شناسایی و حذف عفونت‌های پروتئوس وولگاریس و کلپسیلا پنومونیا در موش‌های نود

Introduction

Immunodeficient mice are introduced as principal sources of animal models in a wide range of biomedical experiments, especially in oncology researches (1). Nude (nu) mouse harbors a single-gene mutation in Foxn1 gene, winged-helix/forkead transcription factor, which causes the lack of body hair. In 1966, it was unveiled that homozygote nu mutations contribute to a rudimentary thymus, called athymic state. An athymic state generally leads to inappropriate T-cell-mediated functions, including rejection of transplantation and deficiency, or lack of defense against infections. On the other hand, upon their different strains and microbiological status, nude mice possess natural killer (NK) cells and macrophages with different variety of numbers and activities. Xenograft transplants can be successfully generated from nude mice (2). However, because of their inhibited immune system, nudes are extremely vulnerable to infection and, in spite of the advanced monitoring systems and high level of expertise applied for nude mice maintenance, development of infectious agents is unavoidable. Along with diverse mice strains (3-5), several papers have introduced nude mice as mouse models of bacterial or viral infections (6, 7). However, there is a lack of information for guidelines preventing unwanted infections, and developing in-vivo experiments with least mice fatality. One of the few researches performed to solve this problem is the study of Burr and colleagues. They  introduced a method of treatment for Corynebacterium-associated murine hyperkeratosis (8).  Giving a particular emphasis to the impact of close monitoring of mice health status, the current study presents a guideline for the detection and eradication of bacterial infections, Proteus vulgaris and Klebsiella pneumoniae, in nude mice.

Materials and Methods

Chemicals and reagents: BACTEC automated blood culture and pediatric vials were purchased from Becton Dickinson, Inc (Towson, USA). Ciprofloxacin (500 mg) and cefixime (400 mg) were obtained from Ariya (Tehran, Iran) and Loghman (Tehran, Iran), respectively.

Mice, maintenance and xenographt transplantation: 48 athymic nude mice (C57BL/6 Nude, 4–6 week-old female, 12.7 ± 0.17 gr (mean ± SEM) were purchased from Pasteur Institute (Amole, Iran). Mice were placed in a standard specific-pathogen-free (SPF) room and xenograft transplantation was performed at week 2 according to the approved national patent number IR.UI.REC.1396.056 (for more information, related protocols and procedures for confirmatory tests are introduced in the supplementary information). The study was in accordance with the ethical standards of the responsible Ethics Committee of the University of Isfahan (ethics number: IR.UI.REC.1396.056 for animal handling). Food, beddings, water bottles and cages were sterilized and replaced twice weekly.

Detection of infectious agents and antimicrobial susceptibility tests: Four weeks followed by transplantation, mice started losing weight and 7 mice died during 4 days. Autopsies were performed and automated blood cultures were performed using radiometric BACTEC system.  200 µl of mice heart-blood samples were subjected into separate BACTEC tubes and sent to a pathology reference laboratory (Isfahan, Iran) for investigating the origin of infection. Antimicrobial susceptibility tests were performed using disc diffusion method on Mueller-Hinton agar culture media. The diameter of the inhibition zone around each disc was measured and the degree of sensitivity was interpreted as susceptible, intermediate or resistant. Same procedure was performed while we encountered a second infection at week 6 post- transplantation. Selected antibiotic tabs were dissolved in 2% dextrose in water then filtered. Dextrose was added to the solvent because of the antibiotics bitter taste. Water bottles containing antibiotics were replaced twice a week.

Mice isolation and precautionary managements: Followed by each time identification of infectious pathogens, cages, food and water bottles were replaced with newly disinfected resources and daily change of beddings were carried out in order to prevent any possible spread of infection through feces. Mice showing torpid or aggressive behavior, hunched posture or sudden weight loss were considered as suspected carriers of infection and transferred to separate cages.

Results

Post-mortem examination of infected mice: Four weeks followed by transplantation (Figure 1S), body weight was decreased in 7 mice (-2.2 ± 0.75 gr, mean ± SEM) resulting in their sudden death during 4 days. Autopsies showed a couple of abscesses in corpse abdominal cavities (Figure 1).

Fig. 1- Comparison between the infected and healthy mice. Followed by ventral dissections, the abdominal cavities of infected mice were compared with that of a healthy mouse. Infected nudes showed several abscesses (indicated by arrows). Corps are representatives of infected (A) and healthy (B) mice.

.Demand-treatment and low-dose prophylaxis: At week 4 post-transplantation, BACTEC analyses determined the presence of Proteus vulgaris in the heart-blood samples of infected mice. Antibiogram tests demonstrated bacteria susceptibility to several antibiotics, among which, ciprofloxacin was chosen (Figure 2A, B). Mice with altered behavior were kept in separate cages from healthy mice and given 132 mg/kg oral ciprofloxacin (9) for 2 weeks. Blood culture analyses showed negative contamination post demand-treatment.  At week eight, Klebsiella pneumoniae was identified in the blood samples of two dead mice. Antibiotic susceptibility tests showed Klebsiella susceptibility to cephalosporins (Figure 3A, B). Mice with signs of infection were treated with 500 mg/kg oral cefixime, for 1 week. At the same time, 132 mg/kg cefixime was given to the remained mice as prophylaxis. At week nine, automated blood culture analysis results showed no bacterial contamination, indicating complete elimination of bacteria. Subsequently, the prophylactic treatment was terminated and no more infection was observed until the end of the experiment (Figure 4). According to the protocol introduced in this study, the infection rate was decreased from 29.16 to 0 percent in the SPF room.

Fig. 2- Representatives of an automated blood culture (A) and an antimicrobial susceptibility test (B) followed by first time murine infection. BACTEC analyses detected Proteus vulgaris in the heart-blood samples of dead mice. Antimicrobial susceptibility tests determined a wide range of antibiotics, among which the injectable antibiotics were ignored to prevent any mouse skin damage and stressful conditions. Oral ciprofloxacin was therefore selected and administered to the infected mice.

Fig. 3- Representatives of an automated blood culture (A), and an antimicrobial susceptibility test (B) followed by second time murine infection. BACTEC analyses detected eradication of Proteus vulgaris, but detection of Klebsiella pneumoniae in the heart-blood samples of dead mice. Antimicrobial susceptibility tests determined resistance to the previously administered ciprofloxacin, but susceptibility to the cephalosporin family of antibiotics.

Fig. 4- Guideline for detection and elimination of bacterial infections in nude mice

Discussion and Conclusion

Nude mice can be used as experimental animal models for human diseases, especially in cancer research (1).  Due to the lack of T cells and severely impaired immune response, nudes are susceptible to infection (10, 11) and they mostly have short life span. Weight loss or failure to gain weight is one of the symptoms of infection. Considering that the immune system is not completely inactive in nude mice, providing a specific, but not absolute, pathogen-free living condition for these animals is adequate for maintaining them in good health, in order to perform in-vivo experiments (12). However, keeping nude mice in an SPF room must be accompanied with the closely monitoring of mice health conditions, in order to prevent any possible infections during the examinations. In the current study, after confirmation of the bacterial infection in blood cultures using radiometric BACTEC system, specific antibiotic was selected amongst those suggested by antimicrobial susceptibility tests. The rationale behind this selection was the absence of any interference between the chosen antibiotic and the chemotherapy regimen applied during the study. Furthermore, the feasibility of administering the anti-bacterial treatment was concerned. Therefore, oral antibiotics were preferred in this regard. Eventually, complete bacterial elimination was reported and the experiment was terminated at the predicted time.

The bacterial infections identified in this study were P. vulgaris and K. pneumoniae. Proteus vulgaris is a rod-shaped, gram-negative bacterium, which belongs to the family of Enterobacteriaceae, and can cause gastrointestinal infections (13, 14). K. pneumoniae is a gram-negative bacterium inducing pneumonia, urinary tract infections and liver abscesses (15). K. pneumoniae is considered as a fatal, virulent bacterium for nude mice. Our results demonstrated that Proteus vulgaris was sensitive to ciprofloxacin. Chauhan and Pathiranage found similar sensitivity pattern for Proteus vulgaris (16, 17). However, Bilal and Akerele claimed that Proteus vulgaris was resistant to ciprofloxacin (18, 19). On the other hand, according to the present study Klebsiella pneumonia is susceptible to cefixime. This information is not in compliance with previous studies performed by Bokaeian and Gurung (20, 21). Variation in bacteria resistance patterns may be attributed to the antibiotics manufacturers’ protocols of application and their possible different formulations.

The prophylactic antimicrobial treatment introduced in this study helped decrease the infection rate from 29.16 to 0 percent. Prophylaxis is defined as a preventive care which is recommended in different vulnerable conditions including surgical procedures (22), cancer chemotherapy-induced neutropenia (23) and several hematological disorders such as hemophilia (24). The immune system of nude mice is impaired; hence these animals are easily prone to various infections, even while maintained in SPF laboratories. Prophylaxis was previously administered to prevent streptococcus pneumoniae and chikungunya virus infections in mice models (25, 26).

The possible impact of different infections on the biomedical parameters of in-vivo experiments, recommend exclusion of the infected mouse from the study. However, the developed infection may easily spread amongst the whole number of mice, and eventually terminate the entire project. Unless, practical guidelines are used for treating the infected mice and protecting the rest of animals from possible contaminations. The current study introduces a simple, rapid and reliable approach to easily detect the infected mice, eradicate the origin and prevent the spread of infection in nude mice in order to keep the safety of results and ensure cost-effectiveness of the study.

Supplementary information

Protocol for establishing human leukemia xenograft mouse models

Materials: Roswell Park Memorial Institute-1640 (RPMI1640), fetal bovine serum (FBS) and penicillin-streptomycin (Pen/Strep) were from Bioidea (Tehran, Iran). CCRF-CEM (T-ALL) human cell line was obtained from Pasteur Institute (Tehran, Iran).  Cells were cultured in RPMI1640 containing 10% heat-inactivated FBS and 1% Pen/Strep. 48 athymic nude mice (C57BL/6 Nude, 4 to 6-week-old female, 12.7 ± 0.17 gr (mean ± SEM) were purchased from Pasteur Institute (Amole, Iran). Mouse anti-human monoclonal antibodies against human CD3, CD4 and CD7 markers were purchased from Dako (Les Ulis, France).

Methods: After a 2-week delay for giving mice enough time for adaptation, 300 mg/kg cyclophosphamide was injected intraperitoneally. 72h later, mice undergo transplantation receiving 15 × 10⁶ CCRF-CEM cells in 100 μl FBS, subcutaneously.

Immunophenotyping assay: To confirm the xenograft transplantation, heart blood samples were collected from nude mice transplants. Immunophenotyping assays were performed using mouse anti-human monoclonal antibodies against human CD3, CD4 and CD7 markers. 1 × 10⁵ cells were subsequently applied to Partec CyFlow ML Flow Cytometer (Munster, Germany) and data were analyzed using FloMax® software.

Results

Twenty days post-transplantation, followed by the observation of few small nodules at the site of injected grafts, flow cytometry was performed on blood samples. Results demonstrated the presence of human lymphoblasts CCRF-CEM markers (71.50% CD3, 51.34% CD4 and 46.21% CD7) in mice blood samples (Figure 1S).

Fig. 1S- Confirmation of transplantation in nude mice models using flow cytometry. Transplantation confirmatory test was performed on mice heart-blood samples using flow cytometry. Identification of 71.50% CD3, 51.34% CD4 and 46.21% CD7 human markers confirmed transplantation.

Conflict of Interests

Authors declare no conflict of interests.

Ethical considerations

All investigations were conducted in accordance with the principles outlined in the Declaration of Helsinki and approved by the responsible Ethics Committee of the University of Isfahan (agreement number IR.UI.REC.1396.056 for animal studies).

Funding

This work was supported by PhD grants from the University of Isfahan (to M.A and S.N.) and by a research grant from the Medicinal Plants Research Center, Shahed University (grant 15/2517 to S.N)