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.
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.
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.
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
2. Establishment of a mouse CRI model
NOTE: The surgical procedure is shown in Figure 2.
3. Evaluation of the CRI model
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.
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.
The authors have nothing to disclose.
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).
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 |