A method of constructing an animal model of chronic pulmonary infection / colonization with acinetobacter baumannii

Abstract

The application discloses a method for constructing an animal model of chronic pulmonary infection / persistence of Acinetobacter baumannii, and belongs to the technical field of infectious disease animal model construction and microbial preparation. Compared with the acute infection and rapid clearance mode formed by directly inoculating free bacteria, the live bacterial microspheres prepared in the application can prolong the local exposure and detectable time of Acinetobacter baumannii in the lung, so that the Acinetobacter baumannii is more likely to form a low-load, long-term persistence pulmonary infection state, thereby providing a more stable experimental platform for the mechanism of chronic infection formation, host immune dynamic change and related intervention evaluation.

Description

Technical Field

[0001] This invention belongs to the technical field of animal model construction for infectious diseases and preparation of microbial agents, specifically relating to a method for preparing Acinetobacter baumannii alginate live microspheres and their applications. More specifically, this invention relates to a method for constructing / holding animal models of Acinetobacter baumannii chronic lung infection. Background Technology

[0002] Acinetobacter baumannii is a major pathogen of hospital-acquired pneumonia and ventilator-associated pneumonia. Current animal models of pulmonary infection are mostly established using methods such as nasal instillation, tracheal instillation, or intratracheal delivery of free bacterial suspensions. While these models are relatively straightforward to operate, they typically have the following limitations when used to study chronic infections or long-term persistence:

[0003] First, free bacteria often induce a strong acute inflammatory response in a short period of time after inoculation, and the bacterial load in the lungs changes rapidly. In some cases, it may even cause a high mortality rate, which is not conducive to continuous observation of the dynamics of host immune response, tissue remodeling process and chronic inflammation maintenance mechanism over a longer time scale.

[0004] Secondly, some strains of Acinetobacter baumannii are easily and quickly cleared from the host, making it difficult to establish a stable, low-load, long-term detectable pulmonary retention state. This limits research on the mechanisms of chronic infection formation, the process of continuous colonization, and phenomena related to biofilm-like retention.

[0005] To prolong the exposure time of pathogens in the lungs and improve delivery methods, existing technologies have explored the use of polymeric materials to encapsulate or deliver bacteria. For example, Bayes et al. encapsulated the clinical strain of *Pseudomonas aeruginosa* NH57388A in agarose microspheres, establishing a mouse model of chronic lung infection and observing that the bacteria could persist in the lungs for up to 14 days. However, these techniques share the following common limitations:

[0006] Firstly, existing studies mostly optimize processes for pathogens such as Pseudomonas aeruginosa, and the process parameters (such as sodium alginate / agarose concentration, cross-linking strength, emulsification shearing conditions, etc.) are all set based on the cell characteristics of Pseudomonas aeruginosa, rather than for the specific purpose of optimizing the chronic lung infection / persistence model of Acinetobacter baumannii (such as the cell surface characteristics, alginate affinity, and in vivo survival adaptability of Acinetobacter baumannii).

[0007] Secondly, the preparation process parameters of microspheres in existing technologies often lack systematic optimization. In practical applications, problems such as unstable microsphere particle size distribution, large batch-to-batch variations, fluctuations in viable bacterial activity during preparation, improper cross-linking affecting release behavior, and non-specific inflammatory background caused by oil phase or surfactant residues may still exist. For example, the research by Christophersen et al. showed that differences in microsphere particle size directly affect their retention location in the lungs and the type of inflammatory response they trigger, suggesting that particle size control is crucial for model construction. However, a closed-loop quality control system for multi-parameter synergistic optimization of Acinetobacter baumannii characteristics has not yet been established in existing technologies.

[0008] The aforementioned issues will further affect the reproducibility, consistency, and comparability of animal models, and are particularly unfavorable for conducting comparative studies on the chronic persistence characteristics and related mechanisms between the same or different strains.

[0009] Therefore, there is an urgent need to provide a method for preparing alginate live bacterial microspheres optimized for the characteristics of Acinetobacter baumannii, which has the characteristics of clear process parameters, easy particle size control, convenient quality control, stable live bacterial load and low inflammatory background of the carrier, so as to provide a reliable technical means for constructing an animal model of chronic lung infection / persistence of Acinetobacter baumannii with low load and long-term persistence. Summary of the Invention

[0010] The purpose of this invention is to address the problems existing in existing animal models of Acinetobacter baumannii lung infection, such as prominent acute infection characteristics, unstable long-term retention, insufficient model reproducibility, and difficulty in controlling vector delivery interference, by providing a method for constructing an animal model of chronic lung infection / retention of Acinetobacter baumannii.

[0011] A further objective of this invention is to provide a live bacteria microsphere preparation scheme with clear process parameters, easy quality control, and good batch consistency, so that the obtained microspheres have good sphericity, relatively controllable particle size distribution, relatively stable live bacteria loading capacity, and low carrier background inflammatory interference.

[0012] This invention also aims to obtain live Acinetobacter baumannii microspheres suitable for lung delivery by controlling key factors such as microsphere size, cross-linking strength, and washing and purification conditions, thereby establishing a chronic lung infection / persistence animal model with low load and long-term retention characteristics, and providing a stable experimental platform for related pathogenesis research and intervention effect evaluation.

[0013] This invention addresses the challenges of Acinetobacter baumannii's tendency to persist in the host and the difficulty in establishing a stable chronic infection model through conventional low-dose free bacterial inoculation. It provides a method for constructing an animal model of chronic lung infection / persistence caused by Acinetobacter baumannii. The core of this invention lies not in simply employing alginate encapsulation technology, but in systematically optimizing aspects such as internal phase viscosity, emulsification shearing, cross-linking and solidification, washing and purification, and quality control, based on the bacterial survival characteristics of Acinetobacter baumannii, microsphere size control, cross-linking density, in vivo release behavior, and long-term persistence requirements after lung delivery. This results in a live bacterial microsphere formulation suitable for constructing a chronic lung infection / persistence model characterized by "low load—long-term persistence."

[0014] The technical solution provided by this invention is as follows:

[0015] This invention provides a method for constructing an animal model of chronic lung infection / persistence of Acinetobacter baumannii, the method comprising delivering live Acinetobacter baumannii microspheres intratracheally to the lungs of a non-human mammal, the live Acinetobacter baumannii microspheres being prepared by a method comprising the following steps:

[0016] (1) Preparation of internal phase: The logarithmic growth phase of Acinetobacter baumannii bacterial culture was mixed with a sodium alginate solution with a mass-volume concentration of 2.0%-2.5% to obtain the internal phase;

[0017] The above concentration range is used to balance internal phase viscosity, emulsification and droplet formation ability, microsphere structural stability after cross-linking, and uniformity of cell encapsulation. Among them, lower concentrations are beneficial for reducing system viscosity and forming smaller droplets, but may lead to insufficient mechanical strength after cross-linking; higher concentrations are beneficial for enhancing microsphere forming stability and delaying cell release, but may increase particle size dispersion and affect encapsulation uniformity.

[0018] (2) Preparation of oil phase: Prepare liquid paraffin oil phase containing nonionic surfactant;

[0019] (3) Emulsification to form W / O microdroplets: Under stirring conditions, the inner phase of step (1) is added dropwise to the oil phase of step (2) to form a W / O emulsion;

[0020] (4) Calcium ion crosslinking and curing: The W / O emulsion from step (3) is added to the CaCl2 crosslinked aqueous phase and stirred to cure, forming alginate Acinetobacter baumannii live microspheres;

[0021] (5) Collection and washing purification: Collect the microspheres obtained in step (4) and wash and purify them to remove the residual oil phase and surfactant, and obtain live Acinetobacter baumannii microspheres.

[0022] Preferably, the sodium alginate solution in step (1) has a mass-volume concentration of 2.2%;

[0023] The mixing volume ratio of the Acinetobacter baumannii bacterial suspension to the sodium alginate solution is 1:0.5-1.5, preferably 1:1; the OD of the Acinetobacter baumannii bacterial suspension... 600 The value is 0.2-0.3.

[0024] The preferred mixing method is gentle inverting or slow blowing to reduce the introduction of air bubbles and minimize adverse shear effects on cell and droplet formation.

[0025] Further, the nonionic surfactant mentioned in step (2) is Span-80, which has a volume fraction of 0.10%-0.20% in liquid paraffin, preferably 0.15% (v / v).

[0026] The above surfactant concentration is used to adjust the stability of the W / O emulsion interface, so that the Acinetobacter baumannii-sodium alginate internal phase can form more uniform droplets during emulsification, avoiding droplet fusion, widening of particle size distribution or uneven encapsulation due to interface instability.

[0027] Further, the emulsification stirring speed in step (3) is 600-1000 rpm, preferably 700 rpm;

[0028] The dropping rate of the internal phase is ≤1 mL / min; after the dropping is completed, continue stirring for 3-5 min to stabilize the droplets and converge the particle size distribution.

[0029] In this step, the emulsification shear strength and internal phase viscosity synergistically determine the droplet size and subsequent microsphere size distribution. By controlling the sodium alginate concentration, stirring speed, and dropping rate, live bacterial microspheres suitable for lung delivery and conducive to long-term local retention and sustained release of bacteria can be obtained.

[0030] Further, in step (4), the concentration of the CaCl2 crosslinking aqueous phase is 100-200 mM, preferably 120-150 mM, more preferably 120 mM; the crosslinking curing time is 10-20 min, preferably 10-15 min; and the stirring intensity is 200-400 rpm.

[0031] By adjusting the combination of CaCl2 concentration and cross-linking time, the resulting microspheres have sufficient structural stability while avoiding the formation of an excessively dense surface due to overly strong cross-linking, which would affect the in vivo release behavior and the continuous retention characteristics of the bacteria.

[0032] This invention reveals that for the preparation of Acinetobacter baumannii microspheres, a higher degree of cross-linking is not necessarily better. Insufficient cross-linking can cause microspheres to collapse, rupture, or be lost during washing; excessive cross-linking may lead to the formation of a dense surface layer, affecting cell release kinetics and in vivo retention patterns, which is not conducive to constructing targeted chronic infection / retention models.

[0033] Furthermore, the method for collecting microspheres in step (5) is centrifugation, using low to medium centrifugation intensity, with the preferred centrifugation conditions being 500-800 g for 3-5 min;

[0034] To reduce microsphere deformation or structural loosening caused by decalcification during washing, a low concentration of Ca can be used. 2+ PBS was used as the washing system, and the mixture was washed 2-3 times to remove residual oil phase, surfactants, and free calcium as much as possible. 2+ The Ca-containing 2+ The sterile PBS is PBS containing 2 mM CaCl2;

[0035] The washing solution used is sterile PBS; to reduce microsphere aggregation, 0.01%-0.05% Tween-20 can be added to the washing solution.

[0036] After washing, the degree of purification can be judged by observing the supernatant oil film after standing, observing the morphology and dispersion state of the microspheres under a microscope, or by weighing / residual oil detection. When there is no visible oil film in the supernatant and the microspheres are well dispersed, the washing can be considered sufficient.

[0037] This step aims to minimize the interference of residual oil phase and surfactants on subsequent animal experiments, reduce the increase in inflammatory background caused by non-specific stimuli, and thus improve the reliability of the model construction results.

[0038] Furthermore, the preparation method further includes a quality control step after step (5): the obtained microspheres are subjected to microscopic particle size determination, and when the average particle size deviates from the target range, reverse correction is performed by adjusting the emulsification speed, sodium alginate concentration or dropping speed to achieve batch-to-batch consistency control.

[0039] Specifically, 1) Each batch of samples is sampled and the particle size is measured under a microscope. The number of microspheres is no less than 200, and the average particle size and dispersion index are obtained.

[0040] 2) When the particle size is too large, it can be adjusted by increasing the emulsification speed, reducing the dropping speed, or appropriately reducing the sodium alginate concentration;

[0041] 3) When the particle size is too small, the microsphere stability is insufficient, or the bacterial activity is affected, adjustments can be made by reducing the emulsification speed, increasing the sodium alginate concentration, or optimizing the dropping method;

[0042] 4) Batch-to-batch consistency control is achieved through a closed-loop approach of "preparation parameters - particle size results - repeated measurements - reverse correction";

[0043] 5) Optionally, sterile blank microspheres can be used for small-scale in vivo delivery validation to evaluate whether the background inflammatory response and potential non-specific tissue damage of the carrier are within a low background range, thereby excluding non-specific inflammatory responses caused by residual oil, abnormal cross-linking or unstable preparation.

[0044] Furthermore, the preparation method further includes a degellation and release and load verification step after step (5): the obtained microspheres are placed in a degellation solution containing EDTA to dissociate the microspheres and release the embedded bacteria, and the viable bacterial load of the microspheres is evaluated by CFU counting.

[0045] Specifically, the obtained microspheres are placed in a degelatinizing solution containing EDTA and gently shaken at room temperature to dissociate the cross-linked network and release the encapsulated bacteria. The preferred EDTA concentration is 20-50 mM, the preferred pH is approximately 7.4, and the preferred treatment time is 5-10 min. The released bacteria can be used for CFU counting, microscopic observation, viable cell load verification, or other characterization analyses to evaluate the encapsulation efficiency and batch stability of the microspheres.

[0046] Preferably, the non-human mammal is a rodent; the rodent is a mouse or a rat.

[0047] Preferably, the Acinetobacter baumannii strains for modeling chronic lung infections in this invention should simultaneously meet the following conditions:

[0048] (1) It has sufficient colonization and adhesion capabilities to persist in lung tissue;

[0049] (2) The virulence level should not be too strong to avoid rapid death or acute fulminant lung injury in the host in the early stage of infection;

[0050] (3) It can form a persistent, low-level infection state in the host's lungs, rather than a short-term, high-intensity pathogenic process.

[0051] As an example, this invention uses Acinetobacter baumannii XH386 ( Acinetobacter baumannii XH386 is a publicly reported strain, with its source strain number NH57388A. Due to its relatively mild virulence and strong adhesion and persistent colonization capabilities, the Acinetobacter baumannii strain XH386 was able to successfully establish a long-lasting chronic lung infection model.

[0052] The present invention also provides the application of the Acinetobacter baumannii live microspheres prepared by the method in the preparation of formulations for constructing animal models of chronic lung infection / persistence of Acinetobacter baumannii.

[0053] Compared with conventional free bacterial inoculation methods or bacterial embedding methods lacking targeted process optimization, the present invention has at least the following beneficial effects:

[0054] 1. The particle size is relatively easy to control, making it easy to achieve stable preparation.

[0055] This invention achieves better sphericity and a relatively concentrated particle size distribution in the obtained microspheres by synergistically controlling the concentration of sodium alginate, emulsification speed, internal phase dropping rate, and calcium ion crosslinking conditions. It also facilitates quality control through microscopic measurement and parameter adjustment, thereby improving the stability of the preparation process and batch-to-batch consistency.

[0056] 2. It helps maintain the activity of live bacteria and obtain a more stable loading state.

[0057] This invention employs a gentle mixing method to prepare the internal phase and optimizes the shear strength and curing conditions during emulsification and crosslinking processes. This reduces the decrease in viable bacterial activity caused by mechanical shearing or improper crosslinking conditions during preparation, thereby helping to maintain good viable bacterial activity and a relatively stable microsphere loading state.

[0058] 3. The microspheres have good dispersibility, and the purified formulation has little interference with subsequent experiments.

[0059] This invention effectively reduces interference from residual oil phase, surfactants, and excess free calcium ions by combining centrifugal collection with multiple rounds of washing and purification. At the same time, low concentrations of anti-agglomeration components can be added as needed to improve microsphere dispersibility and reduce aggregation, thereby improving the operability and reproducibility of the formulation in animal delivery.

[0060] 4. The vector itself has a low inflammatory background.

[0061] The results of the sterile blank microsphere control show that the blank microspheres prepared under the process conditions of this invention, when delivered to the lungs, only caused a mild and transient local inflammatory response, with a low overall degree of pathological damage, and were close to the PBS control. This indicates that the carrier itself does not significantly increase the basal inflammation level of the animal model, which is beneficial to improving the interpretability of subsequent infection model evaluation.

[0062] 5. It is more conducive to building a low-burden, long-term chronic lung infection / persistence model.

[0063] Compared with the rapid clearance mode after acute infection formed by direct inoculation of free bacteria, the live bacterial microspheres prepared in this invention can prolong the local exposure and detectability time of Acinetobacter baumannii in the lungs, making it easier to form a low-load, long-term persistent lung infection state, thereby providing a more stable experimental platform for the formation mechanism of chronic infection, dynamic changes in host immunity and related intervention evaluation. Attached Figure Description

[0064] Figure 1A schematic diagram of the process for constructing an animal model of chronic lung infection / holding Acinetobacter baumannii (inner phase-emulsification-crosslinking-washing-delivery).

[0065] Figure 2 The image below shows the particle size distribution under a microscope; the top image shows blank microspheres, and the bottom image shows live bacterial microspheres at 400x magnification.

[0066] Figure 3 The bacterial load in the lungs of acute and chronic models.

[0067] Figure 4 For lung pathology scoring.

[0068] Figure 5 The results of immunofluorescence combined with FISH staining in lung tissue of a mouse model of chronic lung infection on days 11, 13, and 15 are shown; the scale bar of each left image is 50 μm, and the scale bar of each right image is 10 μm.

[0069] Figure 6 The image below shows the microscopic distribution of the microspheres prepared in Example 2.

[0070] Figure 7 The image below shows the microscopic distribution of the microspheres prepared in Example 3.

[0071] Figure 8 The image below shows the microscopic distribution of the microspheres prepared for Comparative Example 2.

[0072] Figure 9 The pathological scores of mice after the microspheres prepared for Comparative Example 2 were delivered to the lungs were evaluated over time.

[0073] Figure 10 The image below shows the microscopic distribution of the microspheres prepared for Comparative Example 3.

[0074] Figure 11 The pathological scores of the microspheres prepared for Comparative Example 3 after delivery to the lungs of mice over time.

[0075] Figure 12 The image below shows the microscopic size distribution of the microspheres prepared for Comparative Example 4.

[0076] Figure 13 The pathological scores of the microspheres prepared for Comparative Example 4 after delivery to the lungs of mice over time.

[0077] Figure 14 The lung inflammatory changes are represented by sterile blank microspheres.

[0078] Figure 15 Bright field image of LS01 live bacterial microspheres.

[0079] Figure 16 The changes in body weight of XH386 and LS01 live bacteria with the same bacterial count.

[0080] Figure 17 The body temperature changes were measured for the same bacterial count of XH386 and LS01 live bacteria.

[0081] Figure 18 The behavior scores for XH386 and LS01 live bacteria with the same bacterial count were given. Detailed Implementation

[0082] The following embodiments are used to illustrate the technical solutions of the present invention, but do not limit the scope of protection of the present invention. Unless otherwise specified, specific conditions in the embodiments are performed according to conventional conditions in the art. Unless otherwise stated, the raw materials, reagents, and instruments used are all commercially available conventional products in the art.

[0083] Example 1: Preparation of Acinetobacter baumannii XH386 live bacterial microspheres using 2.2% sodium alginate, emulsification at 700 rpm, and cross-linking with 120 mM CaCl2.

[0084] This embodiment illustrates the preparation method of Acinetobacter baumannii live microspheres under the preferred conditions of the present invention, and the effects of the obtained microspheres on microscopic morphology, long-term lung retention, and maintenance of chronic inflammation.

[0085] (1) Preparation of sodium alginate solution

[0086] Weigh 0.11 g of sodium alginate, add sterile water to a final volume of 5.0 mL, and stir magnetically overnight at 4°C until completely dissolved to obtain a sodium alginate solution with a mass-volume concentration of 2.2% (w / v). This solution is then sterilized using a 0.22 µm filter membrane before use.

[0087] (2) Internal phase configuration

[0088] Acinetobacter baumannii XH386 in the logarithmic growth phase was taken. Acinetobacter baumannii XH386, a publicly reported strain (source strain number NH57388A), 5.0 mL of bacterial suspension, OD 600 The concentration was 0.25, and it was mixed with 5.0 mL of the above 2.2% sodium alginate solution. The mixture was then gently inverted 10 times to obtain an internal phase with a total volume of 10.0 mL.

[0089] (3) Oil phase configuration

[0090] Measure 100 mL of liquid paraffin, add 150 µL of Span-80, mix thoroughly to obtain an oil phase containing 0.15% (v / v) Span-80.

[0091] (4) Emulsification

[0092] The oil phase was placed in a beaker and stirred at 700 rpm on a magnetic stirrer. Using a 23G syringe, 10.0 mL of the internal phase was slowly added dropwise to the oil phase at a rate of approximately 1.0 mL / min. After the addition was complete, stirring was continued for 4 min to form a W / O emulsion.

[0093] (5) Crosslinking curing

[0094] Weigh 1.76 g of CaCl2•2H2O and add sterile water to a final volume of 100 mL to obtain a 120 mM CaCl2 crosslinking solution. Place this crosslinking solution in a beaker and stir at 300 rpm. Then, slowly add the above emulsion along the beaker wall to the crosslinking solution over a period of 90 s, and continue stirring for 15 min to complete the crosslinking and solidification, forming live Acinetobacter baumannii microspheres.

[0095] (6) Collection and washing

[0096] The cross-linked microsphere suspension was aliquoted into sterile centrifuge tubes and centrifuged at 600 g for 4 min to collect the microspheres. After discarding the supernatant, the microspheres were gently resuspended and washed three times with sterile PBS containing 2 mM CaCl2. Finally, the microspheres were resuspended in sterile PBS to obtain a uniformly dispersed suspension of viable bacteria.

[0097] (7) Microscopic morphology and particle size quality control

[0098] 80 µL of the microsphere suspension was dropped onto a glass slide, covered with a coverslip, and observed under a microscope. Images were acquired at 400x magnification. The particle size of 200 microspheres was statistically analyzed. The average particle size of the obtained microspheres was within the preferred target particle size range (approximately 100-200 µm), with relatively regular morphology, intact boundaries, and good dispersibility.

[0099] like Figure 1 As shown, this embodiment establishes a clear preparation process, namely, "internal phase configuration—oil phase emulsification—Ca..." 2+ Crosslinking—washing purification—delivery application.

[0100] like Figure 2 As shown in the figure below, the live bacterial microspheres obtained in this embodiment appear as spherical or near-spherical particles with clear outlines and complete structures under a 400x microscope, indicating that the preferred parameters of this invention can stably encapsulate Acinetobacter baumannii without significant collapse or breakage.

[0101] (8) Animal delivery and lung retention testing

[0102] The above-mentioned live bacterial microspheres were delivered into the lungs of mice via the airway, and lung tissue was collected at predetermined time points for CFU assay and pathological observation. Results are as follows: Figure 3 As shown, after using the live bacterial microspheres prepared in this embodiment, the bacterial load in the lungs remained at approximately 10 after day 5.3 The CFU / lung level was observed and remained detectable until day 15-18. Compared to the acute infection group with free bacteria, bacterial clearance was significantly delayed, exhibiting a long-term lung retention characteristic.

[0103] (9) Pathological evaluation of the lungs

[0104] Lung tissue was stained with hematoxylin and eosin (HE) at the same time points and semi-quantitative pathological scoring was performed. Results are as follows: Figure 4 As shown, the live bacterial microsphere group in this embodiment maintained a moderate level of fluctuation in lung pathology scores over a long observation period, suggesting that the model does not cause acute fulminant injury, but rather maintains a persistent and controllable local inflammatory response over a long period of time.

[0105] (10) Analysis of bacterial colonization morphology in the lungs and their interaction with immune cells

[0106] To further clarify the distribution characteristics of bacteria in the lungs and their interaction with host cells, lung tissue was harvested on days 11, 13, and 15 post-infection for immunofluorescence combined with FISH staining. The results are as follows: Figure 5 The results showed that Acinetobacter baumannii positive signals (in red) were detected in lung tissue at all time points, indicating that the bacteria persisted in the later stages of infection. Morphological observation showed that most bacterial signals were isolated and scattered, with only a few bacterial signals (in green) associated with CD68. + Macrophages are closely associated, showing a distribution that surrounds the lesion; meanwhile, Ly6G is visible around the lesion. + Neutrophil infiltration (yellowish). These results indicate that persistent bacteria remain in the lung tissue during the chronic infection phase, most of which do not show clear phagocytic encapsulation, with only a small portion exhibiting localized association with macrophages.

[0107] (10) Conclusion

[0108] This embodiment demonstrates that Acinetobacter baumannii XH386 live bacterial microspheres prepared using 2.2% sodium alginate, emulsification at 700 rpm, and cross-linking with 120 mM CaCl2 exhibit good sphericity, morphological integrity, and long-term retention capacity in the lungs, and can be used to construct a "low-load-long-term retention" chronic lung infection / retention model.

[0109] Example 2: Preparation of Acinetobacter baumannii XH386 live bacterial microspheres using 2.0% sodium alginate

[0110] This embodiment illustrates that, under the condition of sodium alginate concentration of 2.0% (w / v), the emulsification dispersion-calcium ion crosslinking solidification-washing purification process described in this invention can be used to prepare live Acinetobacter baumannii microspheres, thereby supporting the implementation of the lower limit of the sodium alginate concentration range described in this invention.

[0111] (1) Preparation of sodium alginate solution

[0112] Weigh 0.10 g of sodium alginate, add sterile water to a final volume of 5.0 mL, and stir magnetically overnight at 4°C to ensure complete dissolution, yielding a sodium alginate solution with a mass-volume concentration of 2.0% (w / v). The resulting solution is then sterilized using a 0.22 µm filter membrane before use.

[0113] (2) Internal phase configuration

[0114] Take 5.0 mL of Acinetobacter baumannii XH386 bacterial suspension in the logarithmic growth phase, and measure the OD of the bacterial suspension. 600 =0.25, and mixed with 5.0 mL of the above 2.0% sodium alginate solution, and mixed 10 times by gentle inversion to obtain an internal phase with a total volume of 10.0 mL.

[0115] (3) Oil phase configuration

[0116] Measure 100 mL of liquid paraffin, add 150 µL of Span-80, mix thoroughly to obtain an oil phase containing 0.15% (v / v) Span-80.

[0117] (4) Emulsification

[0118] The oil phase was placed in a beaker and stirred at 700 rpm using a magnetic stirrer. Using a 23G syringe, 10.0 mL of the inner phase was slowly added dropwise to the oil phase at a rate of 1.0 mL / min. After the addition was complete, stirring was continued for 4 min to form a W / O emulsion.

[0119] (5) Crosslinking curing

[0120] Weigh 1.76 g of CaCl2•2H2O and add sterile water to a final volume of 100 mL to obtain a 120 mM CaCl2 crosslinking solution. Place the crosslinking solution in a beaker and stir at 300 rpm. Then, slowly add the emulsion along the beaker wall to the crosslinking solution over a period of 90 s, and continue stirring for 15 min to complete the crosslinking process and form live microspheres.

[0121] (6) Collection and washing

[0122] The cross-linked microsphere suspension was aliquoted into sterile centrifuge tubes and collected by centrifugation at 600 g for 4 min. After discarding the supernatant, the microspheres were gently resuspended and washed three times with sterile PBS containing 2 mM CaCl2. Finally, the microspheres were resuspended in sterile PBS to obtain a live Acinetobacter baumannii XH386 microsphere suspension.

[0123] (7) Microscopic morphology and particle size observation

[0124] 80 µL of the microsphere suspension was dropped onto a glass slide, covered with a coverslip, and observed under a microscope. Multiple images were acquired at 400x magnification, and the diameter of 200 microspheres was measured. The results are as follows: Figure 6 The results show that the microspheres obtained in this embodiment can form relatively complete spherical or near-spherical particles with clear boundaries, which can meet the needs of subsequent delivery and observation of live bacteria microspheres.

[0125] (8) Conclusion

[0126] This embodiment demonstrates that Acinetobacter baumannii live microspheres can be successfully prepared using the process described in this invention under conditions of 2.0% (w / v) sodium alginate, indicating that the lower limit of the sodium alginate concentration range described in this invention is feasible.

[0127] Example 3: Preparation of Acinetobacter baumannii XH386 live bacterial microspheres using 2.5% sodium alginate

[0128] This embodiment illustrates that, under the condition of sodium alginate concentration of 2.5% (w / v), the emulsification dispersion-calcium ion crosslinking solidification-washing purification process described in this invention can be used to prepare live Acinetobacter baumannii microspheres, thereby supporting the implementation of the upper limit of the sodium alginate concentration range described in this invention.

[0129] (1) Preparation of sodium alginate solution

[0130] Weigh 0.125 g of sodium alginate, add sterile water to a final volume of 5.0 mL, and stir magnetically overnight at 4°C to ensure complete dissolution, yielding a sodium alginate solution with a mass-volume concentration of 2.5% (w / v). The resulting solution is then sterilized using a 0.22 µm filter membrane before use.

[0131] (2) Internal phase configuration

[0132] Take 5.0 mL of Acinetobacter baumannii XH386 bacterial suspension in the logarithmic growth phase, and measure the OD of the bacterial suspension. 600 =0.25, and mixed with 5.0 mL of the above 2.5% sodium alginate solution, and mixed 10 times by gentle inversion to obtain an internal phase with a total volume of 10.0 mL.

[0133] (3) Oil phase configuration

[0134] Measure 100 mL of liquid paraffin, add 150 µL of Span-80, mix thoroughly to obtain an oil phase containing 0.15% (v / v) Span-80.

[0135] (4) Emulsification

[0136] The oil phase was placed in a beaker and stirred at 700 rpm using a magnetic stirrer. Using a 23G syringe, 10.0 mL of the inner phase was slowly added dropwise to the oil phase at a rate of 1.0 mL / min. After the addition was complete, stirring was continued for 4 min to form a W / O emulsion.

[0137] (5) Crosslinking curing

[0138] Weigh 1.76 g of CaCl2•2H2O and add sterile water to a final volume of 100 mL to obtain a 120 mM CaCl2 crosslinking solution. Place the crosslinking solution in a beaker and stir at 300 rpm. Then, slowly add the emulsion along the beaker wall to the crosslinking solution over a period of 90 s, and continue stirring for 15 min to complete the crosslinking process and form live microspheres.

[0139] (6) Collection and washing

[0140] The cross-linked microsphere suspension was aliquoted into sterile centrifuge tubes and collected by centrifugation at 600 g for 4 min. After discarding the supernatant, the microspheres were gently resuspended and washed three times with sterile PBS containing 2 mM CaCl2. Finally, the microspheres were resuspended in sterile PBS to obtain a live Acinetobacter baumannii XH386 microsphere suspension.

[0141] (7) Microscopic morphology and particle size observation

[0142] 80 µL of the microsphere suspension was dropped onto a glass slide, covered with a coverslip, and observed under a microscope. Multiple images were acquired at 400x magnification, and the diameter of 200 microspheres was measured. The results are as follows: Figure 7 As shown, the microspheres obtained in this embodiment can also form relatively complete spherical or near-spherical particles with clear boundaries, possessing basic morphological characteristics that can be used for subsequent delivery and observation.

[0143] (8) Conclusion

[0144] This embodiment demonstrates that Acinetobacter baumannii live microspheres can also be successfully prepared using the process described in this invention under conditions of 2.5% (w / v) sodium alginate, indicating that the upper limit of the sodium alginate concentration range described in this invention is also feasible.

[0145] Supplementary notes related to Example 1

[0146] Examples 1, 2, and 3 demonstrate that, within a sodium alginate concentration range of 2.0%-2.5% (w / v), the emulsification dispersion, calcium ion crosslinking solidification, and washing purification processes described in this invention can all prepare live Acinetobacter baumannii microspheres. 2.2% (w / v) is a preferred embodiment of this invention.

[0147] Those skilled in the art will understand that the technical effect of the present invention does not simply come from the minute changes in sodium alginate concentration within the aforementioned small range, but rather from the synergistic optimization of multiple parameters such as sodium alginate concentration, emulsification shear conditions, crosslinking strength, washing and purification methods, and quality control strategies.

[0148] Comparative Example 1: Acute pulmonary infection model of free Acinetobacter baumannii XH386

[0149] This comparative example illustrates that without the alginate-encapsulated microspheres of the present invention, Acinetobacter baumannii is less likely to establish long-term lung retention and is more likely to establish a model of rapid clearance after acute infection.

[0150] (1) Preparation of bacterial culture

[0151] Take a bacterial suspension of Acinetobacter baumannii XH386 in the logarithmic growth phase and adjust it to an inoculation level equivalent to the total amount of live bacteria recovered during the delivery of the live microspheres obtained in Example 1.

[0152] (2) Animal delivery

[0153] The above-mentioned free bacterial solution was delivered into the lungs of mice via the airway without alginate encapsulation.

[0154] (3) Detection of bacterial load in the lungs

[0155] Lung tissue homogenates were collected on days 1, 2, 3, 4, 5, 6, 7, and 8 post-infection for CFU testing. Results were as follows... Figure 3 As shown, the pulmonary bacterial load in the acute infection group decreased rapidly after infection, dropping to extremely low levels or near clearance by day 5-6, but failed to maintain the long-term detectable state as shown in Example 1.

[0156] (4) Pathological evaluation of the lungs

[0157] like Figure 4 As shown, the pathological scores of the acute infection group with free bacteria were high in the early stage of infection, and then gradually decreased as the bacteria were cleared, suggesting that it caused strong but short-lived acute inflammatory damage, rather than a persistent chronic inflammatory state.

[0158] (5) Conclusion

[0159] This comparative example shows that without the alginate live bacteria microsphere encapsulation method described in this invention, Acinetobacter baumannii XH386 is difficult to establish long-term lung retention, and only exhibits the bacterial dynamics characteristics of rapid decline after acute infection, which cannot achieve the effect of the chronic lung infection / retention model of this invention.

[0160] Comparative Example 2: Live Acinetobacter baumannii microspheres prepared under low cross-linking strength conditions

[0161] This comparative example illustrates that when the crosslinking strength is lower than the preferred conditions of this invention, the structural stability of the obtained microspheres and the performance of subsequent models will be affected.

[0162] (1) Preparation of sodium alginate solution

[0163] Weigh 0.11 g of sodium alginate, add sterile water to make up to 5.0 mL, and stir magnetically overnight at 4℃ to obtain a sodium alginate solution with a mass-volume concentration of 2.2% (w / v). After sterilization by filtering through a 0.22 µm filter membrane, it is ready for use.

[0164] (2) Internal phase configuration

[0165] Take 5.0 mL of Acinetobacter baumannii XH386 bacterial suspension in the logarithmic growth phase, and measure the OD of the bacterial suspension. 600 =0.25, and mixed with 5.0 mL of the above 2.2% sodium alginate solution, gently inverted and mixed 10 times to obtain an internal phase with a total volume of 10.0 mL.

[0166] (3) Oil phase configuration

[0167] Measure 100 mL of liquid paraffin, add 150 µL of Span-80, and mix well to obtain the oil phase.

[0168] (4) Emulsification

[0169] Place the oil phase in a beaker and stir at 700 rpm. Using a 23G syringe, add the internal phase dropwise to the oil phase at a rate of 1.0 mL / min. After the addition is complete, continue stirring for 4 min to form a W / O emulsion.

[0170] (5) Low crosslinking curing

[0171] Weigh 1.18 g of CaCl2•2H2O and add sterile water to a final volume of 100 mL to obtain an 80 mM CaCl2 crosslinking solution. Place the crosslinking solution in a beaker and stir at 300 rpm. Then, slowly add the emulsion along the beaker wall to the crosslinking solution over a period of 90 s, and continue stirring for 8 min to complete the crosslinking process.

[0172] (6) Collection and washing

[0173] After aliquoting the obtained microsphere system, the microspheres were collected by centrifugation at 600 g for 4 min. The supernatant was discarded, and the microspheres were washed three times with sterile PBS containing 2 mM CaCl2, and finally resuspended in sterile PBS.

[0174] (7) Microscopic observation

[0175] Prepare a slide using 80 µL of the obtained microsphere suspension and observe it under a microscope. The results are as follows: Figure 8The results showed that some particle boundaries were not very clear, and some particles were slightly deformed or stuck together after washing, suggesting that the low cross-linking strength would have a certain impact on the stability of the microsphere structure.

[0176] (8) Observation of animal delivery effect

[0177] The obtained microspheres were delivered into the lungs of mice via the airway, and the bacterial load in the lungs was detected and pathological changes were observed at subsequent time points. Results are as follows: Figure 9 The results showed that although this group could be delivered intrapulmonaryly, its long-term retention and model consistency were not as good as the preferred condition group shown in Example 1.

[0178] (9) Conclusion

[0179] This comparative example shows that although microspheres can be formed under the crosslinking conditions of 80 mM CaCl2 and 8 min, the structural stability and model performance are affected, indicating that the appropriate crosslinking strength plays an important role in achieving the effect of the present invention.

[0180] Comparative Example 3: Live Acinetobacter baumannii microspheres prepared under high cross-linking strength conditions

[0181] This comparative example illustrates that when the crosslinking strength is higher than the preferred conditions of this invention, although the resulting microspheres can be formed, their structure and subsequent model effects will deviate.

[0182] (1) Preparation of sodium alginate solution

[0183] Weigh 0.11 g of sodium alginate, add sterile water to make up to 5.0 mL, and stir magnetically overnight at 4℃ to obtain a sodium alginate solution with a mass-volume concentration of 2.2% (w / v). After sterilization by filtering through a 0.22 µm filter membrane, it is ready for use.

[0184] (2) Internal phase configuration

[0185] Take 5.0 mL of Acinetobacter baumannii XH386 bacterial suspension in the logarithmic growth phase, and measure the OD of the bacterial suspension. 600 =0.25, and mixed with 5.0 mL of the above 2.2% sodium alginate solution, gently inverted and mixed 10 times to obtain an internal phase with a total volume of 10.0 mL.

[0186] (3) Oil phase configuration

[0187] Measure 100 mL of liquid paraffin, add 150 µL of Span-80, and mix well to obtain the oil phase.

[0188] (4) Emulsification

[0189] Place the oil phase in a beaker and stir at 700 rpm. Using a 23G syringe, add the internal phase dropwise to the oil phase at a rate of 1.0 mL / min. After the addition is complete, continue stirring for 4 min to form a W / O emulsion.

[0190] (5) High cross-linking curing

[0191] Weigh 2.94 g of CaCl2•2H2O and add sterile water to a final volume of 100 mL to obtain a 200 mM CaCl2 crosslinking solution. Place the crosslinking solution in a beaker and stir at 300 rpm. Then, slowly add the above emulsion along the beaker wall to the crosslinking solution over a period of 90 s, and continue stirring for 20 min to complete the crosslinking process.

[0192] (6) Collection and washing

[0193] After aliquoting the obtained microsphere system, the microspheres were collected by centrifugation at 600 g for 4 min. The supernatant was discarded, and the microspheres were washed three times with sterile PBS containing 2 mM CaCl2, and finally resuspended in sterile PBS.

[0194] (7) Microscopic observation

[0195] Prepare a slide using 80 µL of the microsphere suspension and observe it under a microscope. The results are as follows: Figure 10 The results show that the microspheres are generally intact in shape, but the particle structure is relatively dense, suggesting that a relatively tight alginate network was formed under high cross-linking conditions.

[0196] (8) Observation of animal delivery effect

[0197] The obtained microspheres were delivered into the lungs of mice via the airway, and the bacterial load in the lungs was detected and pathological changes were observed at subsequent time points. Results are as follows: Figure 11 The results showed that although some intrapulmonary retention was observed in this group, the overall model performance differed from that of Example 1, and the combined effect of long-term retention and model consistency shown in Example 1 was not reflected.

[0198] (9) Conclusion

[0199] This comparative example shows that although microspheres can be formed under the crosslinking conditions of 200 mM CaCl2 and 20 min, excessively high crosslinking strength will affect the structural state of the microspheres and the subsequent model performance, indicating that stronger crosslinking conditions are not necessarily better.

[0200] Comparative Example 4: Large-diameter live bacterial microspheres formed under low emulsification shear conditions

[0201] This comparative example illustrates how the microsphere size control and model consistency are affected when the emulsification shear conditions deviate from the preferred settings of this invention.

[0202] (1) Preparation of sodium alginate solution

[0203] Weigh 0.11 g of sodium alginate, add sterile water to make up to 5.0 mL, and stir magnetically overnight at 4℃ to obtain a sodium alginate solution with a mass-volume concentration of 2.2% (w / v). After sterilization by filtering through a 0.22 µm filter membrane, it is ready for use.

[0204] (2) Internal phase configuration

[0205] Take 5.0 mL of Acinetobacter baumannii XH386 bacterial suspension in the logarithmic growth phase, and measure the OD of the bacterial suspension. 600 =0.25, and mixed with 5.0 mL of the above 2.2% sodium alginate solution, gently inverted and mixed 10 times to obtain an internal phase with a total volume of 10.0 mL.

[0206] (3) Oil phase configuration

[0207] Measure 100 mL of liquid paraffin, add 150 µL of Span-80, and mix well to obtain the oil phase.

[0208] (4) Low-shear emulsification

[0209] Place the oil phase in a beaker and stir at 400 rpm. Using a 23G syringe, add the internal phase dropwise to the oil phase at a rate of 2.0 mL / min. After the addition is complete, continue stirring for 4 min to form a W / O emulsion.

[0210] (5) Crosslinking curing

[0211] Weigh 1.76 g of CaCl2•2H2O and add sterile water to a final volume of 100 mL to obtain a 120 mM CaCl2 crosslinking solution. Place the crosslinking solution in a beaker and stir at 300 rpm. Then, slowly add the emulsion along the beaker wall to the crosslinking solution over a period of 90 s, and continue stirring for 15 min to complete the crosslinking process.

[0212] (6) Collection and washing

[0213] After aliquoting the obtained microsphere system, the microspheres were collected by centrifugation at 600g for 4 min. The supernatant was discarded, and the microspheres were washed three times with sterile PBS containing 2 mM CaCl2, and finally resuspended in sterile PBS.

[0214] (7) Microscopic observation

[0215] Prepare a slide using 80 µL of the microsphere suspension, observe it under a microscope, and measure the diameter of 200 microspheres. The results are as follows: Figure 12 The results show that the particle size of this group is generally large and the distribution is relatively dispersed, indicating that it is difficult to achieve the preferred particle size control effect of the present invention under low shear emulsification conditions.

[0216] (8) Observation of animal delivery effect

[0217] The obtained microspheres were delivered into the lungs of mice via the airway, and the bacterial load in the lungs was detected and pathological changes were observed at subsequent time points. Results are as follows: Figure 13 The results showed that there were significant differences among the animals in this group, and the model consistency was not as good as in Example 1.

[0218] (9) Conclusion

[0219] This comparative example shows that under the conditions of emulsification at 400 rpm and dripping at 2.0 mL / min, the resulting microspheres have a larger particle size and a more dispersed distribution, which affects the consistency of the model. This indicates that particle size control is an important factor in achieving the effect of this invention.

[0220] Comparative Example 5: PBS airway delivery control

[0221] (1) Processing method

[0222] Sterile PBS was delivered into the lungs of mice via the airway; it contained no microspheres or bacteria.

[0223] (2) Results

[0224] like Figure 4 As shown, the PBS group had a lower overall pathological score during the observation period, exhibiting only mild, focal operation-related reactions. Serum TNF-α was virtually undetectable, and IL-6 was also within the low background range.

[0225] (3) Conclusion

[0226] PBS delivery itself only causes a mild operational background response, which is insufficient to produce the persistent lung infection / retention effect described in this invention.

[0227] Example 1: Evaluation of the background of lung inflammation using sterile blank microspheres

[0228] (1) Preparation method

[0229] Alginate microspheres were prepared according to the method in Example 1, but Acinetobacter baumannii was not added to the inner phase; instead, an equal volume of sterile culture medium was used.

[0230] (2) Microscopic morphology

[0231] like Figure 2 As shown in the image above, the blank microspheres exhibit a relatively complete spherical or near-spherical structure with clear boundaries.

[0232] (3) Animal delivery and pathological results

[0233] Blank microspheres were delivered into the lungs of mice, and lung tissue was harvested on days 2 and 4 post-drug administration for HE staining and pathological scoring. Results are as follows: Figure 4As shown, the overall score of the blank microsphere group remained at a low level, similar to that of the PBS control, with no obvious necrosis, hemorrhage or severe congestion or other damaging changes observed.

[0234] (4) Detection of inflammatory factors

[0235] The results are as follows Figure 14 As shown, TNF-α and IL-6 in BALF showed only a slight and transient increase, which gradually decreased thereafter.

[0236] (5) Conclusion

[0237] The results indicate that the microsphere carrier of the present invention has a low inflammatory background and is not the main cause of the persistent infection effect. This further illustrates that the chronic model effect obtained in Example 1 mainly comes from the combined effect of the live bacteria load and the process parameters of the present invention.

[0238] Example 2: Comprehensive comparison of Example 1 and various comparative examples

[0239] Examples 1, 2, and 3 demonstrate that, within a sodium alginate concentration range of 2.0%-2.5% (w / v), the emulsification dispersion, calcium ion crosslinking solidification, and washing purification processes described in this invention can all prepare live Acinetobacter baumannii microspheres, thus indicating the feasibility of this concentration range. 2.2% (w / v) is a preferred embodiment of this invention.

[0240] Comparative Examples 2, 3, and 4 further demonstrate that the technical effect of this invention does not simply stem from minor variations in sodium alginate concentration within the aforementioned range, but rather relies on the synergistic optimization of key parameters such as sodium alginate concentration, emulsification shear conditions, crosslinking strength, washing and purification methods, and quality control strategies. In particular, low or high crosslinking strength, or deviations in particle size control from the preferred conditions, all negatively impact the microsphere structure, particle size distribution, or consistency in the animal model.

[0241] Example 3

[0242] In establishing animal models of chronic lung infection caused by Acinetobacter baumannii, it was found that not all clinical isolates of Acinetobacter baumannii are suitable for constructing chronic infection models. For chronic lung infection models, the ideal modeling strain is not necessarily the more virulent it is. If the strain is too virulent, the host is prone to severe inflammatory response, serious lung damage, or even death in the early stages of infection. The resulting model is more similar to acute infection than chronic infection, which is not conducive to subsequent long-term observation and stability evaluation. Conversely, if the strain has moderate pathogenicity and strong lung adhesion and persistent colonization capabilities, it is more conducive to establishing a long-term persistent infection state in the host, thereby achieving stable construction of a chronic lung infection model.

[0243] Based on the above research findings, this invention further proposes that Acinetobacter baumannii strains suitable for modeling chronic lung infections should preferably meet the following conditions simultaneously:

[0244] (1) It has sufficient colonization and adhesion capabilities to persist in lung tissue;

[0245] (2) The virulence level should not be too strong to avoid rapid death or acute fulminant lung injury in the host in the early stage of infection;

[0246] (3) It can form a persistent, low-level infection state in the host's lungs, rather than a short-term, high-intensity pathogenic process.

[0247] Based on this, the present invention, through screening, found that the Acinetobacter baumannii XH386 strain, due to its relatively mild virulence and strong adhesion and persistent colonization capabilities, can successfully establish a long-lasting chronic lung infection model. In contrast, the LS01 strain is more virulent and tends to cause acute severe infection, making it difficult to establish a stable and sustainable chronic infection process. Therefore, XH386 is more suitable for constructing the "low-load-long-term persistence" chronic lung infection / persistence model described in this invention.

[0248] 1. Source of strains and basis for setting up comparison ratios:

[0249] The Acinetobacter baumannii XH386 used in this embodiment of the invention is a publicly reported clinical isolate, and its source strain number is NH57388A.

[0250] The Acinetobacter baumannii LS01 used in the comparative example is a clinical isolate obtained by our research team from blood samples of patients with community-acquired severe pneumonia, and related research has been completed and published by our team. Previous studies have shown that LS01 has strong virulence characteristics, exhibiting rapid growth and high lethality in insect infection models in previous experiments. Based on the above research foundation, this application selects LS01 as a comparative example strain to evaluate the applicability of highly virulent clinical isolates in constructing a chronic lung infection model under alginate live bacterial microsphere delivery conditions, and to illustrate that not all Acinetobacter baumannii strains are suitable for establishing the model described in this invention.

[0251] 2. Comparative Example: Live bacterial microspheres were prepared using strain LS01 under the same or similar conditions as in Example 1, and modeling was performed.

[0252] This comparative example is used to illustrate that, under the same or similar microsphere preparation and delivery conditions as in Example 1, if the embedded strain is replaced with Acinetobacter baumannii LS01, although live microspheres can be formed, the resulting animal infection outcome is significantly different from the "low-load-long-term retention" chronic lung infection / retention model required by the present invention. This indicates that not all Acinetobacter baumannii strains are suitable for constructing the model of the present invention.

[0253] The steps for preparing LS01 live bacterial microspheres are the same as those in Example 1.

[0254] (1) Microscopic morphological observation

[0255] 80 µL of the prepared microsphere suspension was dropped onto a glass slide, covered with a coverslip, and observed under a microscope. The results are as follows: Figure 15 The results show that, under the same or similar process conditions as in Example 1, LS01 can also form spherical or near-spherical microspheres, indicating that the alginate encapsulation process described in this invention has certain applicability to spheroidization and is not only applicable to a single strain.

[0256] (2) Animal delivery and observation of infection outcomes

[0257] The aforementioned LS01 live bacterial microspheres were delivered intratracheally to the lungs of mice, and the animals' general condition, survival, bacterial load in lung tissue, and pathological changes were observed at predetermined time points. Results are as follows: Figure 16-18 The results showed that, compared with the XH386 strain used in Example 1, animals in the LS01 group were more prone to obvious acute stress symptoms in the early stage of infection, including weight loss, reduced activity, piloerection, and worsening respiratory status; some animals experienced rapid disease progression, leading to a shortened observation window. Further results from lung tissue bacterial load testing showed that the LS01 group could exhibit a high bacterial load in the early stage of infection, but it was difficult to subsequently achieve the low-level, long-term stable retention state required by this invention.

[0258] (3) Pathological evaluation of the lungs

[0259] Lung tissues were stained with hematoxylin and eosin (HE) at the same time points, and semi-quantitative pathological scores were performed. The results showed that the LS01 group exhibited more pronounced acute inflammatory damage in the early stages of infection, including extensive infiltration of inflammatory cells, increased alveolar structural destruction, and elevated pathological scores. Compared to Example 1, this model more closely resembled an acute, high-intensity infection process rather than a sustained, controllable state of chronic inflammation.

[0260] (4) Results Analysis

[0261] This comparative example shows that although the LS01 strain can also be encapsulated into live microspheres under the same or similar process parameters as in Example 1, its infection outcome after delivery to the animal lungs differs significantly from that of XH386. LS01 tends to induce acute severe pathological changes in the early stages of infection, making it difficult to achieve the "low-load-long-term retention" chronic lung infection / retention technology effect shown in Example 1. Therefore, not all Acinetobacter baumannii strains are suitable for constructing the chronic model described in this invention; in comparison, XH386, due to its relatively mild virulence and greater ease of continuous colonization, is more suitable for achieving the objectives of this invention.

[0262] 3. Explanation of Comparison Results

[0263] Comparison shows that, under the same or similar conditions of alginate encapsulation, emulsification, cross-linking, and delivery, simply changing the encapsulated bacterial strain can lead to significantly different animal infection outcomes. The XH386 strain used in Example 1 can establish a long-term, low-level persistent bacterial presence in lung tissue and maintain a relatively controllable chronic inflammatory state; while the LS01 strain used, although it can complete microsphere encapsulation, its intrapulmonary infection process tends to exhibit acute, highly pathogenic manifestations, making it difficult to establish a stable chronic persistence model.

[0264] This demonstrates that the technical effectiveness of this invention depends not only on the microsphere preparation parameters but also on the biological characteristics of the selected Acinetobacter baumannii strain. In other words, this invention does not obtain the chronic lung infection model simply by embedding any Acinetobacter baumannii strain; rather, it requires selecting a specific strain with moderate virulence and strong sustained colonization ability. The XH386 strain precisely meets these requirements, therefore its selection is clearly necessary, targeted, and irreplaceable.

[0265] Therefore, the core of this invention does not lie in the isolated setting of a single parameter, but in the systematic optimization of the live microsphere preparation process around the specific purpose of constructing a chronic lung infection / persistence model of Acinetobacter baumannii, thereby achieving a comprehensive effect of low load, long-term persistence and low carrier background interference.

Claims

1. A method for constructing an animal model of chronic lung infection / holding of Acinetobacter baumannii, characterized in that, The method includes delivering live Acinetobacter baumannii microspheres via the airway to the lungs of a non-human mammal, wherein the Acinetobacter baumannii is Acinetobacter baumannii XH386, and the live Acinetobacter baumannii microspheres are prepared by a method comprising the following steps: (1) Preparation of internal phase: The logarithmic growth phase of Acinetobacter baumannii bacterial culture was mixed with a sodium alginate solution with a mass-volume concentration of 2.0%-2.5% to obtain the internal phase; (2) Preparation of oil phase: Prepare liquid paraffin oil phase containing nonionic surfactant; (3) Emulsification to form W / O microdroplets: Under stirring conditions, the inner phase of step (1) is added dropwise to the oil phase of step (2) to form a W / O emulsion; the stirring speed for emulsification is 600-1000 rpm; The dropping rate of the internal phase is ≤1 mL / min; (4) Calcium ion crosslinking and curing: The W / O emulsion from step (3) is added to the CaCl2 crosslinking aqueous phase and stirred to cure, forming alginate Acinetobacter baumannii live microspheres; the concentration of the CaCl2 crosslinking aqueous phase is 100-200 mM; the crosslinking and curing time is 10-20 min; (5) Collection and washing purification: Collect the microspheres obtained in step (4) and wash and purify them to remove the residual oil phase and surfactants, and obtain live Acinetobacter baumannii microspheres; The washing and purification process uses a Ca-containing method. 2+ Wash 2-3 times with sterile PBS.

2. The method according to claim 1, characterized in that, The sodium alginate solution in step (1) has a mass-volume concentration of 2.2%. The volume ratio of the Acinetobacter baumannii bacterial suspension to the sodium alginate solution is 1:0.5-1.5; OD of the Acinetobacter baumannii bacterial culture 600 The value is 0.2-0.

3.

3. The method according to claim 1, characterized in that, The nonionic surfactant mentioned in step (2) is Span-80, which has a volume fraction of 0.10%-0.20% in liquid paraffin.

4. The method according to claim 1, characterized in that, After the addition in step (3) is complete, continue stirring for 3-5 minutes.

5. The method according to claim 1, characterized in that, The stirring intensity in step (4) is 200-400 rpm.

6. The method according to claim 1, characterized in that, The method for collecting microspheres in step (5) is centrifugation, with centrifugation conditions of 500-800 g for 3-5 min; The Ca-containing 2+ The sterile PBS is PBS containing 2 mM CaCl2; Add 0.01%-0.05% Tween-20 to the washing solution.

7. The method according to claim 1, characterized in that, The preparation method further includes a quality control step after step (5): the microspheres are subjected to microscopic particle size determination, and when the average particle size deviates from the target range, reverse correction is performed by adjusting the emulsification speed, sodium alginate concentration or dropping speed to achieve batch-to-batch consistency control.

8. The method according to claim 1, characterized in that, The preparation method further includes a degellation and release and load verification step after step (5): the obtained microspheres are placed in a degellation solution containing EDTA to dissociate the microspheres and release the embedded bacteria, and the viable bacterial load of the microspheres is evaluated by CFU counting.

9. The method according to any one of claims 1-8, characterized in that, The non-human mammals mentioned are rodents; The rodents referred to are mice or rats.

10. The use of Acinetobacter baumannii live microspheres prepared by the method according to any one of claims 1-8 in the preparation of formulations for constructing animal models of chronic lung infection / persistence of Acinetobacter baumannii.

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