A kit for constructing an iron-dependent growth environment for acinetobacter baumannii and a method of use thereof

Abstract

This invention discloses a kit for constructing an iron-dependent growth environment for Acinetobacter baumannii and its application method, belonging to the field of biotechnology. The kit is specifically designed for Acinetobacter baumannii, and its core components include a 50 mmol / L sterile stock solution tube of 2,2′-bipyridine, a 2000 μmol / L sterile stock solution tube of FeCl3, and a user manual. It does not include general-purpose LB medium, thus avoiding the problem of high-temperature and high-pressure deterioration of core reagents and highlighting the innovative combination of dedicated reagents. This invention also provides the application method of the kit. By adding the kit stock solution to the autoclaved and cooled LB medium in a specific ratio, standardized growth environments with normal iron, iron deficiency, and iron enrichment can be rapidly constructed. Dynamic monitoring and verification of the reversibility of growth inhibition-recovery ensure that the iron environment is precise and controllable. The kit is easy to operate, has good reproducibility, and can achieve standardized construction of the iron environment for Acinetobacter baumannii. It is suitable for iron metabolism-related research such as functional analysis of iron uptake-related proteins, development of iron deprivation antibacterial strategies, and screening of iron-deficient strains.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, specifically to a kit for constructing an iron-dependent growth environment for Acinetobacter baumannii, and a standardized application method for the kit. Background Technology

[0002] Acinetobacter baumannii is a common multidrug-resistant opportunistic pathogen in clinical practice. Its iron metabolism homeostasis is closely related to bacterial proliferation, virulence expression, and drug resistance regulation. Iron deficiency is a typical microenvironmental feature of the host infection site. Acinetobacter baumannii needs to take up iron ions through specific iron transport systems (such as the TonB1 and TonB2 dependent systems) to maintain its survival. Therefore, constructing a standardized iron gradient growth environment is the core foundation for elucidating its iron metabolism mechanism and developing iron deprivation antibacterial strategies.

[0003] In existing technologies, conventional methods for constructing bacterial iron environments often involve using iron chelating agents and exogenous iron agents to regulate the iron content of the culture medium. However, their application to Acinetobacter baumannii has significant drawbacks: First, there is a lack of specific iron concentration thresholds tailored to this bacterium. Commonly used iron-deficient and iron-rich concentrations are often borrowed from other bacteria, failing to accurately simulate the iron-dependent growth characteristics of Acinetobacter baumannii. Second, iron chelating agents (such as 2,2′-bipyridine) and FeCl3 are prone to decomposition, hydrolysis, and precipitation under high temperature and pressure, and direct mixing into the culture medium for sterilization can lead to reagent failure. Third, there is no standardized procedure for reagent preparation, resulting in significant differences in operation among different researchers, leading to poor reproducibility of iron environment construction and making it difficult to compare experimental results across different researchers.

[0004] Although there are general methods for regulating the iron environment in microbial experiments, there is no dedicated kit for Acinetobacter baumannii, and the iron deficiency gradient range of 100-500 μmol / L 2,2′-bipyridine and the iron enrichment gradient range of 20-300 μmol / L FeCl3 are not clearly defined, which cannot meet the needs of refined and standardized research on iron metabolism of this bacterium.

[0005] In response to this problem, this application proposes a kit for constructing an iron-dependent growth environment for Acinetobacter baumannii and its application method, in order to solve the above-mentioned problems. Summary of the Invention

[0006] This invention aims to provide a kit and its application method for constructing an iron-dependent growth environment for Acinetobacter baumannii, addressing the problems of existing technologies such as the lack of reagents for constructing a specific iron environment for Acinetobacter baumannii, unclear concentration thresholds, non-standard operation, and poor experimental reproducibility. Through standardized reagent combinations and precise concentration gradient design, it achieves rapid and stable construction of normal iron, gradient iron-deficient, and gradient iron-rich environments for this bacterium, providing a unified experimental system for related basic research. To achieve the above objectives, this invention provides the following technical solution:

[0007] Firstly, a kit for constructing an iron-dependent growth environment for Acinetobacter baumannii.

[0008] The kit is a reagent combination specifically for Acinetobacter baumannii and does not include basic LB medium. The core consists of reagent tube 1 (sterile stock solution of 2,2′-bipyridine), reagent tube 2 (sterile stock solution of FeCl3), and the accompanying instruction manual. The specifications, characteristics, and functions of each component are clearly defined as follows:

[0009] Reagent tube 1: 2,2′-Bipyridine (Bipy) sterile stock solution, concentration 50 mmol / L; accurately weighed using an electronic balance, dissolved in sterile double-distilled water, and sterilized by filtration through a 0.22 μm sterile filter membrane to avoid reagent decomposition caused by high-temperature sterilization; 10 mL / tube, store at 4℃ protected from light, shelf life 7 days; core function is to specifically chelate free Fe²⁺ in the culture medium. + This provides core reagents for constructing a gradient iron-deficient environment of 100-500 μmol / L for Acinetobacter baumannii.

[0010] Reagent tube 2: Sterile stock solution of ferric chloride (FeCl3) with a concentration of 2000 μmol / L; prepared by dilution with standard FeCl3 solution, sterilized by filtration through a 0.22 μm sterile filter membrane, and used immediately after preparation; the volume is 10 mL / tube; its core function is to supplement iron ions in the culture medium and provide a core reagent for constructing a 20-300 μmol / L gradient iron-rich environment for Acinetobacter baumannii, and its physicochemical properties are consistent with the state of iron in the host environment.

[0011] Accompanying instruction manual: As the core technology carrier of the reagent kit, the following key contents are clearly recorded: stock solution dilution calculation formula, specific concentration thresholds for iron environment construction (iron-deficient 100-500μmol / L Bipy, iron-rich 20-300μmol / L FFeCl3), standardized operating procedures, iron environment quality verification standards, Acinetobacter baumannii culture conditions, reagent storage precautions and solutions to common problems.

[0012] Secondly, the application method of the above-mentioned reagent kit

[0013] The iron-dependent growth environment for Acinetobacter baumannii was constructed using the kit, following a standardized procedure of "sterilization of basal culture medium - cooling - addition of reagents - inoculation and culture - validation". The specific steps are as follows:

[0014] Basic preparation

[0015] Prepare LB liquid culture medium according to standard methods, autoclave at 121℃ for 20 min, cool naturally to room temperature (25±2℃), and dispense into sterile centrifuge tubes or culture flasks for later use;

[0016] Remove reagent tube 1 from the 4°C refrigerator and allow it to warm to room temperature for 10 minutes; prepare reagent tube 2 fresh and allow it to return to room temperature before use.

[0017] Standardized construction of iron-dependent growth environment

[0018] Normal iron environment: Cooled sterile LB liquid medium was used as the normal iron growth system for Acinetobacter baumannii.

[0019] Gradient iron deficiency environment: Add the 2,2′-bipyridine stock solution from reagent tube 1 to the cooled sterile LB liquid medium, and dilute by volume to a final concentration of 100 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L, and 500 μmol / L, and mix thoroughly by inverting to obtain iron deficiency media of different degrees;

[0020] Gradient iron-rich environment: Add the FeCl3 stock solution from reagent tube 2 to the cooled sterile LB liquid medium, and dilute it to a final concentration of 20 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, and 300 μmol / L by volume ratio, and mix thoroughly by inversion to obtain iron-rich medium of different degrees.

[0021] Note: All reagents were added after LB medium was cooled to completely avoid the problems of high-temperature decomposition of 2,2′-bipyridine and high-temperature hydrolysis and precipitation of FeCl3.

[0022] Bacterial inoculation and standardized culture

[0023] After resuscitating the Acinetobacter baumannii strain, it was cultured to the logarithmic growth phase, diluted with sterile physiological saline, and calibrated to a McFarland (McF) concentration of approximately 3 × 10⁻⁶. 8 (CFU / mL)

[0024] The standardized bacterial suspension was inoculated into each of the above iron environment culture systems at a volume ratio of 1:50, with 3 or more biological replicates set up for each group;

[0025] Incubate in a constant temperature shaking incubator at 37℃ and 180 rpm, supplemented with a 5% CO2 environment to promote the recovery of the strain and ensure the consistency of the bacterial growth status.

[0026] Dynamic monitoring and stability verification in the iron environment

[0027] Dynamic growth monitoring: During the culture period, the OD of the bacterial culture was dynamically measured using an enzyme-linked immunosorbent assay (ELISA) reader or spectrophotometer. 600 Values ​​were measured every 0.5 hours from 0 to 6 hours and every 1 hour from 6 to 24 hours. Colony forming units (CFU) were counted simultaneously on plates, and the amount of bacterial precipitate after centrifugation was recorded to quantify the differences in growth under different iron environments.

[0028] Stability verification criteria: In an iron-deficient environment (100-500 μmol / L Bipy), the growth inhibition rate of Acinetobacter baumannii is ≥80%; in an iron-rich environment (20-300 μmol / L FeCl3), the bacterial cell growth is stable with no obvious toxic inhibition. The difference in growth between groups is P<0.05 according to one-way ANOVA, which means that the iron environment construction is qualified.

[0029] Thirdly, the application scenarios of the reagent kit and application methods.

[0030] The kit and application method of this invention are specifically designed for basic research on iron metabolism in Acinetobacter baumannii. Applicable strains include wild-type Acinetobacter baumannii, the ATCC 17978 standard strain, and its genetically modified mutant strains with TonB gene knockout (ATCC17978△tonB1, ATCC17978△tonB2). Specific applications include:

[0031] Functional identification and mechanistic analysis of the iron uptake regulation system (TonB1 and TonB2-dependent iron transport system) of Acinetobacter baumannii;

[0032] Research and in vitro validation of the synergistic effect of iron deprivation combined with antimicrobial drugs;

[0033] Research on the regulatory mechanism of iron metabolism-related virulence genes in Acinetobacter baumannii;

[0034] Construction of in vivo and in vitro infection models of iron-environment adaptability of Acinetobacter baumannii;

[0035] Screening and typing of iron uptake deficiency in clinical Acinetobacter baumannii isolates.

[0036] Beneficial effects:

[0037] Compared with the prior art, the core innovations and beneficial effects of this invention are reflected in the following points:

[0038] 1. Precise concentration targeting, filling a technological gap: For the first time, the core concentration thresholds for iron environment suitable for Acinetobacter baumannii have been clearly defined—100-500 μmol / L Bipy for iron deficiency and 20-300 μmol / L FeCl3 for iron enrichment, covering the full gradient range from mild to severe iron deficiency and from trace amounts to saturated iron enrichment. This enables refined analysis of iron-dependent growth of bacteria and solves the problem of poor concentration adaptability of traditional methods.

[0039] 2. Standardized reagent combinations, convenient and efficient operation: The core reagents are prepared as quantitative sterile stock solutions and individually packaged into physical reagent kits, eliminating the need for experimenters to prepare high-concentration reagents themselves; only dilution according to the instructions is required to quickly construct the required iron environment, greatly reducing operational errors and improving the repeatability of experiments in different laboratories and different batches.

[0040] 3. Rationalized reagent protection to ensure stable activity: The "LB medium is sterilized separately + core reagents are added after filtration sterilization and cooling" mode is adopted to completely avoid the problems of high-temperature decomposition of 2,2′-bipyridine and high-temperature hydrolysis precipitation of FeCl3, thus ensuring the stability of reagent activity and iron environment.

[0041] 4. Diverse application scenarios and significant scientific research value: It can not only be used for the study of iron metabolism mechanisms, but also realize the rapid screening of iron-deficient strains and the in vitro verification of antibacterial strategies. It provides a brand-new standardized experimental tool for the study of multidrug resistance in Acinetobacter baumannii and has broad prospects for promotion and application. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0043] Figure 1 Iron environment and Ab growth OD provided for embodiments of the present invention 600 The relationship diagram shows that (A) Bipy concentration is related to Ab growth OD. 600 (A) Relationship diagram; (B) Analysis diagram of differences in different growth states of Ab under iron-deficient environment; (C) FeCl3 concentration and Ab growth OD 600 Relationship diagram (D) Analysis of differences in different growth states of Ab under iron-rich environment;

[0044] Figure 2The figure shows the analysis results of the Bipy iron chelation verification provided in the embodiments of the present invention, where Group A: 0 μmol / L FeCl3 + 400 μmol / L Bipy; Group B: 500 μmol / L FeCl3 + 400 μmol / L Bipy; Group C: 1000 μmol / L FeCl3 + 400 μmol / L Bipy; Group D: 2000 μmol / L FeCl3 + 400 μmol / L Bipy; (a) After compensation with different concentrations of FeCl3, the growth OD of Ab... 600 (a) Bar chart of differential analysis; (b) Bacterial precipitate after centrifugation of culture tubes after FeCl3 compensation;

[0045] Figure 3 This is a diagram illustrating the effect of different iron environments on the growth of Acinetobacter baumannii strain ATCC17978, as provided in an embodiment of the present invention.

[0046] Figure 4 The effect of different iron environments on the growth of Acinetobacter baumannii ATCC17978 tonB1 gene knockout strain provided in the embodiments of the present invention;

[0047] Figure 5 The figure shows the effect of different iron environments on the growth of Acinetobacter baumannii ATCC17978 tonB2 gene knockout strain, as provided in the embodiments of the present invention. Detailed Implementation

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0049] The technical solution of the present invention will be described in detail below with reference to specific embodiments, in order to help those skilled in the art understand the present invention, but it does not constitute a limitation on the present invention.

[0050] As attached Figure 1 To be continued Figure 5 As shown:

[0051] Example 1:

[0052] This embodiment is specifically applied to a validation experiment of the iron environment of Acinetobacter baumannii based on reversible iron regulation:

[0053] I. Experimental Materials

[0054] (a) Strains:

[0055] Acinetobacter baumannii ATCC 17978 wild-type strain (WT, normal iron uptake function);

[0056] (II) Instruments:

[0057] McLeod turbidimeter (Grant DEN-1B type);

[0058] Microplate reader (Thermo Wellwash type);

[0059] Desktop air-controlled constant temperature oscillator (Taicang City Science and Education Equipment Hz-9211K model);

[0060] High-speed refrigerated centrifuge (Eppendorf 5810R model);

[0061] Electronic balance (Mettler PL2002 model);

[0062] 15 mL sterile centrifuge tubes (Nice, catalog number 602052-1);

[0063] 200 μL / 1000 μL Yellow pipette tips (in the center, part number P0200-H-ST / P01000-H-ST);

[0064] Sterile 96-well plate (Corning, catalog number 3599).

[0065] II. Experimental Methods:

[0066] (I) Preparation of key reagents:

[0067] Preparation of FeCl3 solution: Take 1.2966 mL of 1% FeCl3 standard solution and dilute to 40 mL with LB liquid medium to prepare a 2000 μmol / L stock solution (prepare immediately before use). Based on the stock solution, perform serial dilutions in LB liquid medium to establish 24 concentration gradients of 0, 20, 40, ..., 460 μmol / L, thus constructing the FeCl3 concentration gradient group (iron-rich environment construction group).

[0068] Bipy solution preparation: Accurately weigh 312.38 mg Bipy using an electronic balance, dissolve in 40 mL of sterile double-distilled water, and sterilize by filtration through a 0.22 μm sterile filter to obtain a 50 mmol / L stock solution (store at 4℃ protected from light, shelf life 7 days). Based on the stock solution, perform serial dilutions in LB liquid medium, setting concentration gradients of 0, 100, 200, ..., 1000 μmol / L (11 concentration points in 100 μmol / L increments) and 1200, 1400, ..., 2000 μmol / L (5 concentration points in 200 μmol / L increments) to construct Bipy concentration gradient groups (iron-deficient environment construction groups).

[0069] (II) Standardized preparation of bacterial suspensions:

[0070] The Acinetobacter baumannii ATCC 17978 standard strain was taken from the -80℃ cryopreservation tube, thawed at room temperature, and a small amount of bacterial solution was taken with a sterile inoculation loop and inoculated onto LB Lennox agar plates using the four-zone streak method. The plates were then incubated in a 5% CO2, 35℃ incubator for 12 hours for recovery.

[0071] Select a single, uniformly shaped colony from the plate and inoculate it into 5 mL of LB broth. Incubate at 37°C with shaking at 180 rpm for 8 hours until the logarithmic growth phase (OD200). 600 ≈0.6);

[0072] The bacterial concentration was verified by plate counting: Logarithmic phase bacterial suspension was serially diluted and inoculated onto LB agar plates, incubated at 35°C for 12 h, and colony forming units (CFU) were counted.

[0073] Dilute the bacterial suspension with sterile saline and calibrate to a McFarland turbidimeter concentration of 1.0 McFarland (verified by plate count, corresponding to approximately 3 × 10⁻⁶). 8 (CFU / mL), for later use.

[0074] (III) Experimental Grouping and Cultivation (Clear Group Design):

[0075] This experiment consisted of two experimental groups, each with a blank control (LB liquid medium only) and a negative control (LB liquid medium + bacterial suspension, no reagents added). Each concentration gradient in each group had three biological replicates, and two independent experiments were conducted in parallel.

[0076] Bipy concentration gradient group (iron-deficient environment construction): Take a 15mL sterile centrifuge tube, add 10mL of LB liquid medium with different concentrations of Bipy, add 1.0 McF bacterial suspension at a volume ratio of 1:50 (bacterial suspension to medium), mix thoroughly and seal.

[0077] FeCl3 concentration gradient group (construction of iron-rich environment): Take 15mL sterile centrifuge tubes, add 10mL of LB liquid medium with different concentrations of FeCl3, add bacterial suspension according to the same volume ratio as above, mix thoroughly and seal.

[0078] All centrifuge tubes were placed in a constant temperature shaking incubator and shaken at 37°C and 180 rpm for 12 hours.

[0079] (iv) Iron supplementation reversal experiment:

[0080] An iron-deficient system was constructed based on LB liquid medium containing 400 μmol / L Bipy, and 1.0 McF bacterial suspension was added at a volume ratio of 1:50 to prepare a 40 mL culture system.

[0081] The system was divided into 4 groups (3 biological replicates per group): Group A (control): 0 μmol / L FeCl3 + 400 μmol / L Bipy; Group B: 500 μmol / L FeCl3 + 400 μmol / L Bipy; Group C: 1000 μmol / L FeCl3 + 400 μmol / L Bipy; Group D: 2000 μmol / L FeCl3 + 400 μmol / L Bipy.

[0082] The culture was carried out at 35℃ and 180 rpm for 12 hours, followed by OD value detection and centrifugation sedimentation observation.

[0083] (v) Testing methods:

[0084] OD 600 Value determination: After the culture is completed, use a pipette to take 200 μL of bacterial culture into a sterile 96-well plate, and measure the absorbance at a wavelength of 600 nm using an ELISA reader. Calculate the mean ± standard deviation (x ± s) of the three replicates for each group.

[0085] Centrifugation precipitation observation: Take 5 mL of bacterial culture, centrifuge at 12000 rpm for 10 min, discard the supernatant, observe the precipitation volume and density with the naked eye and take pictures for recording;

[0086] Bacterial Count: Plate counts were performed on key concentration groups (e.g., 200 μmol / L Bipy, 600 μmol / L Bipy, 280 μmol / L FeCl3, 400 μmol / L FeCl3) to calculate CFU / mL and verify OD. 600 Correlation between the value and the number of bacteria.

[0087] (vi) Statistical analysis:

[0088] Data processing was performed using Graphpad Prism 8.0 software. One-way ANOVA was used to analyze differences between groups, and t-tests were used for pairwise comparisons. P < 0.05 was considered statistically significant, "***" indicated P < 0.001, and "ns" indicated no statistical difference.

[0089] III. Experimental Results

[0090] (I) Results of Bipy concentration gradient group (iron-deficient environment):

[0091] In the negative control group (without Bipy supplementation), Acinetobacter baumannii grew vigorously, with OD... 600 The value was 0.63±0.03, and the plate count result was 2.8×10⁸ CFU / mL;

[0092] When the Bipy concentration is between 0 and 200 μmol / L, bacterial growth is slightly inhibited, but OD... 600 The value remained above 0.40 (P>0.05);

[0093] When the Bipy concentration increases to 300~600 μmol / L, OD 600 The value decreases sharply with increasing concentration: OD value of 200 μmol / L Bipy group 600 The value was 0.21±0.02, which was statistically significant compared with the negative control group (t=18.98, P=0.006), and the plate count was 3.5×10⁷ CFU / mL (partial inhibition); OD in the 600μmol / L Bipy group 600 The value was 0.08±0.01, which was statistically significant (t=88.61, P=0.003), and the plate count was < 1×106 CFU / mL (complete growth arrest).

[0094] When the Bipy concentration exceeds 600 μmol / L, OD 600 The values ​​tended to stabilize (0.07~0.09), with no significant changes (P>0.05).

[0095] (II) Results of FeCl3 concentration gradient group (iron-rich environment):

[0096] When the FeCl3 concentration is between 0 and 300 μmol / L, bacterial growth exhibits fluctuating changes, and OD... 600 The values ​​were between 0.60 and 0.65, showing no statistically significant difference compared to the negative control group (0.63 ± 0.03) (P > 0.05); the OD values ​​of the 280 μmol / L FeCl3 group were... 600 The highest value was (0.64±0.02), but the difference was not statistically significant (t=1.251, P=0.561), and the plate count was 2.7×10⁻⁶. 8 CFU / mL, similar to the negative control group;

[0097] When the FeCl3 concentration exceeds 300 μmol / L, bacterial growth is gradually inhibited: at 320~380 μmol / L, OD... 600 The value decreased to 0.40~0.25; OD of the 400μmol / L FeCl3 group 600The value was 0.10±0.01, which was statistically significant compared with the negative control group (t=17.52, P=0.007), and the plate count was 1.2×10⁻⁶. 7 CFU / mL (significant growth inhibition);

[0098] When the FeCl3 concentration increases to 420~460 μmol / L, OD 600 When the value further decreased to below 0.08, bacterial growth completely stopped (P<0.001).

[0099] (III) Results of the iron supplementation reversal experiment:

[0100] OD 600 Value changes: Group A (0 μmol / L FeCl3 + 400 μmol / L Bipy) OD 600 The value was 0.09±0.01, indicating complete inhibition of bacterial growth; OD values ​​for group B (500 μmol / L FeCl3) and group C (1000 μmol / L FeCl3) were... 600 The values ​​were 0.10±0.01 and 0.18±0.02, respectively, with no significant difference compared to group A (t=0.434, P=0.975; t=5.537, P=0.090); OD of group D (2000μmol / L FeCl3) was significantly different. 600 The value was 0.61±0.03, which was significantly different from group A (t=62.500, P<0.001), but there was no statistically significant difference from the negative control group (0.63±0.03) (P>0.05).

[0101] Centrifugation precipitation observation: No visible bacterial precipitate was observed after centrifugation in groups A, B, and C; after centrifugation, group D formed a dense and uniform precipitate layer (volume approximately 0.25 mL), which was close to the precipitate volume of the negative control group, visually verifying the recovery of bacterial growth;

[0102] Plate count verification: The plate count in group D was 2.6 × 10⁸ CFU / mL, which was not significantly different from the negative control group (2.8 × 10⁸ CFU / mL), confirming that bacterial growth was completely restored after iron supplementation.

[0103] IV. Results Analysis:

[0104] Growth of Ab cells under different iron conditions: Ab cells grew vigorously in LB medium without the addition of Bipy and FeCl3. When the Bipy concentration was in the range of 300-600 μmol / L, the chelating ability of iron was significantly enhanced with increasing Bipy concentration, and the optical density (OD) of Ab cells increased. 600The concentration of Bipy decreased sharply. When the concentration exceeded 600 μmol / L, bacterial growth was completely inhibited, and OD... 600 tending to stabilize Figure 1 A);

[0105] To further quantify the effect of the iron-deficient environment constructed with Bipy on bacterial growth, three groups were selected: no Bipy added as the control group, 200 μmol / L Bipy as the moderate iron-deficient group, and 600 μmol / L Bipy as the severe iron-deficient group. Inter-group comparisons were then performed. The OD values ​​of the three groups were... 600 Comparison revealed that, compared to the control group, Ab growth was partially inhibited in the 200 μmol / L Bipy medium, with a statistically significant difference between groups (t=18.98, P=0.006), while Ab growth was completely halted in the 600 μmol / L Bipy medium, with a statistically significant difference (t=88.61, P=0.003). Figure 1 B). Within the FeCl3 concentration range of 0-300 μmol / L, Ab growth only showed a slight increase or decrease with increasing FeCl3 concentration, exhibiting fluctuating changes, and the promotion and inhibition phenomena were not obvious; when the concentration exceeded 300 μmol / L, Ab growth was gradually inhibited, and growth stopped at 400 μmol / L. Figure 1 C). Intergroup comparisons were conducted using no FeCl3 as the control group, 280 μmol / L FeCl3 as the moderately iron-rich group, and 400 μmol / L FeCl3 as the highly iron-rich group. Compared with the control group, the OD of Ab growth in the 280 μmol / L FeCl3 environment was significantly higher. 600 The increase was slight, but the difference was not statistically significant compared to the control group (t=1.251, P=0.561). However, in an iron environment of 400 μmol / LFeCl3, Ab growth was significantly inhibited (t=17.52, P=0.007). Figure 1 D);

[0106] Results of the iron supplementation reversal experiment: Iron-deficient LB liquid medium containing 400 μmol / L Bipy was supplemented with FeCl3 working solution to prepare culture systems of 500 μmol / L FeCl3, 1000 μmol / L, and 2000 μmol / L FeCl3, respectively, named groups B, C, and D. The 400 μmol / L Bipy medium without FeCl3 solution was used as the control group (group A). ​​After 12 hours of culture, the OD of group B was significantly lower than that of the control group. 600 There was no significant difference (t=0.434, P=0.975), but the OD of group C compared to the control group was... 600No significant difference was observed (t=5.537, P=0.090), indicating complete inhibition of bacterial growth. When the LB liquid medium containing 400 μmol / L Bipy was compensated to 2000 μmol / L FeCl3 solution, the OD of the bacterial culture... 600 The value increased significantly (t=62.500, P<0.001), and the bacteria resumed growth. Figure 2 a). No bacterial precipitate was found in groups A, B, and C after centrifugation, while a visible bacterial precipitate layer appeared in group D, indicating bacterial regeneration. Figure 2 b)

[0107] As shown above, this invention utilizes 200 μmol / L Bipy to construct a moderately iron-deficient environment (partial bacterial inhibition) and 600 μmol / L Bipy to construct a highly iron-deficient environment (complete bacterial cessation). This concentration threshold provides a standardized model for subsequent research on the function of the Acinetobacter baumannii iron uptake system (such as TonB-dependent transporters). Clinically, iron chelating agents are a potential direction for combating drug-resistant bacteria. The effective inhibitory concentration of Bipy determined in this experiment can provide reference data for the dosage screening of iron-deprivation antibacterial drugs.

[0108] 20~300μmol / L FeCl3 is the "iron tolerance concentration range" of Acinetobacter baumannii, and 400μmol / L is the significant inhibitory concentration. This result can be used to study the mechanism of bacterial iron tolerance—excessive iron can induce oxidative stress damage. This model can be used to screen mutant strains of iron tolerance-related genes, providing a tool for elucidating the environmental adaptation mechanism of drug-resistant bacteria.

[0109] 2000 μmol / L FeCl3 can completely reverse the antibacterial effect of 400 μmol / L Bipy, confirming that the inhibitory effect of Bipy is specifically due to iron chelation (rather than direct bactericidal action). This closed-loop validation system can be used to distinguish between "iron-deficient strains" and "non-specific growth-deficient strains", providing an efficient method for screening and validating gene knockout strains related to iron uptake function in Acinetobacter baumannii, avoiding the cumbersome process of traditional molecular validation.

[0110] Example 2: Assembly and Preparation of the Reagent Kit

[0111] This embodiment provides a method for large-scale preparation of the reagent kit, ensuring accurate reagent concentration and standardized packaging:

[0112] Preparation of Reagent Tube 1 (2,2′-Bipyridine Stock Solution): Accurately weigh 312.38 mg of 2,2′-bipyridine powder, add 40 mL of sterile double-distilled water, and stir magnetically until completely dissolved; sterilize by filtration through a 0.22 μm aqueous sterile filter membrane to obtain a 50 mmol / L stock solution; dispense 10 mL / tube into sterile polypropylene reagent tubes, tighten the caps, and label: "Acinetobacter baumannii-specific 2,2′-bipyridine stock solution 50 mmol / L; iron-deficient construction concentration: 100-500 μmol / L; store at 4℃ protected from light, shelf life 7 days."

[0113] Preparation of reagent tube 2 (FeCl3 stock solution): Take 1.2966 mL of 1% (w / v) FeCl3 standard solution, and dilute to 40 mL with sterile LB liquid medium. Mix thoroughly to obtain a 2000 μmol / L stock solution. Filter and sterilize using a 0.22 μm aqueous sterile filter membrane. Dispense 10 mL / tube into sterile polypropylene reagent tubes, tighten the caps, and label with: "Acinetobacter baumannii-specific FeCl3 stock solution 2000 μmol / L; iron-rich construction concentration: 20-300 μmol / L; prepare fresh before use."

[0114] Instructions for Use: Printed on A4 paper, clearly stating the following core contents: ① Stock solution dilution formula: V (stock solution) = [C (final concentration) × V (culture medium)] / C (stock solution concentration); ② Recommended addition volume table for graded iron deficiency / iron enrichment (taking 10mL LB medium as an example); ③ Complete operating procedures and quality verification standards; ④ Reagent storage and disposal requirements.

[0115] Reagent kit assembly: Package two reagent tubes (Reagent tube 1 and Reagent tube 2) and one instruction manual together in a paper reagent kit shell labeled: "Reagent kit for constructing iron-dependent growth environment for Acinetobacter baumannii; Core concentration: 100-500 μmol / L Bipy for iron deficiency, 20-300 μmol / L FeCl3 for iron enrichment."

[0116] Results: Instructions for use of the kit for constructing an iron-dependent growth environment for Acinetobacter baumannii:

[0117] Scope of application: This instruction manual applies to the use, storage and disposal of the Acinetobacter baumannii iron-dependent growth environment construction kit (hereinafter referred to as "this kit"). It is specifically for in vitro studies of iron metabolism related to wild-type, standard, and genetically modified mutant strains of Acinetobacter baumannii and is not intended for clinical diagnosis or treatment.

[0118] I. Reagent Kit Composition and Core Parameters

[0119] This kit is a reagent combination specifically for Acinetobacter baumannii and does not contain basic LB liquid medium. The core components are shown in the table below. All reagents are sterile and should be kept sealed before opening.

[0120] Table 1. Reagent kit composition and core parameters

[0121] Reagent Name reagent tube number Concentration Specifications Filling volume sterilization method Storage conditions Validity period 2,2'-Bipyridine sterile stock solution reagent tube 1 50 mmol / L 10 mL / tube 0.22 μm aqueous filter membrane filtration sterilization 4℃, lightproof and sealed 1 year <![CDATA[Sterile mother liquor of FeCl3]]> Reagent tube 2 2000 μmol / L 10 mL / tube 0.22 μm aqueous filter membrane filtration sterilization 4℃, lightproof and sealed 1 year This instruction manual 1 serving / box Store in a dry place at room temperature Long-term effective

[0122] II. Core Calculation Formulas

[0123] The core reagents in this kit need to be diluted proportionally before being added to cooled, sterile LB liquid medium. The formula for calculating the volume of stock solution added is as follows, to ensure that the final concentration accurately matches the iron environment construction requirements of Acinetobacter baumannii:

[0124]

[0125] Symbol explanation:

[0126] V (stock solution): The volume of stock solution to be added to reagent tube 1 or reagent tube 2 (μL / mL).

[0127] C (final concentration): The final concentration of the reagent in the target iron environment (μmol / L);

[0128] V (culture medium): Volume (mL) of sterile LB liquid culture medium to be treated;

[0129] C (mother liquor concentration): The concentration of the mother liquor in reagent tube 1 (50 mmol / L) or reagent tube 2 (2000 μmol / L).

[0130] III. Recommended Addition Volumes for Gradient Iron Environments (using 10 mL LB liquid medium as an example)

[0131] Based on the specific iron environment concentration thresholds for Acinetobacter baumannii (iron deficiency 100-500 μmol / L 2,2'-bipyridine, iron enrichment 20-300 μmol / L FeCl3), the following recommended addition volume table is formulated. It can be used directly without additional calculations to accurately construct a gradient iron environment.

[0132] (a) Construction of a gradient iron-deficient environment (reagent tube 1)

[0133] Table 2. Addition Table for Gradient Iron-Deficient Environment Construction

[0134] Target final concentration (2,2'-bipyridine) Mother liquor concentration (50 mmol / L) Add the stock solution volume to 10 mL LB medium 100 μmol / L 50 mmol / L 20 μL 200 μmol / L 50 mmol / L 40 μL 300 μmol / L 50 mmol / L 60 μL 400 μmol / L 50 mmol / L 800 μL 500 μmol / L 50 mmol / L 100 μL

[0135] Note: The target concentration can be constructed by yourself according to the core calculation formula.

[0136] (II) Construction of a gradient iron-rich environment (2 reagent tubes)

[0137] Table 3. Gradient Iron-Rich Environment Construction Addition Table

[0138] <![CDATA[Target final concentration (FeCl3)]]> Mother liquor concentration (2000 μmol / L) Add the stock solution volume to 10 mL LB medium 20 μmol / L 2000 μmol / L 100 μL 50 μmol / L 2000 μmol / L 250 μL 100 μmol / L 2000 μmol / L 500 μL 200 μmol / L 2000 μmol / L 1000 μL 300 μmol / L 2000 μmol / L 1500 μL

[0139] Note: The target concentration can be constructed by yourself according to the core calculation formula.

[0140] IV. Standardized Operating Procedures

[0141] This kit must be used in a sterile laboratory environment, strictly following the aseptic operating procedures for microbiological experiments. The core process is "basal culture medium preparation → reagent warming → iron environment construction → bacterial inoculation and culture → quality verification," and the specific steps are as follows:

[0142] Step 1: Preparation of basic LB liquid culture medium

[0143] 1. Prepare LB liquid culture medium according to standard laboratory formula, dissolve it thoroughly, and dispense it into suitable sterile culture containers (such as sterile centrifuge tubes, culture flasks, 96-well sterile culture plates).

[0144] 2. Place the LB liquid culture medium in an autoclave and autoclave it at 121°C for 20 min;

[0145] 3. After sterilization, remove and allow to cool naturally to room temperature (25±2℃), maintaining a sterile state throughout the process, for later use.

[0146] Step 2: Reagent and reagent warming and preparation

[0147] 1. Reagent tube 1 (2,2'-bipyridine stock solution): Remove from the 4°C refrigerator, allow to warm to room temperature in the dark for 10 min, gently invert to mix, avoiding vigorous shaking;

[0148] 2. Reagent tube 2 (FeCl3 stock solution): Confirmed to be a freshly prepared product of the day, warm it to room temperature to about 25°C, gently invert and mix well before use;

[0149] 3. All reagent operations must be performed in a sterile laminar flow hood, using sterile pipettes and tips to avoid cross-contamination.

[0150] Step 3: Construction of an iron-dependent growth environment for Acinetobacter baumannii

[0151] (1) Construction of normal iron environment

[0152] Take the cooled sterile LB liquid culture medium directly, without adding any reagents, as the normal iron growth control system.

[0153] (2) Construction of gradient iron-deficient environment

[0154] 1. Take 10 mL of cooled sterile LB liquid culture medium and place it in a sterile laminar flow hood;

[0155] 2. Referring to the "Addition Table for Constructing a Gradient Iron-Deficient Environment", use a sterile pipette to draw the corresponding volume of stock solution from reagent tube 1 and slowly add it to the LB medium;

[0156] 3. Gently invert or gently blow to mix, avoiding the formation of a large number of air bubbles, to obtain iron-deficient culture media of different gradients.

[0157] (3) Construction of gradient iron-rich environment

[0158] 1. Take 10 mL of cooled sterile LB liquid culture medium and place it in a sterile laminar flow hood;

[0159] 2. Referring to the "Gradient Iron-Rich Environment Construction Addition Table", use a sterile pipette to draw the corresponding volume of stock solution from reagent tube 2 and slowly add it to the LB medium;

[0160] 3. Gently invert or gently blow to mix, and you will obtain iron-rich culture media of different gradients.

[0161] Step 4: Bacterial inoculation and standardized culture

[0162] 1. Preparation of Acinetobacter baumannii bacterial suspension: After resuscitation of the target strain, culture it to the logarithmic growth phase, dilute with sterile physiological saline, and calibrate to a McFarland (McF) concentration of approximately 3 × 10⁻⁶. 8 (CFU / mL)

[0163] 2. Inoculation: Inoculate the standardized bacterial suspension into each iron environment culture system at a volume ratio of 1:50, and set up 3 or more biological replicates for each iron environment;

[0164] 3. Incubation: Place the inoculated culture system in a constant temperature shaking incubator at 35℃ and 180 rpm, and provide a 5% CO2 environment to promote the recovery of the strain. The incubation time is set according to the experimental requirements (24 h is recommended).

[0165] Step 5: Iron Environmental Quality Verification

[0166] During the cultivation period, dynamic monitoring and stability verification should be carried out according to the following methods to ensure that the iron environment is qualified before it can be used for subsequent experimental analysis:

[0167] 1. Dynamic growth monitoring:

[0168] 0-6 h incubation period: OD of bacterial culture was measured every 0.5 h. 600 value;

[0169] 6-24 h incubation period: OD of bacterial culture was measured every 1 h. 600 value;

[0170] Colony forming units (CFU) were counted simultaneously using the plate coating method, and the amount of bacterial precipitate after centrifugation was recorded to quantify the differences in growth.

[0171] 2. Conformity verification criteria:

[0172] Iron-deficient environment (100-500 μmol / L 2,2'-bipyridine): Acinetobacter baumannii growth inhibition rate ≥80%;

[0173] Iron-rich environment (20-300 μmol / L FeCl3): The strains showed stable growth with no obvious iron toxicity inhibition and no significant toxic effects compared to the normal iron group.

[0174] The differences in growth between groups were analyzed by one-way ANOVA, and the result was P<0.05, indicating that the iron environment construction was qualified.

[0175] V. Requirements for Reagent Storage and Management

[0176] To ensure the accuracy of reagent activity and iron environment construction, the following storage and management guidelines must be strictly followed:

[0177] (a) Storage of unopened reagents

[0178] 1. Reagent tubes: Store at 4℃ in a sealed container, protected from light, high temperatures, strong light, and oxidizing substances. Do not freeze.

[0179] 2. Reagent tube 2: This is a reagent to be prepared and used immediately. The kit is only provided with empty tubes and preparation instructions. The reagent must be prepared and used on the same day by the experimenter using qualified FeCl3 standard solution.

[0180] 3. The entire kit should be stored in a dry, well-ventilated laboratory refrigerated area, and should be kept away from toxic and hazardous chemicals.

[0181] (II) Reagent Management After Opening

[0182] 1. Reagent tube 1: Once opened, it must be used within 48 hours. After each use, immediately tighten the cap and continue to store it in the cool, dark place. Do not use if it exceeds the expiration date or if the solution changes color, becomes cloudy, or precipitates.

[0183] 2. Reagent tube 2: After opening, it must be used within 4 hours. Any remaining portion must not be retained and should be disposed of as hazardous waste.

[0184] (III) Explanation of Validity Period

[0185] 1. Reagent tube 1: Based on the production date indicated on the kit, the shelf life is 1 year when stored at 4℃ protected from light; it will expire after this date.

[0186] 2. Reagent tube 2: Based on the production date indicated on the reagent kit, the shelf life is 1 year when stored at 4℃ protected from light; it will expire after this date.

[0187] 3. This instruction manual has no expiration date. If the technical parameters of the reagent kit are updated, a new version of the instruction manual will be released simultaneously with the new product.

[0188] VI. Waste Disposal Requirements

[0189] All reagents and laboratory waste related to this kit must be disposed of in strict accordance with the "Laboratory Hazardous Waste Disposal Standards" and relevant national environmental protection regulations. They must be classified and disposed of properly, and indiscriminate disposal is strictly prohibited. Specific requirements are as follows:

[0190] (a) Reagent waste disposal

[0191] 1. Expired or ineffective reagent tube 1 mother liquor: This is chemical reagent waste. It should be collected in a special chemical waste liquid container (labeled "containing 2,2'-bipyridine waste liquid"), and after neutralization and degradation treatment, it should be transferred and disposed of by a qualified hazardous waste treatment agency.

[0192] 2. The remaining mother liquor from reagent tube 2: This is a chemical waste liquid containing heavy metal iron ions. It should be collected in a special heavy metal waste liquid container (labeled "iron ion waste liquid"), and after precipitation and solidification treatment, it should be handed over to a qualified institution for disposal.

[0193] 3. Empty test tubes: After being disinfected by soaking in 75% ethanol for 30 minutes, they should be disposed of as medical waste or recyclable plastic waste.

[0194] (II) Waste disposal in the experimental system

[0195] 1. Acinetobacter baumannii bacterial culture and culture medium after cultivation: These are pathogenic microbial wastes and must first be sterilized and inactivated by autoclaving at 121℃ for 20 minutes. After confirming sterility, they should be disposed of as chemical waste or domestic waste.

[0196] 2. Pipette tips, culture containers, gloves, and other consumables that have come into contact with bacterial solutions: After being sterilized and inactivated by autoclaving, they shall be treated as medical hazardous waste, sealed in special yellow medical waste bags, and disposed of centrally by designated institutions.

[0197] (III) Handling Responsibilities

[0198] Laboratory operators are primarily responsible for waste disposal and must properly collect, classify, and record waste. Laboratory managers must regularly check the disposal process to ensure it meets safety and environmental protection requirements.

[0199] VII. Precautions

[0200] 1. This kit is intended for in vitro studies of iron metabolism in Acinetobacter baumannii and is prohibited for use with other bacteria, fungi, or for clinical diagnostic purposes.

[0201] 2. The 2,2'-bipyridine in reagent tube 1 is toxic. When handling it, wear disposable gloves, a mask and goggles to avoid skin contact, inhalation or ingestion. If contact occurs, immediately rinse the contact area with plenty of water and seek medical attention if necessary.

[0202] 3. FeCl3 mother liquor is easily hydrolyzed, so it needs to be prepared quickly to avoid prolonged contact with air. It should be used immediately after dilution to prevent precipitation.

[0203] 4. When constructing an iron environment, reagents must be added after LB medium has cooled to room temperature. Adding them at high temperatures is strictly prohibited to avoid reagent decomposition and inactivation.

[0204] 5. All data records during the experiment must be true and complete. Qualified iron environment verification data are an important basis for the validity of the experimental results.

[0205] Example 3: Constructing an iron environment using a kit and analyzing the function of the TonB gene.

[0206] This embodiment uses Acinetobacter baumannii ATCC 17978 wild-type strain, ATCC17978△tonB1 strain, and ATCC17978△tonB2 strain as research subjects to verify the practicality and effectiveness of the kit.

[0207] Experimental materials

[0208] Strain: Acinetobacter baumannii ATCC 17978 wild-type strain (WT, normal iron uptake function);

[0209] Acinetobacter baumannii ATCC 17978 △tonB1 gene knockout strain and ATCC17978 △tonB2 (iron uptake defect, gene deletion strain constructed by plasmid homologous recombination technology and verified by gene sequencing and PCR).

[0210] Reagent kit: The dedicated reagent kit prepared according to this invention;

[0211] Basic reagents and instruments: LB liquid culture medium, sterile physiological saline, McFarland turbidimeter, ELISA reader, constant temperature shaking incubator, sterile 96-well plate, sterile centrifuge tubes (50 mL).

[0212] Experimental methods

[0213] Preparation of basic LB medium: Prepare LB liquid medium, autoclave and cool to room temperature, then dispense into sterile centrifuge tubes, 40 mL per tube.

[0214] Construction of gradient iron environment (follow the kit instructions):

[0215] Normal iron group: 40 mL sterile LB medium, without adding any reagents;

[0216] Iron-deficient group: Add the stock solution from reagent tube 1 to 40 mL of LB medium and dilute to a final concentration of 100 μmol / L, 300 μmol / L, and 500 μmol / L, respectively;

[0217] Iron-rich group: Add the stock solution from reagent tube 2 to 40 mL of LB medium and dilute to final concentrations of 20 μmol / L, 100 μmol / L, and 300 μmol / L, respectively.

[0218] Preparation and inoculation of bacterial suspensions: Three bacterial suspensions (ATCC17978, ATCC17978△tonB1, and ATCC17978△tonB2) at a concentration of 0.5 Mcf were prepared and diluted to 40 mL at a 1:50 volume ratio with LB liquid medium. Unadulterated LB liquid medium tubes were used as blank controls. Each culture system was performed in triplicate, and cultured at 37°C and 180 rpm with shaking. The optical density (OD) at 600 nm was measured using a spectrophotometer. 600 The bacterial culture system was used. Measurements were taken every 0.5 hours for the first 0-6 hours using a microplate reader, and then every hour for the next 6-24 hours. OD values ​​were recorded. 600 Results were obtained and bacterial growth curves were plotted.

[0219] Culture and detection: Incubate at 35℃ and 180 rpm for 24 h with shaking, and dynamically measure OD according to the kit requirements. 600 Values ​​are used to plot growth curves.

[0220] Experimental results

[0221] TonB gene functional analysis: The growth inhibition rate of ATCC17978△tonB1 strain was significantly higher than that of wild-type strain in all iron-deficient groups (P<0.01), and growth could not be restored in the 300 μmol / L FeCl3 iron-rich group; the growth phenotype of ATCC17978△tonB2 strain was not significantly different from that of wild-type strain (P>0.05), confirming that TonB1 protein is the core iron uptake regulatory protein of Acinetobacter baumannii under iron-deficient environment, which is consistent with the previous research conclusions.

[0222] Results Analysis

[0223] The kit of this invention can rapidly and stably construct a gradient iron environment adapted to Acinetobacter baumannii, with good experimental reproducibility. The kit allows for intuitive analysis of functional differences in the TonB gene without the need for cumbersome molecular biology verification, greatly improving research efficiency and fully demonstrating the practicality and scientific validity of the kit.

[0224] In summary, this invention, relying on a specific iron concentration threshold tailored to Acinetobacter baumannii and a standardized reagent combination, constructs iron-deficient, normal iron, and iron-rich growth environments that are both precise and controllable, as well as stable and reproducible. It allows for the standardized construction of gradient iron environments as needed, effectively solving the technical pain points of traditional methods, such as the difficulty in quantifying iron content in culture media, the complexity of iron chemical forms, and the inability to standardize experimental conditions. This establishes a unified and reproducible experimental foundation for iron-related research in Acinetobacter baumannii. Furthermore, through the synergistic application of iron chelation and exogenous iron supplementation, this invention can specifically reverse the growth-inhibiting effect of iron chelators on bacteria, accurately verifying the iron-dependent specificity of the inhibition mechanism. This provides an intuitive and reliable basis for distinguishing between iron uptake defects and non-specific growth abnormalities in Acinetobacter baumannii, significantly simplifying the cumbersome mechanism verification process in traditional research and improving research efficiency.

[0225] This invention provides a highly efficient and dedicated experimental tool for studying the iron metabolism mechanism of Acinetobacter baumannii. It can be directly applied to the functional analysis of iron uptake-related genes and proteins, and the exploration of iron homeostasis regulatory pathways. Simultaneously, it establishes a standardized experimental platform for cutting-edge research on iron toxicity tolerance and environmental adaptation of drug-resistant bacteria, assisting researchers in deeply understanding the pathogenic mechanisms and environmental adaptation mechanisms of Acinetobacter baumannii. The iron environment model constructed in this invention has significant practical reference value for the development of clinical antibacterial strategies. The iron-deficient environment model can accurately simulate the iron-limited microenvironment at the site of infection in the host, providing a realistic experimental scenario for studying the mechanism of action and dosage screening of iron-deprived antibacterial drugs. The iron-rich environment model can be used to explore the toxicity regulation mechanism related to iron metabolism, providing new ideas and experimental support for the clinical control of multidrug-resistant Acinetobacter baumannii infection and the development of novel antibacterial strategies.

[0226] The present invention has been described in detail above with reference to exemplary embodiments only. Those skilled in the art should understand that, without departing from the scope of protection defined by the claims of the present invention, partial adjustments, equivalent substitutions, or detailed optimizations can be made to the technical solutions of the present invention, and all such modifications should fall within the scope of protection of the present invention. Therefore, the accompanying drawings and specific embodiments of the present invention are merely illustrative examples and are not intended to limit the scope of protection of the present invention.

Claims

1. A kit for constructing an iron-dependent growth environment for Acinetobacter baumannii, characterized in that, The core consists of reagent tube 1, reagent tube 2 and accompanying instruction manual, but does not include basic LB medium. The reagent tube 1 contains a sterile stock solution of 2,2′-bipyridine with a concentration of 50 mmol / L. It is sterilized by filtration through a 0.22 μm sterile filter membrane, stored at 4°C in the dark, and has a shelf life of 7 days. The volume is 10 mL / tube. The reagent tube 2 contains sterile FeCl3 stock solution with a concentration of 2000 μmol / L. It should be prepared and used immediately, and the volume is 10 mL / tube. The accompanying instruction manual specifies the dilution ratio of the mother liquor, the core concentration threshold for constructing the iron environment, the operating steps, the quality verification standards, the bacterial culture conditions, and the precautions for reagent storage. The core concentration threshold for constructing the iron environment is 100-500 μmol / L 2,2′-bipyridine for iron deficiency and 20-300 μmol / L FeCl3 for iron enrichment.

2. The reagent kit according to claim 1, characterized in that, The sterile stock solution of 2,2′-bipyridine in reagent tube 1 is used for specific chelation of intracellular and extracellular Fe. 2+ The reagent provides core reagents for constructing a 100-500 μmol / L gradient iron-deficient environment for Acinetobacter baumannii; the FeCl3 sterile mother liquor in reagent tube 2 has physicochemical properties that match the state of iron in the human host environment, and is used to provide core reagents for constructing a 20-300 μmol / L gradient iron-rich environment for Acinetobacter baumannii.

3. A method for constructing an iron-dependent growth environment for Acinetobacter baumannii using the kit described in any one of claims 1-2, characterized in that, Includes the following steps: 1) Basic preparation: Autoclave LB liquid culture medium according to routine procedures and cool it to room temperature for later use; bring reagent tube 1 and reagent tube 2 from the kit, which have been refrigerated at 4°C, to room temperature for later use. 2) Iron environment construction: Normal iron environment: directly use cooled sterile LB liquid culture medium as the bacterial culture system; Iron-deficient environment: Add the sterile stock solution of 2,2′-bipyridine from reagent tube 1 to the cooled sterile LB liquid medium, dilute to a final concentration of 100-500 μmol / L, mix thoroughly and use as a bacterial culture system; Iron-rich environment: Add the sterile FeCl3 stock solution from reagent tube 2 to the cooled sterile LB liquid medium, dilute to a final concentration of 20-300 μmol / L, mix thoroughly and use as a bacterial culture system; 3) Bacterial inoculation and culture: The Acinetobacter baumannii suspension was calibrated to a concentration of 1.0 McF and inoculated into each iron environment culture system prepared in step 2) at a volume ratio of 1:

50. The culture was carried out under constant temperature shaking at 35-37℃ and 180 rpm, while 5% CO2 environment was used to assist in the recovery of the strain. 4) Iron environment detection and stability verification: Dynamically measure the OD of bacterial cultures in different iron environment culture systems at different culture time points. 600 The colony-forming units and the amount of precipitate after centrifugation were recorded simultaneously. The validation standard for the successful construction of an iron-deficient environment was that the growth inhibition rate of Acinetobacter baumannii in the iron-deficient environment was ≥80%, and the difference between groups was P<0.05 by one-way ANOVA.

4. The application method according to claim 3, characterized in that, In step 2), the final concentration of 100-500 μmol / L 2,2′-bipyridine is the specific iron-deficient concentration range adapted to Acinetobacter baumannii, achieving gradient iron inhibition of Acinetobacter baumannii growth; the final concentration of 20-300 μmol / L FeCl3 is the specific iron-rich concentration range adapted to Acinetobacter baumannii, matching the iron tolerance and physiological growth requirements of Acinetobacter baumannii, and achieving refined analysis of the iron-dependent growth of the strain.

5. The application method according to claim 3, characterized in that, In step 4), the OD of the bacterial culture is dynamically measured. 600 The specific method for determining the values ​​is as follows: measurements are taken every 0.5 hours during the 0-6 hour incubation period and every 1 hour during the 6-24 hour incubation period. Based on the measurement results, growth curves of Acinetobacter baumannii are plotted to quantify the differences in growth of Acinetobacter baumannii under different iron environments.

6. The application method according to claim 3, characterized in that, In step 3), the Acinetobacter baumannii includes wild-type Acinetobacter baumannii, the Acinetobacter baumannii ATCC 17978 standard strain, and the genetically modified Acinetobacter baumannii mutant strain with TonB gene knockout.

7. The application method according to claim 3, characterized in that, In step 2), each iron environment culture system was prepared with three or more biological replicates. In step 3), bacterial inoculation and culture, and in step 4), iron environment detection and stability verification were carried out simultaneously in the corresponding biological replicate system to ensure the reliability and reproducibility of the experimental results.

8. The application of the kit according to any one of claims 1-2 or the application method according to any one of claims 3-7 in basic research on iron metabolism in Acinetobacter baumannii, characterized in that, The application is at least one of the following: Functional analysis of iron uptake-related proteins or regulatory systems in Acinetobacter baumannii; Research and validation of iron deprivation-related antibacterial strategies for Acinetobacter baumannii; Research on the regulatory mechanism of iron metabolism-related virulence in Acinetobacter baumannii; Construction and study of in vivo and in vitro infection models of iron environment-related Acinetobacter baumannii; Screening and validation of iron-deficient strains of Acinetobacter baumannii.

9. The application according to claim 8, characterized in that, The iron-related proteins or regulatory systems mentioned are TonB1 and TonB2-dependent iron transport systems.

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