Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging (Video) | JoVE

Chen Yang; Fei Mo; Jiaxue Zhang; Peipei Zhang; Qingqing Li; Jiye Zhang

doi:10.3791/65307

Immunology and Infection

A Catheter-Related Candida albicans Infection Model in Mouse

Published: March 22, 2024 doi: 10.3791/65307

Chen Yang¹, Fei Mo², Jiaxue Zhang¹, Peipei Zhang¹, Qingqing Li¹, Jiye Zhang¹

¹School of Pharmacy, Health Science Center, Xi’an Jiaotong University, ²Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

Experimental animals, male BALB/c mice (12-16 g), were purchased from the Laboratory Animal Center, Xi'an Jiaotong University Health Science Center. All the procedures were approved by the Institutional Animal Ethical Committee of Xi'an Jiaotong University with the license number SCXK (Shaanxi) 2021-103.

1. Buffer and equipment preparation

Transfect C. albicans strains with a high-expression plasmid pCaExp.
1. 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.
Culture the transfected C. albicans strains.
1. Select monoclonal colonies of C. albicans fluorescent strain from the yeast extract peptone dextrose medium (YPD) plate and culture overnight (30 °C and 220 rpm) in 5 mL of YPD liquid medium (YPD + 50 µg/mL carbenicillin).
2. Resuspend the C. albicans in normal saline after centrifugation at 400 x g for 5 min at RT.
3. Adjust the concentration of C. albicans suspension to 1 x 10⁸ cells/mL by comparing the turbidity to 0.5 McFarland standard.
Prepare the surgical instruments.
1. Ensure to autoclave all the surgical instruments (scissors, forceps, hemostatic forceps, needle holders, suture needles) at 121°C for 30 min. Sterile 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.

2. Establishment of a mouse CRI model

NOTE: The surgical procedure is shown in Figure 2.

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.
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).
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.
Leave the mice in the normal control group without any treatment and provide free access to food and water.
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.
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.
For the mice in the model group, pipette 20 µL of C. albicans suspension onto the back-depilation area to simulate the commensal C. albicans on the skin.
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.
Pipette another 20 µL of C. albicans suspension along the catheter to the tissue to simulate C. albicans in the external environment.
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.

3. Evaluation of the CRI model

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.
Observe C. albicans and biofilms on the catheter by scanning electron microscopy.
1. Immerse the catheters into 2.5% glutaraldehyde solution at 4 °C for 48 h. Rinse the catheters with sterile PBS three times.
2. Fix the catheters with 1% osmic acid for 3 h and rinse them with sterile PBS three times.
3. Dehydrate the cells on the catheters in gradient ethanol solution with ascending concentrations (50%, 70%, 80%, 90%, and 100%, 15 min/gradient).
4. Immerse the catheters in tert-butyl alcohol three times (30 min each time).
5. Quickly freeze the catheters in liquid nitrogen, and freeze-dry the sample in a freeze-dryer as per the manufacturer's instructions.
6. Sputter-coat the catheter samples with a 10 nm gold by an ion beam deposition.
7. 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.
Observe C. albicans on the catheter by fluorescence microscopy.
1. Immerse the catheters in 4% paraformaldehyde solution for fixation at 4 °C for 48 h.
2. 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.
Observe C. albicans residing in the mouse skin.
1. Immerse the dorsal skin tissues of mice into 4% paraformaldehyde solution for fixation at 4 °C for 48 h.
2. Dehydrate the dorsal skin tissues in a gradient ethanol solution with ascending concentrations (50%, 70%, 80%, 90%, and 100%, 15 min/gradient).
3. Embed the dehydrated dorsal skin tissues in paraffin at 55-60 °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).
4. Section the dorsal skin tissues (thickness = 5 µm) with a microtome.
5. Dewax the paraffin sections by immersing the slides in xylene twice for 20 min.
6. Rehydrate the sections via eluting with gradient ethanol (absolute ethanol, 90% ethanol, 75% ethanol, water) for 5 min each time.
7. 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.
8. Stain the sections with a chevron staining solution (per the manufacturer's instructions) for 30 min in the dark and rinse the sections under running water for 5 min.
9. 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.
10. Immerse the sections in ethanol three times ( 5 min each) and xylene two times ( 5 min each) before sealing the section with neutral gum.
11. 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.
Observe the histopathological changes in the dorsal skin tissues.
1. Immerse the dorsal skin tissues into 4% paraformaldehyde solution for fixation at 4 °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).
2. Embed the dehydrated dorsal skin tissues in paraffin as described in step 3.4.3.
3. Section the dorsal skin tissues (thickness = 5 mm) with a microtome.
4. Dewax the paraffin sections by immersing the slides in xylene twice for 20 min.
5. Rehydrate the sections via eluting with gradient ethanol (absolute ethanol, 90% ethanol, 75% ethanol, water) for 5 min each time.
6. Stain the sections with hematoxylin for 4 min before rinsing with tap water to remove float color.
7. Differentiate the specimen with 1% hydrochloric acid-ethanol solution before rinsing the slides with running water.
8. Immerse the specimen in 85% and 95% ethanol for 5 min, and stain them with eosin solution for 3 min.
9. Dehydrate the specimen by immersing them in gradient ethanol (70%, 90%, 95%, and 100%) and xylene for 2 min each.
10. Seal the specimen with neutral resin.
11. Observe the images of the specimen with a microscope and analyze the pathological changes.

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Representative Results

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.

Figure 1: pCaExp plasmid atlas. Please click here to view a larger version of this figure.

Figure 2: Schematic showing the procedure of the C.albicans-associated CRI mice model. Please click here to view a larger version of this figure.

Figure 3: SEM on the surface of the catheter in each group. (A) Catheter group; (B) Model group (1000x, scale bar = 50 µm; 5000x, scale bar = 10 µm). This figure has been modified with permission from Mo et al.²². Please click here to view a larger version of this figure.

Figure 4: Catheter surface fluorescence microscopy in each group. (A) Catheter group; (B) Model group (scale bar = 100 µm). This figure has been modified with permission from Mo et al.²². Please click here to view a larger version of this figure.

Figure 5: H&E staining of the back skin of mice in each group. (A) Catheter group; (B) Model group; (C) Control group, (40x, scale bar = 400 µm; 100x, scale bar = 200 µm). This figure has been modified with permission from Mo et al.²². Please click here to view a larger version of this figure.

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 µm; 100x, scale bar = 200 µm). 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. Please click here to view a larger version of this figure.

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Discussion

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 × 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 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'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.

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Disclosures

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.

Acknowledgments

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).

Materials

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

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Immunology and Infection