Name: a catheter-related candida albicans infection model in mouse
Uploaded: 2024-03-22T12:30:00.000+00:00
Duration: 3 min 24 s
Description: Watch this Scientific Journal Video about Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging at JoVE.com

We are grateful for the financial support from the Natural Science Foundation of Shaanxi Province (grant number 2021SF-118) and the National Natural Science Foundation of China (grant numbers 81973409, 82204631).

Experimental animals, male BALB/c mice (12-16 g), were purchased from the Laboratory Animal Center, Xi&#39;an Jiaotong University Health Science Center. All the procedures were approved by the Institutional Animal Ethical Committee of Xi&#39;an Jiaotong University with the license number SCXK (Shaanxi) 2021-103.
1. Buffer and equipment preparation
<ol>
	<li>Transfect C. albicans strains with a high-expression plasmid pCaExp.
	<ol>
		<li>Purchase C. albicans (SC5314) from ATCC. Obtain the EGFP high-expression fluorescent strain22 by transfecting C. albicans with a high-expression plasmid pCaExp containing the complete open reading frame of EGFP gene (the plasmid map is shown in Figure 1) and use this for subsequent experiments.</li>
	</ol>
	</li>
	<li>Culture the transfected C. albicans strains.
	<ol>
		<li>Select monoclonal colonies of C. albicans fluorescent strain from the yeast extract peptone dextrose medium (YPD) plate and culture overnight (30 &#176;C and 220 rpm) in 5 mL of YPD liquid medium (YPD + 50 &#181;g/mL carbenicillin).</li>
		<li>Resuspend the C. albicans in normal saline after centrifugation at 400 x g for 5 min at RT.</li>
		<li>Adjust the concentration of C. albicans suspension to 1 x 108 cells/mL by comparing the turbidity to 0.5 McFarland standard.</li>
	</ol>
	</li>
	<li>Prepare the surgical instruments.
	<ol>
		<li>Ensure to autoclave all the surgical instruments (scissors, forceps, hemostatic forceps, needle holders, suture needles) at 121&#176;C for 30 min. Sterile&#160;polyethylene catheters (inner diameter: 0.28 mm; outer diameter: 0.63 mm) are used. 
		NOTE: The catheters used in this study were sterilized with ethylene oxide gas and the packaging was opened in an ultra-clean table exposed to UV for more than 30 min. Prior to implantation in mice, the catheters were immersed in 75% ethanol to prevent contamination.</li>
	</ol>
	</li>
</ol>
2. Establishment of a mouse CRI model
NOTE: The surgical procedure is shown in Figure 2.
<ol>
	<li>Acclimatize 30 BALB/c mice (12-16 g, male) in specific-pathogen-free (SPF) conditions with free access to water and food and 12 h-12 h alternating light and dark cycle.</li>
	<li>Randomly divide 30 BALB/c mice into three groups (n = 10 mice/group): (A) normal control group; (B) catheter group (catheters implanted without C. albicans); (C) Model group (catheters implanted with C. albicans).</li>
	<li>Anesthetize the mice with 1-4% isoflurane and place the mice on an operating table in a prone position. The loss of righting reflex and no response to toe stimulation confirms the successful anesthesia. Remove the hair and sterilize the surgical site with three alternating rounds of iodophor or chlorhexidine and alcohol scrubs.</li>
	<li>Leave the mice in the normal control group without any treatment and provide free access to food and water.</li>
	<li>For the mice in the catheter and model groups, maintain anesthesia at 3% isoflurane. Confirm adequate anesthetic depth by the absence of a response to toe pinch and adjust isoflurane concentration as needed.</li>
	<li>For the mice in the catheter group, intradermally insert a 1 mL sterile syringe needle into the back-depilated area to make a hole. Insert a catheter (about 1 cm in length) into the hole after removing the syringe needle.</li>
	<li>For the mice in the model group, pipette 20 &#181;L of C. albicans suspension onto the back-depilation area to simulate the commensal C. albicans on the skin.</li>
	<li>After the solution gets absorbed by the skin, insert a catheter in the back-depilated area with the same procedures as described in step 2.5.</li>
	<li>Pipette another 20 &#181;L of C. albicans suspension along the catheter to the tissue to simulate C. albicans in the external environment.</li>
	<li>Fix the catheters with tape and gauze and return the mice to cages for feeding. At the end of the treatment, subcutaneously inject the mice with meloxicam (4 mg/kg) as analgesia for three consecutive days. 
	NOTE: After surgery, mice were carefully fed with water and food. The mice were monitored twice a day. Mice were euthanized by an IACUC-approved method if they experienced feeding difficulties, significant weight loss (10-20%), and hypothermia.</li>
</ol>
3. Evaluation of the CRI model
<ol>
	<li>After 3 days, anesthetize the mice with 3% isoflurane and sacrifice them by cervical dislocation. Collect the catheters and skin tissue samples from the back of the mice.</li>
	<li>Observe&#160;C. albicans and biofilms on the catheter by scanning electron microscopy.
	<ol>
		<li>Immerse the catheters into 2.5% glutaraldehyde solution at 4 &#176;C for 48 h. Rinse the catheters with sterile PBS three times.</li>
		<li>Fix the catheters with 1% osmic acid for 3 h and rinse them with sterile PBS three times.</li>
		<li>Dehydrate the cells on the catheters in gradient ethanol solution with ascending concentrations (50%, 70%, 80%, 90%, and 100%, 15 min/gradient).</li>
		<li>Immerse the catheters in tert-butyl alcohol three times (30 min each time).</li>
		<li>Quickly freeze the catheters in liquid nitrogen, and freeze-dry the sample in a freeze-dryer as per the manufacturer&#39;s instructions.</li>
		<li>Sputter-coat the catheter samples with a 10 nm gold by an ion beam deposition.</li>
		<li>Observe the presence of C. albicans and its biofilm on the catheter surface of each group under a scanning electron microscope (under high vacuum, 1.5 kV conditions) and record the images in each group.</li>
	</ol>
	</li>
	<li>Observe C. albicans on the catheter by fluorescence microscopy.
	<ol>
		<li>Immerse the catheters in 4% paraformaldehyde solution for fixation at 4 &#176;C for 48 h.</li>
		<li>Observe the presence of C. albicans and its biofilm on the catheter surface of each group by a fluorescence microscope under 484 nm excitation and record the images in each group. 
		NOTE: The magnification is 400x. The fluorescence of Candida albicans can be observed with excitation at 490 nm and emission at 510 nm.</li>
	</ol>
	</li>
	<li>Observe C. albicans residing in the mouse skin.
	<ol>
		<li>Immerse the dorsal skin tissues of mice into 4% paraformaldehyde solution for fixation at 4 &#176;C for 48 h.</li>
		<li>Dehydrate the dorsal skin tissues in a gradient ethanol solution with ascending concentrations (50%, 70%, 80%, 90%, and 100%, 15 min/gradient).</li>
		<li>Embed the dehydrated dorsal skin tissues in paraffin at 55-60 &#176;C. Pay attention to the temperature to avoid brittle tissues. To remove as many impurities as possible, repeat this step three times (30 min each).</li>
		<li>Section the dorsal skin tissues (thickness = 5 &#181;m) with a microtome.</li>
		<li>Dewax the paraffin sections by immersing the slides in xylene twice for 20 min.</li>
		<li>Rehydrate the sections via eluting with gradient ethanol (absolute ethanol, 90% ethanol, 75% ethanol, water) for 5 min each time.</li>
		<li>Stain the sections with periodic acid by immersing the section in the periodic acid solution for 15 min before washing with running water once and distilled water twice.</li>
		<li>Stain the sections with a chevron staining solution (per the manufacturer&#39;s instructions) for 30 min in the dark and rinse the sections under running water for 5 min.</li>
		<li>Immerse the sections in hematoxylin solution for 3-5 min before washing with running water (2-3 min), differentiated solution (5-10 min), and running water, respectively.</li>
		<li>Immerse the sections in ethanol three times ( 5 min each) and xylene two times ( 5 min each) before sealing the section with neutral gum.</li>
		<li>Observe the images of the specimen with a microscope and analyze the C. albicans residues in mouse skin. 
		NOTE: The magnification is 10x for the eyepiece and 4x or 10x for the objective lens.</li>
	</ol>
	</li>
	<li>Observe the histopathological changes in the dorsal skin tissues.
	<ol>
		<li>Immerse the dorsal skin tissues into 4% paraformaldehyde solution for fixation at 4 &#176;C for 48 h. Dehydrate the dorsal skin tissues in gradient ethanol solution with ascending concentrations (50%, 70%, 80%, 90%, and 100%, 15 min/gradient).</li>
		<li>Embed the dehydrated dorsal skin tissues in paraffin as described in step 3.4.3.</li>
		<li>Section the dorsal skin tissues (thickness = 5 mm) with a microtome.</li>
		<li>Dewax the paraffin sections by immersing the slides in xylene twice for 20 min.</li>
		<li>Rehydrate the sections via eluting with gradient ethanol (absolute ethanol, 90% ethanol, 75% ethanol, water) for 5 min each time.</li>
		<li>Stain the sections with hematoxylin for 4 min before rinsing with tap water to remove float color.</li>
		<li>Differentiate the specimen with 1% hydrochloric acid-ethanol solution before rinsing the slides with running water.</li>
		<li>Immerse the specimen in 85% and 95% ethanol for 5 min, and stain them with eosin solution for 3 min.</li>
		<li>Dehydrate the specimen by immersing them in gradient ethanol (70%, 90%, 95%, and 100%) and xylene for 2 min each.</li>
		<li>Seal the specimen with neutral resin.</li>
		<li>Observe the images of the specimen with a microscope and analyze the pathological changes.</li>
	</ol>
	</li>
</ol>

Establishing a Mouse Model for Candida albicans-Associated Catheter-Related Infection

<ol>
	<li>Kojic, E. M., Darouiche, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+infections+of+medical+devices.">Candida infections of medical devices.</a> microbiology reviews. 17 (2), 255-267 (2004).</li><li>Giri, S., Kindo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+Candida+species+causing+blood+stream+infection.">A review of Candida species causing blood stream infection.</a> Indian Journal of Medical Microbiology. 30 (3), 270-278 (2012).</li><li>Weinstein, R. A., Darouiche, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Device-associated+infections:+A+macroproblem+that+starts+with+microadherence.">Device-associated infections: A macroproblem that starts with microadherence.</a> Clinical Infectious Diseases. 33 (9), 1567-1572 (2001).</li><li>Mermel, L. A., 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=Guidelines+for+the+management+of+intravascular+catheter-related+infections.">Guidelines for the management of intravascular catheter-related infections.</a> Clinical Infectious Diseases. 32 (9), 1249-1272 (2001).</li><li>Seidler, M., Salvenmoser, S., M&#252;ller, F. -. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effects+of+micafungin+against+Candida+biofilms+on+polystyrene+and+central+venous+catheter+sections.">In vitro effects of micafungin against Candida biofilms on polystyrene and central venous catheter sections.</a> International Journal of Antimicrobial Agents. 28 (6), 568-573 (2006).</li><li>Chaves, F., 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=Diagnosis+and+treatment+of+catheter-related+bloodstream+infection:+Clinical+guidelines+of+the+Spanish+Society+of+Infectious+Diseases+and+Clinical+Microbiology+and+(SEIMC)+and+the+Spanish+Society+of+Spanish+Society+of+Intensive+and+Critical+Care+Medicine+and+Coronary+Units+(SEMICYUC).">Diagnosis and treatment of catheter-related bloodstream infection: Clinical guidelines of the Spanish Society of Infectious Diseases and Clinical Microbiology and (SEIMC) and the Spanish Society of Spanish Society of Intensive and Critical Care Medicine and Coronary Units (SEMICYUC).</a> Medicina Intensiva. 42 (1), 5-36 (2018).</li><li>Raad, I. I., Bodey, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+complications+of+indwelling+vascular+catheters.">Infectious complications of indwelling vascular catheters.</a> Clinical Infectious Diseases. 15 (2), 197-208 (1992).</li><li>Paul DiMondi, V., Townsend, M. L., Johnson, M., Durkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+catheter+lock+therapy+for+the+management+of+a+persistent+Candida+albicans+bloodstream+infection+in+an+adult+receiving+hemodialysis.">Antifungal catheter lock therapy for the management of a persistent Candida albicans bloodstream infection in an adult receiving hemodialysis.</a> Pharmacotherapy. 34 (7), e120-e127 (2014).</li><li>Bouza, E., Guinea, J., Guembe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+antifungals+against+candida+biofilm+in+catheter-related+candidemia.">The role of antifungals against candida biofilm in catheter-related candidemia.</a> Antibiotics (Basel). 4 (1), 1-17 (2014).</li><li>Raad, I., 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=Ultrastructural+analysis+of+indwelling+vascular+catheters:+a+quantitative+relationship+between+luminal+colonization+and+duration+of+placement.">Ultrastructural analysis of indwelling vascular catheters: a quantitative relationship between luminal colonization and duration of placement.</a> The Journal of Infectious Diseases. 168 (2), 400-407 (1993).</li><li>Yousif, A., Jamal, M. A., Raad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-based+central+line-associated+bloodstream+infections.">Biofilm-based central line-associated bloodstream infections.</a> Advances in Experimental Medicine and Biology. 830, 157-179 (2015).</li><li>Douglas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+biofilms+and+their+role+in+infection.">Candida biofilms and their role in infection.</a> Trends in Microbiology. 11 (1), 30-36 (2003).</li><li>Mack, D., 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=Biofilm+formation+in+medical+device-related+infection.">Biofilm formation in medical device-related infection.</a> International Journal of Artificial Organs. 29 (4), 343-359 (2006).</li><li>Schinabeck, M. K., 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=Rabbit+model+of+Candida+albicans+biofilm+infection:+liposomal+amphotericin+B+antifungal+lock+therapy.">Rabbit model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock therapy.</a> Antimicrobial Agents and Chemotherapy. 48 (5), 1727-1732 (2004).</li><li>Anaissie, E. J., Rex, J. H., Uzun, O., Vartivarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+adverse+outcome+in+cancer+patients+with+candidemia.">Predictors of adverse outcome in cancer patients with candidemia.</a> The American Journal of Medicine. 104 (3), 238-245 (1998).</li><li>Fujimoto, K., Takemoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+liposomal+amphotericin+B+against+four+species+of+Candida+biofilms+in+an+experimental+mouse+model+of+intravascular+catheter+infection.">Efficacy of liposomal amphotericin B against four species of Candida biofilms in an experimental mouse model of intravascular catheter infection.</a> Journal of Infection and Chemotherapy. 24 (12), 958-964 (2018).</li><li>Shuford, J. A., Rouse, M. S., Piper, K. E., Steckelberg, J. M., Patel, R. <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+caspofungin+and+amphotericin+B+deoxycholate+against+Candida+albicans+biofilms+in+an+experimental+intravascular+catheter+infection+model.">Evaluation of caspofungin and amphotericin B deoxycholate against Candida albicans biofilms in an experimental intravascular catheter infection model.</a> The Journal of Infectious Diseases. 194 (5), 710-713 (2006).</li><li>Koh, A. Y., K&#246;hler, J. R., Coggshall, K. T., Van Rooijen, N., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+damage+and+neutropenia+are+required+for+Candida+albicans+dissemination.">Mucosal damage and neutropenia are required for Candida albicans dissemination.</a> PLoS Pathogens. 4 (2), e35 (2008).</li><li>Tucey, T. 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=Glucose+homeostasis+is+important+for+immune+cell+viability+during+candida+challenge+and+host+survival+of+systemic+fungal+infection.">Glucose homeostasis is important for immune cell viability during candida challenge and host survival of systemic fungal infection.</a> Cell Metabolism. 27 (5), 988-1006 (2018).</li><li>Lawrence, E. L., Turner, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+for+urinary+catheters:+a+review+of+their+history+and+development+in+the+UK.">Materials for urinary catheters: a review of their history and development in the UK.</a> Medical Engineering &amp; Physics. 27 (6), 443-453 (2005).</li><li>Schumm, K., Lam, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+of+urethral+catheters+for+management+of+short-term+voiding+problems+in+hospitalized+adults:+a+short+version+Cochrane+review.">Types of urethral catheters for management of short-term voiding problems in hospitalized adults: a short version Cochrane review.</a> Neurourology and Urodynamics. 27 (8), 738-746 (2008).</li><li>Mo, F., 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=Development+and+evaluation+of+a+film+forming+system+containing+myricetin+and+miconazole+nitrate+for+preventing+candida+albicans+catheter-related+infection.">Development and evaluation of a film forming system containing myricetin and miconazole nitrate for preventing candida albicans catheter-related infection.</a> Microbial Drug Resistance. 28 (4), 468-483 (2022).</li><li>Balikci, E., Yilmaz, B., Tahmasebifar, A., Baran, E. T., Kara, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+strategies+for+hemodialysis+catheters+to+prevent+catheter-related+infections:+A+review.">Surface modification strategies for hemodialysis catheters to prevent catheter-related infections: A review.</a> Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 109 (3), 314-327 (2021).</li><li>Mar&#237;a, L. T., Alejandro, G. S., Mar&#237;a Jes&#250;s, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+venous+catheter+insertion:+Review+of+recent+evidence.">Central venous catheter insertion: Review of recent evidence.</a> Best Practice &amp; Research. Clinical Anaesthesiology. 35 (1), 135-140 (2021).</li><li>Kojic, E. M., Darouiche, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+infections+of+medical+devices.">Candida infections of medical devices.</a> Clinical Microbiology Reviews. 17 (2), 255-267 (2004).</li><li>He, Y., 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=Retrospective+analysis+of+microbial+colonization+patterns+in+central+venous+catheters,+2013-2017.">Retrospective analysis of microbial colonization patterns in central venous catheters, 2013-2017.</a> Journal of Healthcare Engineering. 2019, 8632701 (2019).</li><li>Mo, F., 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=In+vitro+and+in+vivo+effects+of+the+combination+of+myricetin+and+miconazole+nitrate+incorporated+to+thermosensitive+hydrogels+on+C.">In vitro and in vivo effects of the combination of myricetin and miconazole nitrate incorporated to thermosensitive hydrogels on C.</a> albicans biofilms. Phytomedicine. 71, 153223 (2020).</li><li>Cant&#243;n-Bulnes, M. L., Garnacho-Montero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+approach+to+the+management+of+catheter-related+bloodstream+infection.">Practical approach to the management of catheter-related bloodstream infection.</a> Revista Espanola de Quimioterapia. 32 Suppl 2 (Suppl 2), 38-41 (2019).</li><li>B&#246;hlke, M., Uliano, G., Barcellos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodialysis+catheter-related+infection:+prophylaxis,+diagnosis+and+treatment.">Hemodialysis catheter-related infection: prophylaxis, diagnosis and treatment.</a> The Journal of Vascular Access. 16 (5), 347-355 (2015).</li><li>Fang, X., 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=Effects+of+different+tissue+specimen+pretreatment+methods+on+microbial+culture+results+in+the+diagnosis+of+periprosthetic+joint+infection.">Effects of different tissue specimen pretreatment methods on microbial culture results in the diagnosis of periprosthetic joint infection.</a> Bone &amp; Joint Research. 10 (2), 96-104 (2021).</li><li>Naumenko, Z. S., Silanteva, T. A., Ermakov, A. M., Godovykh, N. V., Klushin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenging+diagnostics+of+biofilm+associated+periprosthetic+infection+in+immunocompromised+patient:+A+clinical+case.">Challenging diagnostics of biofilm associated periprosthetic infection in immunocompromised patient: A clinical case.</a> Open Access Macedonian Journal of Medical Sciences. 7 (5), 786-790 (2019).</li><li>Cai, Y., 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=Metagenomic+next+generation+sequencing+improves+diagnosis+of+prosthetic+joint+infection+by+detecting+the+presence+of+bacteria+in+periprosthetic+tissues.">Metagenomic next generation sequencing improves diagnosis of prosthetic joint infection by detecting the presence of bacteria in periprosthetic tissues.</a> International Journal of Infectious Diseases. 96, 573-578 (2020).</li><li>Samanipour, A., Dashti-Khavidaki, S., Abbasi, M. R., Abdollahi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+patterns+of+microorganisms+isolated+from+nephrology+and+kidney+transplant+wards+of+a+referral+academic+hospital.">Antibiotic resistance patterns of microorganisms isolated from nephrology and kidney transplant wards of a referral academic hospital.</a> Journal of Research in Pharmacy Practice. 5 (1), 43-51 (2016).</li><li>Huang, G., Huang, Q., Wei, Y., Wang, Y., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+and+diverse+regulation+of+the+Ras/cAMP/protein+kinase+A+pathway+in+Candida+albicans.">Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans.</a> Molecular Microbiology. 111 (1), 6-16 (2019).</li><li>Garlito-D&#237;az, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+antifungal-loaded+sol-gel+can+prevent+candida+albicans+prosthetic+joint+infection.">A new antifungal-loaded sol-gel can prevent candida albicans prosthetic joint infection.</a> Antibiotics (Basel). 10 (6), 711 (2021).</li></ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRI is one of the most common nosocomial infections in clinical practice23. Pathogens in the skin appendages, such as the epidermis, sebaceous glands, and hair follicles, are all possible causes of CRI23,24. Candida is the third largest pathogen that causes CRI, in which Candida albicans was the most common type of biofilm infection25,26. Therefore, we aimed to build a relevant animal model of Candida albicans biofilm-related CRI so as to support the treatment and prevention of related CRI.
To construct the CRI model, a small amount of C. albicans was added to the dorsal skin of mice, which simulates the clinical situation in which part of the C. albicans cannot be fully eradicated in the deep tissues and appendages of the skin by routine sterilization. After the implantation of the catheter, C. albicans was re-inoculated to mimic the presence of C. albicans in the external environment during surgery.
In this study, a 3-day time point was selected for the model construction, which is lower than that of the traditional C. albicans biofilm-related animal models18,27 due to the difficulty in the biofilm formation. Post-infection, C. albicans adhesion and biofilm formation were visible on the catheter surface in this model, which was proved by the SEM and fluorescence microscopy results (Figure 3 and Figure 4). This may be due to the concentration of C. albicans in this study was 1 &#215; 108 CFU/mL, which was much higher than that of other animal models18,27. Besides, the skin around the catheter is in constant contact with the external environment. To simulate the extreme environments that CRI may encounter, C. albicans were inoculated again after the surgery.
The recurrence of infection is often caused by pathogens that remain in surrounding tissues23,28,29. Therefore, the presence or absence of pathogens in tissues is important for CRI. In this paper, PAS staining was undertaken to investigate the residues of C. albicans in the skin tissues. This method could also be used to evaluate the clearance effect of new therapeutic drugs or methods for CRI.
In conclusion, a Candida albicans strain with eGFP was used to construct a mouse CRI model to facilitate the intuitive observation of Candida albicans colonization&#160;on catheters. This strain can also be used to evaluate the interaction between Candida albicans and host cells, for example, the invasion and adhesion of Candida albicans to the host, the anti-Candida albicans effect of therapeutics, and the immune response. Besides, a two-step inoculation method was used to simulate pathogens derived from the external environment and the body. It is worth noting that subsequent microbial culture after infection was not conducted. The presence of biofilms is an important factor in the low sensitivity of cultures30,31,32. Previous reports suggest that microbial culture after infection had low sensitivity, specificity, and accuracy30,31,32,33,34. Instead, the presence of biofilms on the implant is a more reliable index. Therefore, SEM and fluorescence microscopy were used in this study to visualize and identify Candida albicans forming biofilms.
However, this model did not simulate the interaction between the patient&#39;s weakened immunity and the Candida albicans infection observed in clinics. If the model could consider the immunocompromised treatments (such as continuous injections of glucocorticoids)35 before the Candida albicans inoculation, it would be possible to better simulate infections occurring in clinical situations.

In recent years, with the development and application of biomedical materials, implant-related infections are emerging as difficult clinical problems1,2. With the wide application of medical catheters in clinics, the number of related infections and deaths is huge every year3,4. The common infection routes of a catheter-related infection (CRI) include: (1) pathogens on the surface of the skin infiltrate into the body and adhere to the outer surface of the catheter5,6,7; (2) improper aseptic operation-derived pathogens invade, adhere and colonize on the catheter; (3) pathogens in the blood circulation adhere and colonize on the catheter; (4) drugs contaminated by pathogenic microorganism.
Candida is the third most common reason for CRI8,9. It is very likely to cause bloodstream infection and other life-threatening invasive candidiasis after biofilms are formed on the surface of the implant. The prognosis is poor, and the mortality rate is high2. It is reported that biofilms are formed on the surface of the catheter within 2 weeks after central venous insertion and in the lumen of the catheter a few weeks later10,11.
Candida albicans (C. albicans) biofilms formed on medical catheters exhibit a double-layer network composed of yeast, stroma, and mycelium12,13. The formation of C. albicans biofilms is not only a key for drug resistance and immune evasion13 but also vital to produce disseminated spores, which leads to further hematogenous infection2,12 and results in more serious and even life-threatening consequences. C. albicans-associated CRI is a major cause of clinical fungal bloodstream infections7,14, and more than 40% of patients with C. albicans infection in the central venous catheter will develop into bacteremia15.
According to the Infectious Disease Society of America, the recommended treatment of Candida CRI includes (1) removal of the infected catheter; (2) subjecting the patients to a 14 days-systemic antifungal therapy8; (3) reimplanting a new catheter4. However, in clinical applications, catheters cannot be fully removed sometimes. Some patients can only be treated with systemic antibiotics and antimicrobial lock therapy, accompanied by strong side effects16,17.
Existing animal models of C. albicans, such as the oropharyngeal candidiasis model, vaginal candidiasis model, and invasive systemic infection model caused by candidiasis18,19 cannot correlate well with the clinical CRI. Therefore, in this study, a C. albicans-associated CRI model in mice was established. Clinical commonly used polyethylene catheters were used as subcutaneous implants20,21, and C. albicans were inoculated on the skin surface to simulate the adhesion of C. albicans to the medical catheters and the formation of biofilms.
This model has been successfully used in our laboratory to screen the anti-biofilm effect of different therapeutics22. In addition, due to the lag detection of C. albicans after catheter infection, a C. albicans strain containing enhanced green fluorescent protein (EGFP) was constructed and inoculated in mice to facilitate the intuitive observation of the colonies and biofilms of C. albicans on the implanted catheter.

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

a catheter-related candida albicans infection model in mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

The C. albicans and biofilms on the catheters could be observed by the SEM. As shown in Figure 322, the surface of the polyethylene catheters in the catheter group was smooth, and no adhered pathogenic microorganism was observed. However, mature and dense C. albicans biofilms were visible on the surface of the polyethylene catheters in the model group, indicating that C. albicans could successfully colonize and form biofilms on the catheter surface in mice under the experimental conditions. Moreover, fluorescence microscopy results further verified the above conclusions (Figure 4)22. There was no obvious fluorescence on the surface of the polyethylene catheters in the catheter group. However, strong fluorescence emitted by adherent C. albicans cells was visible on the catheter surface in the model group. This indicated that a large number of C. albicans cells adhered to the surface of the catheters, which demonstrated the successful construction of C. albicans biofilm-related CRI models in mice.
In order to verify the infection of mouse skin tissue more intuitively, Sheff Periodate staining analysis was performed. It detects the carbohydrates of the fungal cells, which is commonly used in clinical research (Figure 5)22. The skin tissue in the normal control and catheter group was stained negatively by periodic acid-Schiff (PAS), which indicated the absence of C. albicans cells in the tissues. A small number of positive PAS-stained C. albicans cells were observed in the model group, further validating the successful simulation of C. albicans-related invasion and adhesion.
Next, the pathological changes in mice skin tissues induced by C. albicans were evaluated by histopathological analysis. As shown in Figure 622, the epidermis layer was significantly thickened and extended to the inner part of the skin in the model group. Inflammation infiltration was also visible, indicating that the infection of C. albicans caused obvious pathological changes in mouse skin tissue. The epidermis layer, dermis layer, sebaceous glands, hair follicles, and other structures were clear and complete in the catheter group. No edema and inflammation infiltration were observed, similar to the normal control group. These results indicated that inserting the catheter alone did not cause obvious changes in the skin tissue. The pathological changes in the tissues of the model group resulted from the infection caused by C. albicans. In summary, the results validate the successful establishment of a CRI mouse model associated with C. albicans biofilm.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65307/65307fig01.jpg" /> 
Figure 1: pCaExp plasmid atlas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65307/65307fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65307/65307fig02.jpg" /> 
Figure 2: Schematic showing the procedure of the C.albicans-associated&#160;CRI mice model. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65307/65307fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65307/65307fig03.jpg" /> 
Figure 3: SEM on the surface of the catheter in each group. (A) Catheter group;&#160;(B)&#160;Model group (1000x, scale bar = 50 &#181;m; 5000x, scale bar = 10 &#181;m). This figure has been modified with permission from Mo et al.22. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65307/65307fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65307/65307fig04.jpg" /> 
Figure 4: Catheter surface fluorescence microscopy in each group. (A) Catheter group; (B)&#160;Model group (scale bar = 100 &#181;m). This figure has been modified with permission from Mo et al.22. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65307/65307fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65307/65307fig05.jpg" /> 
Figure 5: H&#38;E staining of the back skin of mice in each group. (A) Catheter group; (B) Model group; (C) Control group, (40x, scale bar = 400 &#181;m; 100x, scale bar = 200 &#181;m). This figure has been modified with permission from Mo et al.22. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65307/65307fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65307/65307fig06.jpg" /> 
Figure 6: PAS staining of the back skin of mice in each group. (A) Catheter group; (B) Model group; (C) Control group, (40x, scale bar = 400 &#181;m; 100x, scale bar = 200 &#181;m). Significant thickening and extension of the epidermis layer to the inner part of the skin can be seen in the model group (red rectangles). This figure has been modified with permission from Mo et al.22. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65307/65307fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

A Catheter-Related Candida albicans Infection Model in Mouse

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

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.4K Views.  Xi’an Jiaotong University. Ons onderzoek is gericht op het opstellen van een muismodel van Candida albicans-geassocieerde kathetergerelateerde infectie, waarbij biofilm zich vormt op de katheter. Dit model is nuttig voor het correleren met de klinische CRI en de interactie tussen Candida albicans en gastheer. De bestaande diermodellen van Candida albicans, zoals het orofaryngeale candidiasismodel, het vaginale candidiasismodel en het invasieve systemische infectiemodel veroorzaakt door candidiasis, kunnen niet goed cor...

Video: Auteur in de schijnwerpers: Verbetering van de detectie van Candida albicans bij katheterinfecties met behulp van fluorescerende eiwittagging 

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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

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

Medicine

Establishment and Characterization of UTI and CAUTI in a Mouse Model

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

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

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

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.remotexs.ntu.edu.sg/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.

1. C. albicans Growth

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

A Catheter-Related Candida albicans Infection Model in Mouse

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video