. 
	NOTE: Standardize the microbial growth curve for each laboratory previously. In the current research, cultures were allowed to incubate for around 8 h, until they reached the mid-log phase of growth as indicated by the optical density at 540 nm (OD540), with a mean value of 0.536 &#177; 0.062 arbitrary units (mean &#177; standard deviation [SD]).</li>
	<li>Centrifuge the culture at 5,000 x g at room temperature for 5 min, discard the supernatant and wash the pellet twice with the same volume of sterile phosphate-buffered saline (PBS; 0.136 M NaCl, 2 mM KCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4).</li>
	<li>Use 100 &#181;L aliquots for performing four serial 10-fold dilutions in PBS, spread dilutions on SDA plates, and incubate at 37 &#176;C for 48 h for colony count. As a standard, fungal suspensions are used at concentrations of approximately 4.5 &#215; 107 colony forming units per milliliter (CFU/mL)].</li>
</ol>

3. Induction of OC in mice
NOTE: The following methodology was previously described by Takakura et al.13 and reproduced by our group10,14, with some modifications.
<ol>
	<li>Randomly distribute mice in nine groups (n = 5), corresponding to the same number of treatments (Supplementary File 1). 
	NOTE: The oral cavity of mice should be negative for Candida growth. This needs to be checked by swabbing the oral cavity and plating the sample on SDA.</li>
	<li>Keep the animals in propylene boxes10(5 mice per box max.),&#160;with wood shavings for floor covering,&#160;in a temperature-controlled vivarium at 23 &#177; 2 &#176;C under a 12/12 h light/dark cycle. No specific enrichment needs to be provided.</li>
	<li>Maintain mice on extruded mouse diet and filtered water ad libitum.</li>
	<li>Subcutaneously inject the immunosuppressive drug, prednisolone&#160;(100 mg/kg body weight, see Table of Materials), for the first time on day one to all the members of groups C+L+ 20, C+L+ 40, C+L+ 80, C+L- 20 &#181;M, C+L- 40, C+L- 80, C-L+, and C-L- (Supplementary File 1). 
	NOTE: Keep mice from the same group in the same box and monitor them daily for any sign of stress and weight loss. Seek veterinary advice for analgesic or altered food/water if stress or weight loss are noted..</li>
	<li>Starting on day one and until the end of the experiment, provide tetracycline hydrochloride in drinking water at 0.83 mg/mL13.</li>
	<li>Inoculate mice tongues on day two, including groups C+L+ 20, C+L+ 40, C+L+ 80, C+L- 20, C+L- 40, C+L- 80, C-L+, and C-L- (Supplementary File 1), with the C. albicans inoculum. Do not inoculate those mice from the negative control group (NCtrl).
	<ol>
		<li>Sedate animals by intramuscular injection of 10 mg/kg chlorpromazine chloride (see Table of Materials) on each thigh muscle&#160;before carrying out inoculation.</li>
		<li>Perform intraoral inoculation of the mice&#160;by soaking a sterile swab (one per animal) into a freshly prepared (i.e., prepared immediately before inoculation) standardized suspension of C. albicans.</li>
		<li>Place sedated mice in a supine position. Gently pull their tongues out of their mouths using a sterile forceps. Rub each mouse&#39;s tongue with a soaked swab for 30 s. Monitor mice until they recover from sedation.</li>
	</ol>
	</li>
	<li>On day five, administer a complementary subcutaneous injection of prednisolone (100 mg/kg body weight) to maintain the immunosuppression. 
	&#8203;NOTE: This protocol is adequate to keep the infection for 7 days after intraoral inoculation. The protocol timeline is shown in Figure 2.</li>
</ol>
4. Antimicrobial Photodynamic Therapy and recovery of C. albicans from oral lesions
<ol>
	<li>On day seven, prior to treatment, anesthetize the animals using an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight) combined with xylazine (10 mg/kg body weight) (see Table of Materials).</li>
	<li>Place each animal in a supine position on a pad device14,15equipped with stainless steel wires that can be looped around the incisors to keep the mouth open. 
	NOTE: Isothermal pads are recommended for warming the mice while they are sedated, preventing hypothermia at room temperature and avoiding mortality related to anesthesia15. Each anesthetized animal should be monitored by the lack of a withdrawal reflex when toes are pinched to confirm the anesthesia depth. One maintenance dose of ketamine at 1/3 the original dose (without xylazine) may be administered if needed.</li>
	<li>With the mandible and cheeks retracted, gently place the tongue outside the mouth.</li>
	<li>For mice from C+L+ groups, pipette 70 &#181;L of the PS on the dorsum of the tongue (at one of the tested concentrations). Maintain the mice in a dark environment for a duration of 20 min as a pre-irradiation period. Ensure that during this time, the tongue of each animal remains positioned inside the oral cavity to prevent the photosensitizer (PS) from being ingested.</li>
	<li>After the photosensitization period, pull the tongue out of the mouth for illumination. Place the LED device onto the dorsum of the tongue and illuminate at 37.5 J/cm2 (7 min with the present device) (Figure 3).</li>
	<li>For animals from the C+L- groups, pipette 70 &#181;L of the PS at one of the tested concentrations on the dorsum of the tongue, and keep these mice in the dark for 27 min.</li>
	<li>For animals from the C-L+ group (treated with light without any previous photosensitization), pipette 70 &#181;L of sterile saline solution on the dorsum of the tongue. Keep the mice in the dark for 20 min and then illuminate their tongues at 37.5 J/cm2 (7 min with the present device).</li>
	<li>For mice from the C-L- group (neither treated with PS nor with light), pipette 70 &#181;L of sterile saline solution on the dorsum of the tongue and keep them in the dark for 27 min.</li>
	<li>Recover C. albicans from the tongues of all mice immediately after treatment. Swab the dorsum of the tongue for 1 min with a sterile cotton swab.</li>
	<li>Place each sample swab in a tube containing 1 mL of sterile saline and vortex for 1 min to resuspend the microbial cells.</li>
	<li>Immediately perform 10-fold serial dilutions in PBS and plate 25 &#181;L aliquots over SDA plates in duplicate. Incubate the plates aerobically at 37 &#176;C for 48 h.</li>
	<li>After 48 h, use a digital colony counter (see Table of Materials) to determine yeast colony counts. Calculate C. albicans load (CFU/mL) for each animal.</li>
	<li>Transform CFU/mL values into log10 and analyze properly according to the design of the experiment and data assumptions. 
	NOTE: In the present investigation, Welch&#39;s one-way ANOVA and Games-Howell post-hoc test (&#945; = 0.05)16 was used.</li>
</ol>
5. Histopathological analyses
<ol>
	<li>On day eight, anesthetize mice with ketamine at 100 mg/kg combined with xylazine at 10 mg/kg&#160;via intraperitonial injection and euthanize them with an overdose of ketamine (200 mg/kg administered via intraperiotonial). Confirm death by checking for the absence of respiratory movement (apnea), and absence of heartbeat (asystole).</li>
	<li>Carefully pinch the tongue and pull it out of the animal&#39;s mouth. Using a manual scalpel, make an incision before the circumvallate papillae to surgically remove the entire tongue without causing any injuries to the anterior and central regions.</li>
	<li>Fix the tongue in 10% buffered formalin (pH 7.2-7.4) for 24 h and wash it in running water for 2 h.</li>
	<li>Proceed with the inclusion in the paraffin process: dehydration, clarification, and impregnation10,14.</li>
	<li>Cut serial sections (5 &#181;m thick) using a microtome, then affix the sections onto glass slides. Proceed to stain these sections using periodic acid-Schiff and hematoxylin (PAS-H) for histopathological examination and identifying fungi through observation under a light microscope.</li>
	<li>Ask a pathologist, preferably blind to the groups studied, to examine the tissue reaction due to C. albicans infection.</li>
	<li>Describe the histological characteristics of the tissue (epithelium and connective tissue), especially local inflammatory responses of varying intensities.</li>
</ol>

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

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C. albicans has been associated with oral and esophageal infections in individuals with an immunocompromised state, diabetes mellitus, prolonged use of antibiotics, and poor oral hygiene1,3. The study of human infectious diseases requires both in vitro and in vivo investigations before clinical trials can be safely and accurately designed. The present study describes a method for establishing a murine model of OC, which can be used to e...

Oral candidiasis (OC) is the main fungal infection of the oral cavity; it is caused by the overgrowth of Candida spp. Predisposing factors for OC include endocrine dysfunction, the use of broad-spectrum antibiotics, radio- and chemotherapy, nutritional deficiencies, xerostomia (low salivary flow), denture use, poor hygiene, and, especially, immunosuppression1. Among Candida species, Candida albicans is the most prevalent and virulent one; it is found as a commensal species in the human body and as an opportunistic pathogen. C. albicans has the ability to change its morphology from commensal yeasts (blastopores) to pathogenic filaments (hyphae and pseudohyphae)2. The filamentous forms, especially the hyphae, can invade the host epithelium by endocytosis or active penetration, causing infection3. Other virulence factors of C. albicans include adhesion, biofilm formation, and secretion of lipolytic and hydrolytic enzymes and toxins, such as lipases, phospholipases, proteinases, and candidalysin4.
OC treatments involve the use of antifungal agents, especially topical polyenes and azoles (nystatin and miconazole)5. However, they only show short-term efficacy, and recurrence is frequent. In addition, the overuse of antifungals has given rise to the problem of antifungal resistance development and spread6. Therefore, alternative therapies are needed, such as antimicrobial Photodynamic Therapy (aPDT), which combines a photosensitizer (PS) and light at an appropriate wavelength (the same as that of the PS absorption) in the presence of oxygen. PSs are bound to or taken up by cells and, when activated by light, produce reactive oxygen species (ROS) that are toxic to sensitized cells7.
In aPDT, one of the photosensitizers (PSs) employed is curcumin (CUR), a naturally occurring compound extracted from the rhizomes of the turmeric plant (Curcuma longa L.). Curcumin possesses numerous therapeutic attributes, including anti-inflammatory, antioxidant, anticancer, and antimicrobial capabilities8,9. A prior investigation found that aPDT utilizing CUR effectively diminished C. albicans in a murine model of oral candidiasis without causing any harm to the host&#39;s tissues10. CUR is the main curcuminoid extracted from turmeric, but other polyphenols, such as demethoxycurcumin and bis-demethoxycurcumin, are also found in this plant. Curcuminoid-mediated aPDT demonstrated antibacterial activity against biofilms of Staphylococcus aureus grown in catheters11. However, to the best of our knowledge, its antifungal activity against C. albicans remains unclear. Therefore, in this study, we evaluated aPDT mediated by a curcuminoid salt against C. albicans in a murine model of OC.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C. albicans</td>
			<td>ATCC (Rockville, Md, USA)</td>
			<td>90028</td>
			<td>Used to prepare the Candida inoculum</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf&#160;Centrifuge 5804/5804R,B. Braun, Melsungen, Hesse, Germany</td>
			<td>022628146 (NA)</td>
			<td>Used to prepare the Candida inoculum</td>
		</tr>
		<tr>
			<td>Chlorpromazine chloride 2 mg/mL</td>
			<td>Compounding pharmacy, Araraquara, SP, Brazil</td>
			<td>&#160;-&#160;&#160;</td>
			<td>Used to sedate animals during candida inoculation</td>
		</tr>
		<tr>
			<td>Curcumin-based water-soluble salt</td>
			<td>PDTPharma, Cravinhos, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Consisting of 53.4% of natural curcumin, and&#160; 46.6% of other curcuminoids (demethoxycurcumin and bis-demethoxycurcumin). Prepared in water and N-MethylD-Glucamine (final average molecular weight of 730.32 g.mol&#8722;1)</td>
		</tr>
		<tr>
			<td>Digital colony counter</td>
			<td>CP 600 Plus, Phoenix Ind Com Equipamentos Cient&#237;ficos Ltda, Araraquara, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used to count colonies on agar plates</td>
		</tr>
		<tr>
			<td>Extruded mouse chow</td>
			<td>Benelab food, Industry Qualy Animal Nutrition and Commerce Ltda., Lind&#243;ia, S&#227;o Paulo State, Brazil.</td>
			<td>&#160;-</td>
			<td>Used for the feeding of the mice</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride 10%</td>
			<td>Ketamina Agener, Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A, Embu-Gua&#231;u, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used to anesthetize animals before&#160; treatments and for euthanasia</td>
		</tr>
		<tr>
			<td>Light-emitting diode handpiece (prototype)</td>
			<td>Instituto de F&#237;sica de S&#227;o Carlos, University of S&#227;o Paulo, S&#227;o Carlos, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Fabricated with LXHL-PR09, Luxeon III Emitter, Lumileds Lighting, San Jose, California, USA</td>
		</tr>
		<tr>
			<td>Methylprednisolone acetate 40 mg</td>
			<td>DEPO-MEDROL, Pfizer, New York</td>
			<td>&#160;-&#160;</td>
			<td>Used as an immunosuppressant</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica&#160;Microsystems, Bannockburn, IL, USA</td>
			<td>SM2500</td>
			<td>Used to cut the serial sections of the tongues</td>
		</tr>
		<tr>
			<td>Propylene boxes (cages housing) H13 x L20 x D30 cm</td>
			<td>Bonther Equipaments, Ribeir&#227;o Preto, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used to keep the animals throughout&#160; the experimental period</td>
		</tr>
		<tr>
			<td>Sabouraud Dextrose Agar with Chloramphenicol</td>
			<td>HiMedia, Mumbai, India</td>
			<td>MM1067-500G</td>
			<td>Culture medium for yeast growth (agar)</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Spectrophotometer Kasvi K37-VIS , S&#227;o Jos&#233; dos Pinhais, PR, Brazil</td>
			<td>K37-VIS&#160;</td>
			<td>Used to standardize the inoculum concentration</td>
		</tr>
		<tr>
			<td>Tetracycline hydrochloride&#160;</td>
			<td>Compounding pharmacy, Araraquara, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Antibiotic given to induce oral dysbiosis</td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>J.R. Wood Shavings, Comerce of Sawdust Ltda., Conchal, S&#227;o Paulo State, Brazil</td>
			<td>&#160;-</td>
			<td>Used for floor covering inside the housing boxes</td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Calmiun, Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A, Embu-Gua&#231;u, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used in combination with ketamine for anesthesia</td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Broth&#160;</td>
			<td>Difco, InterLab, Detroit, MI, USA</td>
			<td>DF0919-07-3&#160;</td>
			<td>Culture medium for yeast growth (broth)</td>
		</tr>
		<tr>
			<td>Yeast Peptone Dextrose Broth</td>
			<td>NutriSelect Basic, Sigma Aldrich</td>
			<td>Y1375</td>
			<td>Culture medium for maintaining the strains at -80&#176;C and grow</td>
		</tr>
	</tbody>
</table>

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.

The murine model of OC showed typical white patches and pseudomembranes on the tongue of all infected mice (Figure 4A). C. albicans recovered from C-L- animals confirmed tissue colonization by this microorganism (values ranged from 1.62 x 104 to 4.80 x 105 CFU/mL). As expected, animals from the NCtr group did not show any tissue alterations or colony growth after sampling (Figure 4B).
aPDT decreased the ...

Watch this Scientific Journal Video about Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis at JoVE.com

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.

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 ...

curcuminoid-mediated-antimicrobial-photodynamic-therapy-on-murine

Research

JoVE Journal

Immunology and Infection

749 Views.  São Paulo State University (UNESP). 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.

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

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

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.

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.

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

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...