Acinetobacter baumannii and application thereof in construction of animal model with mastitis

Abstract

The invention discloses acinetobacter baumannii and application of the acinetobacter baumannii in construction of a mastitis animal model. The acinetobacter baumannii strain is acinetobacter baumannii AbST3475NMG202308, and the preservation number of the acinetobacter baumannii strain is CGMCC (China General Microbiological Culture Collection Center) No.34450 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms). Experiments prove that the acinetobacter baumannii has high pathogenicity and can be used for establishing a typical animal model caused by acinetobacter baumannii infection, the animal model can be specifically a mastitis animal model, and a certain technical support is provided for subsequent research and development of drugs or vaccines; the acinetobacter baumannii can also be used for screening antibacterial drugs and developing diagnostic reagents and vaccines. The method has an important application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Acinetobacter baumannii and its application in constructing an animal model of mastitis. Background Technology

[0002] Acinetobacter baumannii is an important opportunistic pathogen that is widely found in the environment and hospitals. It has a strong ability to adapt to the environment, resist drugs, and form biofilms, making it difficult to treat clinically.

[0003] Bovine mastitis is the most common mammary gland disease in dairy cows, caused by pathogenic microorganism infection or improper environmental management. In recent years, Acinetobacter baumannii has gradually become one of the important pathogens causing bovine mastitis, with the infection rate gradually increasing, seriously affecting the health of dairy cows and the quality of milk production. Mastitis milk refers to the milk produced by dairy cows after they have mastitis. Its physicochemical properties and nutritional composition have changed significantly, including decreased casein and lactose content, increased pH and conductivity, and the presence of pathogens (such as Streptococcus agalactiae, Staphylococcus aureus, and Mycoplasma) and antibiotic residues. When the somatic cell count in the milk exceeds 500,000 / mL, it indicates a deterioration in the health of the cow's udder, a decline in milk quality, and the possibility that residual antibiotics may cause allergies or bacterial resistance in humans. The treatment of mastitis in cows generally involves antibiotic therapy. Due to the high-dose and long-term use of various antibiotics, residual antibiotics are present in the milk. When people consume milk containing antibiotics, it can cause allergic reactions, such as rashes and discomfort, and in severe cases, anaphylactic shock. Many people develop antibiotic resistance in their bodies due to long-term consumption of mastitis milk containing antibiotic residues, making the treatment of the disease more difficult. Therefore, the prevention and control of mastitis in dairy cows is of great importance.

[0004] Currently, research on bovine mastitis caused by Acinetobacter baumannii faces the following challenges: First, there is a lack of standard strains. Existing studies mostly use human isolates, which do not fully match the clinical characteristics of bovine mammary gland infection and are difficult to accurately simulate the natural infection process. Second, clinical isolates are generally resistant to β-lactam, aminoglycoside, and quinolone antibiotics, and there is an urgent need to screen strains with stable resistance profiles for drug development. Third, the pathogenic mechanism is unclear. The adhesion, invasion, and immune escape mechanisms of Acinetobacter baumannii in mammary tissue are still unclear, which limits the development of vaccines and diagnostic reagents. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a strain of Acinetobacter baumannii that can cause severe inflammatory response in mouse mammary glands.

[0006] The technical problems to be solved are not limited to the technical topics described herein, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0007] This invention first protects a strain of Acinetobacter baumannii, named Acinetobacter baumannii (… Acinetobacter baumannii The strain AbST3475NMG202308 was deposited on May 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 34450. Acinetobacter baumannii (… Acinetobacter baumannii Acinetobacter baumannii ST3475NMG202308CGMCC No.34450 is abbreviated as Acinetobacter baumannii ST3475 or Acinetobacter baumannii ( Acinetobacter baumannii )AbST3475NMG202308 or Acinetobacter baumannii AbST3475NMG202308.

[0008] This invention also protects a culture. The culture may be a substance obtained by culturing Acinetobacter baumannii AbST3475NMG202308 in a culture medium.

[0009] In the above-mentioned cultures, the culture medium can be a solid culture medium or a liquid culture medium.

[0010] The culture medium may specifically be the TSA solid culture medium mentioned in the examples.

[0011] The term "culture" refers to the collective term for liquid or solid products (all substances within the culture container) that have been artificially inoculated and cultured to contain Acinetobacter baumannii. That is, it is the product obtained by growing and / or amplifying Acinetobacter baumannii, which can be a biologically pure culture of Acinetobacter baumannii or may contain a certain amount of culture medium, metabolites, and / or other components produced during the culture process. The term "culture" also includes passaged cultures obtained by subculturing Acinetobacter baumannii, which can be a single-generation culture or a mixture of several generations.

[0012] The application of Acinetobacter baumannii AbST3475NMG202308 or any of the above-mentioned cultures in the preparation of inflammatory animal models is also within the scope of protection of this invention.

[0013] In the above applications, the inflammatory animal model may be at least one of the following: mastitis animal model, pneumonia animal model, endocarditis animal model, and conjunctivitis animal model.

[0014] In the above applications, the inflammatory animal model can be an animal model prepared by infection with Acinetobacter baumannii AbST3475NMG202308.

[0015] In the above applications, the mastitis animal model can be established via mammary duct injection. The inoculation amount can be 10...6 -10 8 CFU / breast.

[0016] In any of the above-described applications, the animal may be a mouse.

[0017] The present invention also protects a method for constructing an animal model of mastitis, which may include the following steps: inoculating the mammary gland of a healthy lactating animal with any of the above-described Acinetobacter baumannii AbST3475NMG202308 through a mammary duct, and completing the construction of the animal model of mastitis when an acute inflammatory response appears clinically.

[0018] In the above method, the inoculum size of Acinetobacter baumannii AbST3475NMG202308 can be 10. 6 -10 8 CFU / breast.

[0019] In the above methods, the clinical occurrence of acute inflammatory reactions includes, but is not limited to, increased somatic cell count in breast milk, redness, swelling, heat and pain in the breast, and / or changes in the characteristics of breast milk.

[0020] In any of the methods described above, the animal may be a mouse.

[0021] Any of the above-described animal models may be resistant to streptomycin and / or penicillin G. Any of the above-described animal models may be sensitive to β-lactam antibiotics (specifically ceftriaxone sodium or amoxicillin), amica, fluoroquinolone antibiotics (such as enrofloxacin or ciprofloxacin), macrolide antibiotics (such as azithromycin or erythromycin), tetracycline antibiotics (such as tetracycline or oxytetracycline), and / or amide alcohol antibiotics (such as florfenicol).

[0022] The use of Acinetobacter baumannii AbST3475NMG202308 or any of the above-described cultures in the preparation of drugs for the prevention and / or treatment of Acinetobacter baumannii infection is also within the scope of protection of this invention.

[0023] In the above applications, the drug may be a vaccine, antibiotic, phage preparation, or immunomodulator. The vaccine may contain inactivated Acinetobacter baumannii AbST3475NMG202308. The inactivated Acinetobacter baumannii AbST3475NMG202308 can be obtained by inactivating Acinetobacter baumannii AbST3475NMG202308 using conventional methods (such as formaldehyde inactivation).

[0024] In the above applications, the drug, in addition to Acinetobacter baumannii AbST3475NMG202308 or any of the cultures described above, also includes an adjuvant.

[0025] "Adjuvant" herein may include aluminum hydroxide and aluminum phosphate, saponins such as QuilA, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsions may be particularly based on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oils such as squalane or squalene; oils resulting from the oligomerization of alkenes, particularly isobutene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / decanoate), glyceryl tri-(octanoate / decanoate), or propylene glycol dioleate; esters of branched-chain fatty acids or alcohols, particularly isostearates. Oils are used in combination with emulsifiers to form emulsions. Preferred emulsifiers are nonionic surfactants, especially sorbitan esters, dimannitol esters (such as anhydromannitol oleate, ethylene glycol esters, polyglycerol esters, propylene glycol esters, and oleate esters, isostearates, ricinoleic esters, or hydroxystearates (all of which may be ethoxylated), and polyoxypropylene-polyoxyethylene block copolymers.

[0026] The application of Acinetobacter baumannii AbST3475NMG202308 or any of the above-described cultures in the preparation of diagnostic reagents for Acinetobacter baumannii infection is also within the scope of protection of this invention.

[0027] In the above applications, the Acinetobacter baumannii infection diagnostic reagent may contain any of the aforementioned inactivated Acinetobacter baumannii AbST3475NMG202308. The Acinetobacter baumannii infection diagnostic reagent may use any of the aforementioned inactivated Acinetobacter baumannii AbST3475NMG202308 as the coating antigen, and diagnose Acinetobacter baumannii infection by detecting whether anti-Acinetobacter baumannii antibodies are present in animal serum via ELISA.

[0028] The application of Acinetobacter baumannii AbST3475NMG202308 or any of the above-described cultures in the preparation of Acinetobacter baumannii antibody detection reagents.

[0029] In the above applications, the Acinetobacter baumannii antibody detection reagent may contain any of the aforementioned inactivated Acinetobacter baumannii AbST3475NMG202308. The Acinetobacter baumannii antibody detection reagent may use any of the aforementioned inactivated Acinetobacter baumannii AbST3475NMG202308 as the coating antigen, and is used to detect whether animal serum contains antibodies against Acinetobacter baumannii via ELISA.

[0030] The use of Acinetobacter baumannii AbST3475NMG202308 or any of the above-described cultures in the preparation of drugs for screening or evaluating the treatment of mastitis in animals is also within the scope of protection of this invention.

[0031] This invention also protects a product containing any of the above-described Acinetobacter baumannii AbST3475NMG202308 or any of the above-described cultures; the product may have at least one of the following b1)-b4): b1) Screening or evaluating drugs for treating mastitis in animals; b2) Medications for the prevention and / or treatment of Acinetobacter baumannii infection; b3) Preparation of diagnostic reagents for Acinetobacter baumannii infection; b4) Prepare antibody detection reagents for Acinetobacter baumannii.

[0032] Experiments have shown that the Acinetobacter baumannii AbST3475NMG202308 isolated in this application is resistant to most antibiotics, with a minimum inhibitory concentration of 128 μg / mL against streptomycin. It carries 70 virulence genes, 18 resistance genes, and 4 prophages. Furthermore, Acinetobacter baumannii AbST3475NMG202308 exhibits strong adhesion and invasiveness to bovine mammary epithelial cells (bMECs). After infection of mouse mammary glands, severe edema and congestion of mouse mammary tissues occur, indicating that Acinetobacter baumannii AbST3475NMG202308 can be used to establish a mouse mastitis model. Therefore, the Acinetobacter baumannii AbST3475NMG202308 isolated in this invention exhibits high pathogenicity and can be used to establish typical animal models caused by Acinetobacter baumannii AbST3475NMG202308 infection. These animal models can serve as clinical lesion models, providing technical support for subsequent drug or vaccine development. Acinetobacter baumannii AbST3475NMG202308 can also be used to screen antibacterial drugs and develop diagnostic reagents and vaccines. This invention has significant application value.

[0033] Preservation Instructions Bacterial strain name: Acinetobacter baumannii Latin name: Acinetobacter baumannii Classification and nomenclature: Acinetobacter baumannii Acinetobacter baumannii Strain number: AbST3475NMG202308 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: May 7, 2025 CGMCC Registration Number: CGMCC No. 34450 Attached Figure Description Figure 1 The colony growth status, Gram staining, and scanning electron microscopy results of Acinetobacter baumannii ST3475 are shown.

[0034] Figure 2 Annotation analysis of virulence genes for Acinetobacter baumannii ST3475.

[0035] Figure 3 Annotation analysis of antibiotic resistance genes in Acinetobacter baumannii ST3475.

[0036] Figure 4 The distribution of prophages in the chr of Acinetobacter baumannii ST3475 is shown; where A represents the distribution of prophages in the chr of Acinetobacter baumannii ST3475, and B represents the annotation analysis of prophages.

[0037] Figure 5 To detect the adhesion and invasion rates of Acinetobacter baumannii ST3475 to bMECs.

[0038] Figure 6 The image shows the inflammatory response of mouse mammary tissue infected with Acinetobacter baumannii ST3475. The left image shows the control group, where the mammary tissue, blood vessels, and mammary tissue are all normal. The right image shows the mammary tissue of the infected mice, which is red, swollen, and congested with blood vessels.

[0039] Figure 7 The results of H&E staining (200×) of mammary tissue from mice infected with Acinetobacter baumannii ST3475 are shown. A is a pathological section of mammary tissue from the control group mice, where mammary epithelial cells are neatly arranged and the alveoli and mammary ducts are tightly connected. B is a pathological section of mammary tissue from the infected group mice, where mammary epithelial cells are detached from the alveoli, neutrophils are aggregated, the distance between alveoli and mammary ducts is widened, and lymphocytes and plasma cells are infiltrated within the spaces. Yellow arrows: neutrophils; red arrows: lymphocytes; blue arrows: plasma cells. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0043] The names of the reagents involved in the following examples and the companies from which they were purchased are shown in Table 1.

[0044] Table 1

[0045] The following examples used GraphPad Prism 8 statistical software to process the data. Experimental results are expressed as mean ± standard deviation; one-way ANOVA was used. P <0.05, ** P <0.01 and *** P <0.001 indicates a significant difference.

[0046] TSA solid culture medium: Weigh 40 g TSA, dissolve it in 700 mL of deionized water, sterilize at 121℃ for 30 minutes, and cool to room temperature (23 ± 2 ℃) to obtain the basic culture medium; then add sterile deionized water to make up to 1000 mL, pour into 90 mm petri dishes, 20 mL / dish, cool and solidify at room temperature, and then store at 2-8℃ to obtain TSA solid culture medium.

[0047] TSB liquid culture medium: Weigh 30 g TSB, dissolve in 700 mL deionized water, sterilize at 121℃ for 30 minutes, and after cooling to 56-60℃, add sterile deionized water to make up to 1000 mL; store at 2-8℃ to obtain TSB liquid culture medium.

[0048] Example 1, Acinetobacter baumannii ( Acinetobacter baumannii Isolation, identification and preservation of AbST3475NMG202308 CGMCCNo.34450 I. Separation 1. Sampling and Transportation Data collection time: August 10, 2023 Collection location: Guanmu Ranch, Inner Mongolia Autonomous Region Collection method: First, the type of mastitis in dairy cows was determined by combining clinical symptoms with the California Mastitis Test (CMT). Then, milk samples from cows with clinical mastitis and subclinical mastitis were aseptically collected in 50 mL sterile centrifuge tubes.

[0049] After collection, the samples were placed in a foam box with ice packs and transported to the dairy cow mastitis detection laboratory of the College of Veterinary Medicine, China Agricultural University, within 48 hours for storage.

[0050] 2. Separation Using an inoculation loop, take 0.1 mL of the sample collected in step 1 and streak it onto TSA solid medium. Then, incubate it in a 37 ℃ incubator for 24-48 h. The selected bacterial strain was named ST3475.

[0051] The colony morphology of bacteria ST3475 is shown in [the image / document]. Figure 1 Medium A: Circular, raised, smooth surface, neat edges, moist, with a diameter of 2-3 mm.

[0052] Gram staining was performed on bacteria ST3475, and the results are shown below. Figure 1 B (1000 ×): Gram-negative bacteria, spherical or coccobacillus-like.

[0053] The bacteria ST3475 were subjected to scanning electron microscopy, and the results are shown in the figure. Figure 1 C: Smooth, non-flagellated, uniformly sized, and interconnected spherical vesicles.

[0054] II. Identification Genomic DNA of bacteria ST3475 was extracted using a bacterial DNA extraction kit. The genomic DNA of Acinetobacter baumannii ST3475 was sent to Shanghai Paisenno Biotechnology Co., Ltd. for sequencing to obtain the genomic data of Acinetobacter baumannii ST3475.

[0055] The nucleotide sequence of the 16S rRNA of bacterial ST3475 is shown in SEQ ID No. 1.

[0056] The sequence shown in SEQ ID No. 1 was BLASTed in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to finally determine the genus and species of bacteria ST3475.

[0057] The results showed that bacteria ST3475 and Acinetobacter baumannii ( Acinetobacter baumannii The bacteria ST3475 showed the highest homology with Acinetobacter baumannii. Therefore, it was identified as Acinetobacter baumannii. Acinetobacter baumannii ).

[0058] III. Preservation The bacterium ST3475 isolated in step one was deposited on May 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 34450. The full name of bacterium ST3475 is Acinetobacter baumannii (…). Acinetobacter baumannii Acinetobacter baumannii ST3475 (AbST3475NMG202308 CGMCC No.34450) is also known as Acinetobacter baumannii ST3475.

[0059] Example 2: Multiple sequence typing (MLST) analysis of Acinetobacter baumannii ST3475 1. Obtaining the housekeeping gene sequence of Acinetobacter baumannii ST3475 Using the genomic DNA of Acinetobacter baumannii ST3475 as a template, the housekeeping gene sequences were amplified using the primers shown in Table 2, resulting in seven housekeeping gene sequences of Acinetobacter baumannii ST3475.

[0060]

[0061] 2. Comparison of housekeeping genes and sequence types Log in to the pubMLST database (https: / / pubmlst.org / ) and search for " Acinetobacter baumannii Click "Submit" → "Genomic MLST", and select the protocol: Acinetobacter baumannii (Oxford)" submitted the genomic data of Acinetobacter baumannii ST3475 (obtained in step two of Example 1), compared it with the sequences of the seven housekeeping genes of Acinetobacter baumannii ST3475, obtained the numbers of the seven housekeeping genes, and then determined the sequence type (ST) of Acinetobacter baumannii ST3475. If the housekeeping gene profile is not found in the database, a new ST type is applied for.

[0062] The test results are shown in Table 3. The results indicate that the combination of the seven housekeeping gene numbers of Acinetobacter baumannii ST3475 differs from known Acinetobacter baumannii strains. After verification by the MLST website administrator, a genotype ID was assigned to Acinetobacter baumannii ST3475. The housekeeping gene numbers of Acinetobacter baumannii ST3475 are shown in Table 3. This indicates that Acinetobacter baumannii ST3475 is a novel genotype isolate, and its sequence type is ST3475.

[0063]

[0064] Example 3: Gene function analysis of Acinetobacter baumannii ST3475 I. Virulence Gene Annotation The Virulence Factors of Pathogenic Bacteria (VFDB) database is specifically designed for studying the virulence factors of pathogenic bacteria, chlamydia, and mycoplasma. Based on the core dataset of the virulence factor database (https: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi), genes associated with virulence factors in the Acinetobacter baumannii ST3475 genome were identified through comparison.

[0065] The results show (see Table 4 and...) Figure 2 The chr sequence predicts that Acinetobacter baumannii ST3475 contains 15 types and 70 virulence genes, including Outer membrane protein, Type IV pili biosynthesis (Pseudomonas), and AdeFGH efflux pump / transport autoinducer.

[0066]

[0067] II. Annotation of Drug Resistance Genes The Comprehensive Antibiotic Resistance Database (CARD) contains high-quality molecular basis reference data on bacterial resistance (AMR), focusing on genes, proteins, and mutations. Resistance genes are identified by aligning the Acinetobacter baumannii ST3475 genome to CARD (https: / / card.mcmaster.ca / ). Alignments are selected based on the following criteria: Perfect and Strict hits only, Exclude nudge ≥ 95% identity, Loose hits to Strict, High quality / coverage, and E-value ≤ e-10.

[0068] The results show (see Table 5 and...) Figure 3 ), chr sequence prediction of Acinetobacter baumannii ST3475 contains OXA-344 , abeS , adeG 18 antibiotic resistance genes, etc.

[0069] Table 5. Antibiotic resistance genes of Acinetobacter baumannii ST3475

[0070] III. Prophage Prediction A prophage is a bacteriophage that integrates its nucleic acid into the host bacterial chromosome after a temperate bacteriophage infects the bacteria. This integrated state of the bacteriophage is an important way to reproduce and transmit the bacteriophage's own genetic information. Bacteria carrying the prophage genome are called lysogenic bacteria, which have the ability to resist repeated infection by the same or related bacteriophages. PHASTER (https: / / phaster.ca / ) was used to predict the presence of prophages in the genome of Acinetobacter baumannii ST3475.

[0071] The results show (see Table 6 and...) Figure 4 In the Acinetobacter baumannii ST3475 genome, the chr sequence predicted four prophages.

[0072]

[0073] Example 4: Drug resistance detection of Acinetobacter baumannii ST3475 I. Detection Methods 1. Resuscitation and Purification Take the frozen Acinetobacter baumannii ST3475 strain, streak it onto MH agar plates (Mueller-Hinton Agar), and incubate at 37°C for 18-24 hours. Pick a single colony and streak it again to ensure purity (observe the consistency of colony morphology).

[0074] 2. Preparation and concentration determination of bacterial suspension The purified Acinetobacter baumannii ST3475 from step 1 was inoculated into MH broth (Mueller-Hinton Broth) and incubated overnight at 37°C and 200 rpm; the bacterial suspension was then adjusted to 0.5 McFarland standard using a turbidimeter.

[0075] 3. Preparation of antibiotic gradients (1) Dissolve antibiotics in sterile water or DMSO according to CLSI guidelines and filter to remove bacteria.

[0076] (2) Add 100 μL of MH broth to each well of a 96-well plate; then add 200 μL of antibiotic solution (highest concentration) to the first well, and serially dilute to the 10th well; the 11th well is a positive control, and the 12th well is a negative control. Only MH broth is added to the 11th and 12th wells without antibiotics.

[0077] 4. Inoculation and Culture After completing step 3, add 100 μL of working bacterial solution (i.e., the bacterial suspension prepared in step 2) to each of the 11 wells from well 1 to well 11, with a final inoculation volume of 5 × 10⁻⁶. 5 (CFU / mL); Add 100 μL MH broth to the 12th well. Incubate at 37°C for 16-20 h (cover plate required to prevent evaporation).

[0078] 5. Judgment The lowest drug concentration that completely inhibits the growth of Acinetobacter baumannii ST3475 is the minimum inhibitory concentration (MIC) of Acinetobacter baumannii ST3475 against this antibiotic.

[0079] II. Test Results Susceptibility testing was performed on Acinetobacter baumannii ST3475 with 12 antibiotics, including β-lactam antibiotics: penicillin G, ceftriaxone sodium, amoxicillin; aminoglycoside antibiotics: streptomycin, amikacin; fluoroquinolones: enrofloxacin, ciprofloxacin; macrolides: azithromycin, erythromycin; tetracyclines: tetracycline, oxytetracycline; and amide alcohols: florfenicol.

[0080] The MICs of Acinetobacter baumannii ST3475 against 12 antibiotics are shown in Table 7. The results showed that Acinetobacter baumannii ST3475 was resistant to penicillin G and streptomycin, but sensitive to the other 10 antibiotics.

[0081]

[0082] Example 5: Detection of adhesion and invasion rates of Acinetobacter baumannii ST3475 I. Detection of Acinetobacter baumannii ST3475 adhering to bovine mammary epithelial cells 1. Cell Culture Bovine mammary epithelial cells (bMECs) were seeded into 24-well plates (approximately 10 wells). 5 Cells / well), then add DMEM medium containing 10% FBS, and culture at 37°C and 5% CO2 until monolayer confluence (approximately 80-90% density).

[0083] 2. Infection Adhesion group: After completing step 1, aspirate the cell culture medium and wash gently with PBS 1-2 times; then add Acinetobacter baumannii ST3475 bacterial suspension (MOI=10:1) and incubate at 37℃ for 1-2 h; wash gently with PBS buffer (pH 7.4, 10 mM PBS buffer) 3 times (to remove unadhesed bacteria).

[0084] Control group: After completing step 1, the cell culture medium was aspirated and the cells were gently washed 1-2 times with PBS; then, an equal volume of DMEM medium containing 10% FBS was added to the bacterial suspension of Acinetobacter baumannii ST3475 in the adhesion group, and the cells were incubated at 37°C for 1-2 h.

[0085] 3. Quantitative analysis After completing step 2, add 300 μL of lysis buffer (obtained by mixing 1 mL Triton X-100 and 99 mL PBS) to lyse the cells for 10 min. After serial dilution, count the bacteria using TSA solid medium (CFU / mL) to obtain the bacterial count; further calculate the adhesion rate. Adhesion rate (%) = Number of bacteria in the adhesion group / Number of bacteria in the control group × 100%.

[0086] II. Detection of Acinetobacter baumannii ST3475 invasion of bovine mammary epithelial cells 1. Cell Culture bMECs were deposited on a 24-well plate (approximately 10). 5 Cells / well), then add DMEM medium containing 10% FBS, and culture at 37°C and 5% CO2 until monolayer confluence (approximately 80-90% density).

[0087] 2. Infection Invasion group: After completing step 1, aspirate the cell culture medium and wash gently with PBS 1-2 times; then add Acinetobacter baumannii ST3475 bacterial suspension (MOI=10:1) and incubate at 37°C for 1 h or 2 h; wash gently with PBS buffer 3 times (to remove unattached bacteria).

[0088] Control group: After completing step 1, the cell culture medium was aspirated and the cells were gently washed 1-2 times with PBS; then, an equal volume of DMEM medium containing 10% FBS was added to the bacterial suspension of Acinetobacter baumannii ST3475 in the invasion group, and the cells were incubated at 37°C for 1 h or 2 h.

[0089] 3. Gentamicin treatment (1) After completing step 2, discard the liquid phase in the well and wash once with sterile PBS buffer, 700 μL / well.

[0090] (2) Add PBS buffer containing 400 μg / mL gentamicin, 500 μL / well, and incubate at 37°C and 5% CO2 for 3 h.

[0091] (3) Wash three times with sterile PBS buffer.

[0092] 4. Invasion rate After completing step 3, add lysis buffer to lyse the cells for 10 minutes, then serially dilute and count the bacteria using TSA solid medium (CFU / mL) to obtain the bacterial count; further calculate the invasion rate: Invasion rate (%) = (Number of bacteria in the invaded group / Number of bacteria in the control group) × 100% III. Test Results The adhesion rate of Acinetobacter baumannii ST3475 to bMECs cells is shown in the figure. Figure 5 In the middle A (0h is the control group, 1h is 1h of infection, and 2h is 2h of infection).

[0093] The results of the detection of the invasion rate of Acinetobacter baumannii ST3475 on bMECs cells are shown in [the table below]. Figure 5 B (0h is the control group, 1h is 1h of infection, and 2h is 2h of infection).

[0094] The results showed that the adhesion and invasion rates of Acinetobacter baumannii ST3475 to bMECs cells increased with the increase of infection time, and the invasion rate (4.1%) and adhesion rate (17.0%) at 2 h of infection were significantly higher than those at 1 h of infection.

[0095] The above results indicate that Acinetobacter baumannii ST3475 has strong adhesive and invasive properties.

[0096] Example 6: Damage test of Acinetobacter baumannii ST3475 on mouse mammary glands I. Experimental Methods 1. Preparation of bacterial strains (1) Take the frozen Acinetobacter baumannii ST3475 strain, streak it onto MH agar plates, and incubate at 37°C for 18-24 hours.

[0097] (2) After completing step (1), pick a single colony and inoculate it into 5 mL of MH broth. Incubate at 37°C and 200 rpm to obtain OD. 600nm The culture medium has a concentration of approximately 0.6.

[0098] (3) After completing step (2), take the cultured bacterial solution, centrifuge at 4000 rpm for 10 min, and collect the bacterial cells. Then wash the bacterial cells twice with PBS, and resuspend them in sterile PBS. Calibrate using McFarland's turbidimetric method or plate count method to obtain a concentration of 10. 7 Acinetobacter baumannii ST3475 bacterial suspension at CFU / mL.

[0099] 2. Animal preparation Lactating female BALB / c mice 48-72 hours postpartum with fully developed mammary glands.

[0100] The animal room temperature is 22±2℃, humidity is 50-6%, and the light cycle is 12h. Animals are allowed to acclimatize to the environment 3 days in advance, with free access to water and food.

[0101] 3. Breast infection (1) Anesthesia Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg).

[0102] (2) Injection into the mammary duct Select the fourth pair (groin) mammary glands and wipe the nipple and surrounding skin with a cotton ball soaked in 75% ethanol. Gently dilate the duct at the nipple opening using a 30G fine needle. Insert a microsyringe (Hamilton 50μL) perpendicularly into the duct 1-2 mm. Slowly inject 10μL of Acinetobacter baumannii ST3475 bacterial solution (i.e., 10... 6 CFU Acinetobacter baumannii ST3475 / mammary gland (as infection group) or saline (as control group). Gently press the nipple after injection to prevent reflux.

[0103] 4. Observation and infection assessment Within 24 hours of infection, observe for acute inflammatory reactions (such as redness and swelling, decreased lactation).

[0104] Mice were sacrificed 24 hours after infection, and mammary gland tissue was collected, fixed in 4% paraformaldehyde for 24 hours, and embedded in paraffin. H&E staining was then performed to observe inflammatory cell infiltration and acinar structure destruction.

[0105] II. Experimental Results Compared with the control group, 24 h after infection with Acinetobacter baumannii ST3475, mice showed significant pathological changes in mammary tissue, including severe congestion and edema (see...). Figure 6 CON was the control group, and ST3475 was the infection group.

[0106] Pathological changes in mouse mammary tissue were assessed using H&E staining. Results showed ( Figure 7 Compared with the control group, 24 hours after infection with Acinetobacter baumannii ST3475, mice showed mammary duct edema, increased spacing, macrophage proliferation, and a large number of neutrophil aggregations and epithelial cell shedding in the mammary alveoli. In contrast, no signs of inflammation were observed in the mammary tissue of the control group mice. This indicates that Acinetobacter baumannii ST3475 infection can effectively establish a mouse mastitis model.

[0107] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Acinetobacter baumannii ( Acinetobacter baumannii AbST3475NMG202308 has the accession number CGMCC No.34450 at the China General Microbiological Culture Collection Center.

2. A culture for incorporating the Acinetobacter baumannii (Acinetobacter baumannii) of claim 1. Acinetobacter baumannii The substance obtained by culturing AbST3475NMG202308 in a culture medium.

3. The Acinetobacter baumannii as described in claim 1 ( Acinetobacter baumannii The use of the culture described in claim 2 or AbST3475NMG202308 in the preparation of an inflammatory animal model.

4. The application according to claim 3, characterized in that: The inflammatory animal model is at least one of the following: mastitis animal model, pneumonia animal model, endocarditis animal model, and conjunctivitis animal model.

5. The application according to claim 4, characterized in that: The mastitis animal model was established via mammary duct injection, with an inoculation dose of 10... 6 -10 8 CFU / breast.

6. A method for constructing an animal model of mastitis, comprising the following steps: inoculating the mammary gland of a healthy lactating animal with the Acinetobacter baumannii of claim 1 via mammary ducts. Acinetobacter baumannii Once an acute inflammatory response appears in clinical practice, the animal model of mastitis will be established. (AbST3475NMG202308) 7. The construction method according to claim 6, characterized in that: Acinetobacter baumannii ( Acinetobacter baumannii The inoculation dose of AbST3475NMG202308 was 10. 6 -10 8 CFU / breast tissue; The clinical manifestations of acute inflammatory reactions include, but are not limited to, elevated somatic cell count in breast milk, redness, swelling, heat and pain in the breast, and / or changes in the characteristics of breast milk.

8. The Acinetobacter baumannii as described in claim 1 ( Acinetobacter baumannii The application of AbST3475NMG202308 or the culture of claim 2 is at least one of the following a1)-a4): a1) Prepare drugs for screening or evaluating treatments for mastitis in animals; a2) Prepare drugs for the prevention and / or treatment of Acinetobacter baumannii infection; a3) Preparation of diagnostic reagents for Acinetobacter baumannii infection; a4) Prepare antibody detection reagents for Acinetobacter baumannii.

9. The application according to claim 8, characterized in that: In a2), the drug is a vaccine, antibiotic, phage preparation, or immunomodulator; the vaccine contains inactivated Acinetobacter baumannii (…). Acinetobacter baumannii AbST3475NMG202308; In a3), the Acinetobacter baumannii infection diagnostic reagent contains inactivated Acinetobacter baumannii (… Acinetobacter baumannii AbST3475NMG202308; preferred, inactivated Acinetobacter baumannii ( Acinetobacter baumannii AbST3475NMG202308 was used as the coating antigen; In the a4), the Acinetobacter baumannii antibody detection reagent contains inactivated Acinetobacter baumannii (… Acinetobacter baumannii AbST3475NMG202308; preferred, inactivated Acinetobacter baumannii ( Acinetobacter baumannii AbST3475NMG202308 was used as the coating antigen.

10. A product containing the Acinetobacter baumannii as described in claim 1 ( Acinetobacter baumannii The culture described in claim 2 or AbST3475NMG202308; the product has the function of at least one of the following b1)-b4): b1) Screening or evaluating drugs for treating mastitis in animals; b2) Medications for the prevention and / or treatment of Acinetobacter baumannii infection; b3) Preparation of diagnostic reagents for Acinetobacter baumannii infection; b4) Prepare antibody detection reagents for Acinetobacter baumannii.

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