Multi-drug-resistant klebsiella pneumoniae and application thereof in construction of pneumonia animal model

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to multi-drug-resistant klebsiella pneumoniae and application thereof in construction of a pneumonia animal model. Specifically, the multi-drug-resistant klebsiella pneumoniae BKP919 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.36301, and the multi-drug-resistant klebsiella pneumoniae BKP919 has wide drug resistance to various antibiotics such as amoxicillin, piperacillin and the like. The bacterial liquid of the strain is used for carrying out one-time intratracheal instillation on a mouse in a nasal instillation manner, and a stable severe pneumonia model can be established within 48-72 hours. The model preparation method is simple to operate and high in efficiency, and the model mouse has remarkable pneumonia symptoms, obvious pathological change and good related index stability, and can be used for pathogenesis research and targeted drug development of multi-drug-resistant klebsiella pneumonia infected pneumonia, so that the model has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a multidrug-resistant Klebsiella pneumoniae strain and its application in constructing animal models of pneumonia. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Klebsiella pneumoniae ( Klebsiella pneumoniae Klebsiella pneumoniae (KP) belongs to the Enterobacteriaceae family, along with common pathogens such as Salmonella and Escherichia coli. Since being classified as a pneumonia pathogen in 1882, KP remains one of the most common hospital-acquired infection pathogens worldwide. Infection with Klebsiella pneumoniae not only causes pneumonia symptoms such as lung abscess, high fever, and pleural effusion, but also leads to sepsis or bacteremia after entering the bloodstream. The World Health Organization lists drug-resistant Klebsiella pneumoniae (CRKp), which produces extended-spectrum β-lactamase (ESBL) and carbapenemase, as a major public health pathogen. Statistics show that 790,000 people die annually from Klebsiella pneumoniae infection; in Europe alone, the number of deaths annually from multidrug-resistant Klebsiella pneumoniae (CRKp) is significant. Multidrug-resistant Klebsiella pneumoniae The number of MDRKP (Multidrug-Resistant Klebsiella pneumoniae) infections in Malawi has exceeded 90,000, with over 7,000 deaths, accounting for 25% of all multidrug-resistant bacterial infections. According to a global burden of disease assessment, the incidence of MDRKP infection is on the rise. Currently, over 75% of Klebsiella pneumoniae causing bloodstream infections in Malawi are multidrug-resistant. Studying the epidemiology, transmission, prevention, and treatment of MDRKP-infected pneumonia, as well as the evolution, transmission, and pathogenic mechanisms of drug-resistant genes, is beneficial for long-term monitoring and control of this disease. Therefore, establishing mouse pneumonia models using MDRKP infection has become an indispensable tool in research experiments.

[0004] Currently, the main methods for preparing mouse pneumonia models using MDRKP infection include aerosol formation via bacterial spraying and intratracheal instillation. Aerosol preparation often requires a laryngoscope and a liquid lung nebulizer, which is difficult to operate (often requiring two people) and time-consuming. Intranasal instillation of bacterial solutions prepared using conventional MDRKP strains requires a large bacterial load, resulting in less pronounced pneumonia symptoms and pathological changes, lower morbidity and mortality in mice, and poor model reproducibility, making it unsuitable for further experimental research. Therefore, selecting a suitable MDRKP strain and using it to infect mice to prepare an effective and stable pneumonia model has become an urgent problem to be solved.

[0005] To address the shortcomings of the existing technology, this invention provides a multidrug-resistant Klebsiella pneumoniae strain and its application in constructing a mouse model of pneumonia. Specifically, the multidrug-resistant Klebsiella pneumoniae strain obtained by screening in this invention (…) Klebsiella pneumoniae BKP919 exhibits resistance to multiple antibiotics. Furthermore, experimental verification has shown that, compared to conventional MDRKP strains, mice infected with BKP exhibit typical pneumonia symptoms and pathological changes. The resulting mouse pneumonia model demonstrates better convenience, effectiveness, and stability, making it a suitable strain for preparing MDRKP-infected mouse pneumonia models. Based on these research findings, this invention was completed.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: The first aspect of the present invention provides a multidrug-resistant Klebsiella pneumoniae strain ( Klebsiella pneumoniae BKP919 is deposited at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China) on October 16, 2025, with accession number CGMCCNo. 36301.

[0007] A second aspect of the present invention provides the application of the multidrug-resistant Klebsiella pneumoniae BKP919 in the construction of an animal model of pneumonia.

[0008] The pneumonia is severe pneumonia. The animal may be a mouse.

[0009] A third aspect of the present invention provides a method for constructing a mouse model of severe pneumonia, the method comprising: The bacterial culture of the multidrug-resistant Klebsiella pneumoniae BKP919 was applied to mice to construct a mouse model of severe pneumonia.

[0010] A fourth aspect of the present invention provides the application of the above-described severe pneumonia mouse model in any one or more of the following: (b1) Epidemiological and transmission studies of pneumonia caused by multidrug-resistant Klebsiella pneumoniae; (b2) Screening or preparing drugs for pneumonia caused by multidrug-resistant Klebsiella pneumoniae; (b3) Study on the evolution, spread and pathogenic mechanism of drug resistance genes in multidrug-resistant Klebsiella pneumoniae.

[0011] The beneficial technical effects of the above technical solution are as follows: The multidrug-resistant Klebsiella pneumoniae BKP919 provided by the above technical solution was isolated from blood samples of clinical patients, providing a realistic clinical background. It exhibits broad resistance to multiple commonly used antibiotics, highly consistent with the drug resistance characteristics of clinical MDRKP, thus providing an ideal experimental strain for studying the infection mechanism of clinical MDRKP. Repeated experiments have verified that after infecting mice with the BKP919 strain, key indicators such as mortality, pneumonia symptoms, pathological changes, weight changes, organ weight changes, blood routine indicators, and serum inflammatory factor levels all showed good stability, meeting the model reproducibility requirements for subsequent experimental studies. Compared with conventional MDRKP strains, mice infected with the BKP919 strain exhibited more pronounced pneumonia symptoms, more obvious pathological damage, and higher bacterial load in viscera. Furthermore, the changes in inflammation-related indicators conformed to the pathophysiological characteristics of severe pneumonia, more realistically simulating the pathological process of clinical MDRKP-infected pneumonia, providing a reliable experimental model for studying the pathogenesis of this disease and screening targeted therapies. Meanwhile, traditional aerosol infection methods require complex equipment and are cumbersome to operate, while the above-mentioned technical solution uses nasal drops for one-time intratracheal instillation, which can be completed by a single person without the need for complex instruments, significantly saving manpower and time, while improving the efficiency and success rate of model preparation.

[0012] In summary, the above-mentioned technical solution provides a method for preparing multidrug-resistant Klebsiella pneumoniae BKP919 and a pneumonia model, which solves the problems of complex model preparation and poor stability in existing technologies. It provides an important experimental tool for related research on pneumonia caused by multidrug-resistant Klebsiella pneumoniae and has high scientific research value and practical application prospects. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 The colony morphology (A) and microscopic image (B) of the multidrug-resistant Klebsiella pneumoniae BKP919 of this invention are shown.

[0015] Figure 2 This is the phylogenetic tree of multidrug-resistant Klebsiella pneumoniae BKP919 for this invention.

[0016] Figure 3 Symptoms of pneumonia in mice infected with the multidrug-resistant Klebsiella pneumoniae BKP919 of this invention. (A) The mice are lethargic, have reduced spontaneous and stimuli-induced activity, and are curled up. (B) The eyes are moist and tearful, with yellowish discharge from the corners of the eyes.

[0017] Figure 4Changes in pneumonia markers in mice during a preliminary infection experiment with either conventional Klebsiella pneumoniae BKP901 or the multidrug-resistant Klebsiella pneumoniae BKP919 of this invention. (A) Survival curve, (B) Body weight change, (C) Visceral weight. * indicates significant difference between the two groups. P<0.05 ** indicates a significant difference between the two groups () P<0.01 *** indicates that the difference between the two groups is highly significant. P<0.001 (The same applies below).

[0018] Figure 5 The changes in pneumonia indicators in mice during formal experiments of infection with conventional Klebsiella pneumoniae BKP901 or the multidrug-resistant Klebsiella pneumoniae BKP919 of this invention are shown. (A) Survival curve, (B) Body weight change, (C) Visceral weight, (D) Visceral Klebsiella pneumoniae load.

[0019] Figure 6 The images show the pathological changes in lung tissue sections of mouse pneumonia models prepared by infection with either conventional Klebsiella pneumoniae BKP901 or the multidrug-resistant Klebsiella pneumoniae BKP919 of this invention. (A) Lungs of control mice, (B) Lungs of mice in the BKP901-infected group, (C) Lungs of mice in the BKP919-infected group.

[0020] Figure 7 The changes in serum inflammatory factor levels in a mouse pneumonia model prepared by infection with multidrug-resistant Klebsiella pneumoniae BKP919 according to this invention. (A) TNFα, (B) IL-1β, (C) IL-6, (D) IL-10. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] As mentioned earlier, the main methods for preparing MDRKP-infected mouse pneumonia models include aerosol formation via bacterial spraying and intratracheal instillation. Aerosol preparation often requires a laryngoscope and a liquid lung nebulizer, which is difficult to operate (often requiring two people) and time-consuming. In contrast, conventional MDRKP intranasal instillation requires a large bacterial load, resulting in less pronounced pneumonia symptoms and pathological changes, lower morbidity and mortality in mice, and poor model reproducibility, making it unsuitable for further experimental research.

[0024] In view of this, in a typical embodiment of the present invention, a multidrug-resistant Klebsiella pneumoniae strain is provided. Klebsiella pneumoniae BKP919, deposited at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), on October 16, 2025, with accession number CGMCC No. 36301. This invention reveals that this strain exhibits broad resistance to multiple antibiotics, including amoxicillin, piperacillin, cefoperazone, and cefuroxime, thus falling into the category of multidrug-resistant bacteria.

[0025] In another specific embodiment of the present invention, the application of the multidrug-resistant Klebsiella pneumoniae BKP919 in the construction of an animal model of pneumonia is provided.

[0026] In this invention, the animal is a non-human animal, and more specifically a non-human mammal, such as a rat, mouse, guinea pig, rabbit, dog, pig, orangutan, monkey, etc. In one specific embodiment of this invention, the non-human animal is a mouse.

[0027] The pneumonia in question is severe pneumonia.

[0028] In another specific embodiment of the present invention, a method for constructing a mouse model of severe pneumonia is provided, the method comprising: The bacterial culture of the multidrug-resistant Klebsiella pneumoniae BKP919 was applied to mice to construct a mouse model of severe pneumonia.

[0029] The McFarland turbidity of the bacterial solution is 1.20–1.50. The administration method can be nasal drops, intraperitoneal injection, intrapulmonary injection, or intratracheal injection, with nasal drops being preferred, which facilitates and quickly constructs an animal model. The administration dose is 20–100 μL / animal, preferably 50 μL / animal.

[0030] More specifically, in this invention, a mouse model of severe pneumonia is established by a single intratracheal instillation of 50 μL / mouse via nasal drops, and the model is established after 48 to 72 hours.

[0031] In this invention, the specific symptoms exhibited by the severely pneumoniad mice are any one or more of the following: (a1) Lethargy, reduced spontaneous or stimulating activities, curling up, and reduced food intake; (a2) The eyes are wet and tearing, with white to yellowish discharge from the corners of the eyes; (a3) Weight loss, increased weight of lung and spleen tissues; (a4) Obvious lung inflammation or structural damage can be seen in the pathological staining sections of the lungs; (a5) A decrease in the number of white blood cells, lymphocytes or platelets, and a decrease in hemoglobin content in a complete blood count; (a6) Increased levels of inflammatory factors in serum.

[0032] In (a6), the inflammatory factors include, but are not limited to, TNFα, IL-1β, IL-6 and IL-10.

[0033] In another specific embodiment of the present invention, the above-mentioned severe pneumonia mouse model is provided for use in any one or more of the following: (b1) Epidemiological and transmission studies of pneumonia caused by multidrug-resistant Klebsiella pneumoniae; (b2) Screening or preparing drugs for pneumonia caused by multidrug-resistant Klebsiella pneumoniae; (b3) Study on the evolution, spread and pathogenic mechanism of drug resistance genes in multidrug-resistant Klebsiella pneumoniae.

[0034] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Example 1: Isolation, purification and identification of multidrug-resistant Klebsiella pneumoniae BKP919 Prepare the solid culture medium according to the following formula: 2.5 g / L beef meal, 17.5 g / L acid-hydrolyzed casein, 1.5 g / L soluble starch, and 2.0 g / L agar. Adjust the pH to 7.4 ± 0.2. Autoclave at 121°C for 20 min, cool, pour into sterile petri dishes, and allow to solidify. In a biosafety cabinet, collect 100 μL of patient blood, spread it onto the culture medium, and incubate aerobically at 37°C for 24 h.

[0036] Colony morphology: Klebsiella pneumoniae BKP919 forms grayish-white, moist, raised, round colonies with neat edges. Figure 1 A).

[0037] Staining and microscopic examination: After staining with crystal violet, Klebsiella pneumoniae BKP919 was observed under a 400x microscope as single or chain-like short, thick rods. Figure 1 B).

[0038] Colonies were inoculated onto liquid culture medium (agar-free medium) and cultured at 37°C and 180 r / min for 24 h to obtain bacterial suspension. The suspension was then sent to Sangon Biotech for 16S rDNA sequencing. Based on Genebank alignment and homology analysis in NCBI, combined with colony morphology, post-staining microscopic examination, and phylogenetic tree analysis... Figure 2 BKP919 was identified as a strain of Klebsiella pneumoniae. Klebsiella pneumoniae ).

[0039] Table 1. Sensitivity of Klebsiella pneumoniae BKP919 to 27 antibiotics

[0040] Example 2: Detection of drug resistance in Klebsiella pneumoniae BKP919 using drug susceptibility testing Antimicrobial susceptibility testing was performed using the microbroth dilution method and the agar dilution method. Breakpoints for antimicrobial agents were primarily determined according to standards set by the Clinical and Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and the U.S. Food and Drug Administration (FDA). Results were expressed using the minimum inhibitory concentration (MIC) range, and bacterial susceptibility and resistance rates were calculated.

[0041] As shown in Table 1, the MIC values ​​of amoxicillin, piperacillin, cefoperazone, cefuroxime, cefoxitin, moxifloxacin, gentamicin, and fosfomycin against Klebsiella pneumoniae BKP919 all exceeded 128 μg / mL. Therefore, BKP919 exhibits multidrug resistance characteristics and is a multidrug-resistant Klebsiella pneumoniae strain.

[0042] Example 3: Preliminary experiment on the preparation of a mouse pneumonia model using conventional Klebsiella pneumoniae BKP901 or the multidrug-resistant Klebsiella pneumoniae BKP919 of this invention. BKP901 or BKP919 were inoculated into liquid culture medium and cultured aerobically at 37°C for 24 h. The McFarland turbidity of the bacterial culture was adjusted to 1.35 using PBS buffer, and the culture was stored at 4°C for later use. Six- to eight-week-old C57BL / 6J mice were selected and divided into two groups: a BKP901 infection group and a BKP919 infection group, with eight mice in each group. Mice were anesthetized by intraperitoneal injection of ready-to-use tribromoethanol (1.25% v / v) at a dose of 0.2 mL / 10 g. A single intratracheal instillation of 50 μL / mouse was also administered.

[0043] Observe the mice's condition 24 hours later: 48 hours after BKP919 infection, 3 mice died, and the rest all showed lethargy, reduced spontaneous activity and responsiveness to stimuli, and curling up. Figure 3 A) Eyes are wet and tearing, with yellowish discharge from the corners of the eyes. Figure 3 B); BKP901 infection in mice showed mild symptoms. 48 hours after infection, 2 mice died, and only 2 of the remaining mice showed reduced spontaneous activity and reduced activity when stimulated.

[0044] Mice were observed continuously for 7 days, with the number of deaths recorded daily to plot survival curves. Fasting body weight was recorded on day 0 (experiment start) and day 7, and statistical analysis was performed. After 7 days, the remaining surviving mice were fasted for 12 hours, and blood was collected from the retro-orbital plexus. Mice were euthanized by inhaling excessive amounts of ether in a fume hood. Serum was centrifuged (1500 rpm, 10 min, 4 ℃) and stored at -20 ℃. Lungs, livers, spleens, and kidneys were removed and weighed.

[0045] From the growth curve ( Figure 4 A) shows that the first two days after infection were the peak period for mortality, after which the mortality rate stabilized. The survival curve of mice in the BKP901 infection group was relatively flat, with a final survival rate of 75.00%, while the survival curve of mice in the BKP919 infection group showed a significant downward trend, with a final survival rate as low as 50.00%. From the data on mouse weight changes ( Figure 4 From B) it can be seen that, 7 days after infection, the body weight of mice in the BKP919 infection group was significantly lower than that in the BKP901 infection group. P<0.001 The weight loss reached 10.30%. In terms of internal organ weight ( Figure 4 C), the lung weight of mice in the BKP919 infection group was significantly increased compared to that in the BKP901 infection group. P<0.01 The spleen weight increased significantly. P<0.05 The differences in liver and kidney weight among the groups of mice were not significant. In conclusion, compared with conventional Klebsiella pneumoniae, BKP919 infection resulted in more significant changes in pneumonia symptoms, survival rate, body weight, and lung and spleen weight in mice. Therefore, it is more suitable as an infectious strain for preparing a mouse Klebsiella pneumoniae pneumonia model.

[0046] Example 4: Formal Experiment of Establishing a Mouse Model of Severe Pneumonia Using BKP901 and BKP919 Following the experimental method described in Example 3, 6-8 week old C57BL / 6J mice were selected and divided into three groups: a control group (CT group), a BKP901 infection group, and a BKP919 infection group, with 12 mice in each group. The mice were anesthetized, infected, and dissected. Furthermore, the lungs were immersed in 4% paraformaldehyde solution, embedded in paraffin, and cut into thin sections. Hematoxylin-eosin (HE) staining was used, and the pathological sections were photographed using a microscope.

[0047] From the growth curve ( Figure 5A) It can be seen that no mice died in the control group, while a higher mortality rate was observed within 3 days after infection with BKP901 or BKP919, followed by a more stable mortality rate after 3 days. The survival curve of mice in the BKP901 infection group showed a significant downward trend compared to the pre-experiment (Example 3), with a final survival rate of 58.33%, a significant decrease of 16.67% compared to the pre-experiment (75.00%). The survival curve of mice in the BKP919 infection group was basically consistent with the pre-experiment, with a final survival rate of 41.67%, close to the pre-experiment level (50.00%). From the data on mouse weight changes ( Figure 5 From B) it can be seen that, compared with the control group, the body weight of mice in both infection groups was significantly reduced ( P<0.001 ); 7 days after infection, the body weight of mice in the BKP919 infection group was significantly lower than that in the BKP901 infection group ( P<0.05 In terms of internal organ weight () Figure 5 C), the lung weight of mice in the BKP919 infection group was significantly increased compared with the control group and the BKP901 infection group. P<0.05 Compared to the control group, the spleen weight of mice in the BKP919 infection group was significantly increased. P<0.05 The BKP901 infection group showed little change; the liver and kidney weights of mice in each group did not differ significantly. The Klebsiella pneumoniae load (…) Figure 5 According to D), the Klebsiella pneumoniae viral load in the lungs of mice in the BKP919-infected group was significantly higher than that in the BKP919-infected group. P<0.05 The same trend was also observed in the contents of the cecum and colon.

[0048] From the lung tissue sections of mice, the lungs of control mice not infected with Klebsiella pneumoniae had more alveoli, which were fuller in shape, with thinner alveolar walls and fewer inflammatory cells. Figure 6 A); After mice were infected with BKP901 in their lungs, a small amount of inflammatory cell infiltration was observed around the alveoli and bronchi, and the alveolar walls were slightly thickened. Figure 6 B); In mice infected with BKP919, a large number of inflammatory cells infiltrated the alveoli, the alveolar walls thickened significantly, and the alveoli showed obvious damage. At the same time, a large number of inflammatory cells were recruited around the bronchi, and eosinophils and blood appeared inside. Figure 6 C). Therefore, based on lung pathological sections, mice infected with BKP919 showed more pronounced lung inflammation than those infected with BKP901.

[0049] In summary, in the formal experiment, compared with conventional Klebsiella pneumoniae, BKP919 infection of mice showed more significant changes in pneumonia symptoms, lung pathological changes, survival rate, body weight, and changes in lung and spleen weight, which were basically consistent with the preliminary experimental results. In addition, the viral load of Klebsiella pneumoniae in the viscera of mice infected with BKP919 was significantly higher than that of mice infected with conventional Klebsiella pneumoniae, and the resulting mouse pneumonia model was more significant and stable.

[0050] Example 5: Detection of inflammatory markers in a mouse model of severe pneumonia established using BKP919 To further verify the inflammatory changes in the mouse model of severe pneumonia prepared by BKP919, the changes in whole blood routine indicators and serum inflammatory factor levels in mice in the control group (CT group) and the BKP919-infected group were detected.

[0051] Table 2 Changes in routine blood parameters of mice

[0052] As can be seen from the test results in Table 2, the number of immune cells such as leukocytes and lymphocytes in the blood of mice was significantly reduced after BKP919 infection. P<0.05 Therefore, the immune cells of mice are severely damaged after infection; the decrease in hemoglobin, red blood cell hemoglobin concentration and red blood cell hemoglobin content indicates that the lung function of mice is abnormal after infection; at the same time, the number of platelets, the number of large platelets and the platelet-weighted mass decrease after infection, and the blood clotting speed in mice slows down, resulting in increased bleeding.

[0053] from Figure 7 It can be seen that the levels of pro-inflammatory factors TNFα, IL-1β, IL-6 and anti-inflammatory factor IL-10 in the serum of BKP919-infected mice were significantly increased, indicating that the mice showed obvious inflammatory manifestations.

[0054] Matters not covered in this invention are common knowledge.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multidrug-resistant strain of Klebsiella pneumoniae ( Klebsiella pneumoniae BKP919 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 16, 2025, with accession number CGMCC No. 36301.

2. The application of the multidrug-resistant Klebsiella pneumoniae BKP919 as described in claim 1 in the construction of an animal model of pneumonia.

3. The application as described in claim 2, characterized in that, The animal is a non-human animal, more specifically a non-human mammal, and further specifically, the non-human animal is a mouse.

4. The application as described in claim 2, characterized in that, The pneumonia in question is severe pneumonia.

5. A method for constructing a mouse model of severe pneumonia, characterized in that, The construction method includes: The bacterial culture of the multidrug-resistant Klebsiella pneumoniae BKP919 was applied to mice to construct a mouse model of severe pneumonia.

6. The construction method as described in claim 5, characterized in that, The McFarland turbidity of the bacterial solution is 1.20–1.

50.

7. The construction method as described in claim 5, characterized in that, The application method is nasal drops, and the application dose is 20-100 μL / animal, preferably 50 μL / animal; Furthermore, a mouse model of severe pneumonia was established by a single intratracheal instillation of 50 μL / mouse via nasal drops, and the result was achieved after 48 to 72 hours.

8. The construction method as described in claim 5, characterized in that, The mice with severe pneumonia exhibited any one or more of the following symptoms: (a1) Lethargy, reduced spontaneous or stimulating activities, curling up, and reduced food intake; (a2) The eyes are wet and tearing, with white to yellowish discharge from the corners of the eyes; (a3) Weight loss, increased weight of lung and spleen tissues; (a4) Obvious lung inflammation or structural damage can be seen in the pathological staining sections of the lungs; (a5) A decrease in the number of white blood cells, lymphocytes or platelets, and a decrease in hemoglobin content in a complete blood count; (a6) Increased levels of inflammatory factors in serum.

9. The application as described in claim 8, characterized in that, In (a6), the inflammatory factors include TNFα, IL-1β, IL-6 and IL-10.

10. The use of the severe pneumonia mouse model obtained by the construction method according to any one of claims 5-9 in any one or more of the following: (b1) Epidemiological and transmission studies of pneumonia caused by multidrug-resistant Klebsiella pneumoniae; (b2) Screening or preparing drugs for pneumonia caused by multidrug-resistant Klebsiella pneumoniae; (b3) Study on the evolution, spread and pathogenic mechanism of drug resistance genes in multidrug-resistant Klebsiella pneumoniae.

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