Mucus type pseudomonas aeruginosa and culture method for promoting growth by using IgG (Immunoglobulin G)

Abstract

The invention relates to the field of microbial culture, discloses a culture method for promoting growth of mucus type pseudomonas aeruginosa by using IgG, and belongs to the technical field of microbial culture. According to the method, a specific improved system is constructed aiming at the technical problems that mucus type pseudomonas aeruginosa with the genotype of ST270 is too long in growth retardation period and extremely slow in proliferation in a conventional LB or TSB culture medium. The method comprises the following specific steps: preparing a basic liquid culture medium and sterilizing; non-thermally inactivated immunoglobulin G (IgG) is added in a sterile manner, so that the final concentration of the non-thermally inactivated immunoglobulin G (IgG) is in a range of 5.0 mu g / mL to 5.0 mg / mL; and inoculating a target strain and then carrying out shake culture at 37 DEG C. By utilizing the modification effect of IgG molecules on the mucus layer on the surface of the thallus, physical steric hindrance or metabolic inhibition caused by an extracellular matrix is effectively overcome, the growth adaptation period is remarkably shortened, the biomass of the thallus is greatly improved, and rapid amplification and phenotype recovery of the rare strains difficult to culture are successfully realized.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, specifically to a strain of myxobolus aeruginosa and its culture method using IgG to promote growth. Background Technology

[0002] Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen found in clinical settings. It is widely present in hospital environments and is one of the leading causes of hospital-acquired pneumonia, bloodstream infections, and wound infections. Among numerous circulating clones, ST270 Pseudomonas aeruginosa is considered a high-risk clone, with a high detection rate in intensive care units in East Asia in recent years. It often exhibits multidrug resistance or extensive drug resistance, posing a significant challenge to clinical anti-infective therapy.

[0003] During chronic respiratory infections, *Pseudomonas aeruginosa* often undergoes adaptive mutations, transforming into a mucinous phenotype. These strains oversynthesize and secrete alginate, forming a thick layer of extracellular polysaccharide mucus on the bacterial surface. While this mucus layer helps the bacteria resist clearance by the immune system and antibiotics in the living host, this phenotype presents significant challenges to the culture and analysis of these strains in in vitro studies.

[0004] Due to the high viscosity and negative charge density of the extracellular alginate layer, it forms a physical barrier around the bacterial cell, severely hindering the diffusion and penetration of nutrients from the culture medium into the cell. When cultured in vitro using common laboratory media such as LB broth, the myxotropic ST270 strain typically exhibits an extremely long growth lag phase, often requiring 24 hours or even longer to initiate proliferation, and the final biomass is far lower than that of the non-myxotropic strain. Furthermore, under conventional culture conditions, this type of strain is prone to aggregation and sedimentation, resulting in poor homogeneity of the culture system and making it difficult to obtain bacterial samples with consistent growth states.

[0005] Current technologies primarily focus on improving bacterial growth by increasing nutrient concentration or optimizing the carbon-nitrogen source ratio, or by adding specific enzymes to attempt to degrade the slime layer. However, simply increasing nutrient concentration cannot effectively address the absorption limitations caused by the physical barrier of the slime layer, and excessively high osmotic pressure may actually inhibit bacterial metabolism. While enzyme treatment can remove the slime, it alters the original surface physiological state of the bacteria, hindering subsequent research into their pathogenic or drug resistance mechanisms. Therefore, there is currently a lack of a specialized culture method that can effectively shorten the lag phase and increase biomass accumulation by adjusting the microenvironment and surface state while preserving the slime-like characteristics of the strain. This limits in-depth research and monitoring of these high-risk drug-resistant strains. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain of myxobolus aeruginosa and its culture method for promoting growth using IgG, which solves the problems of excessively long growth lag phase, slow proliferation rate, and low final biomass of certain myxobolus aeruginosa under conventional in vitro culture conditions.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for promoting the growth of a strain of *Pseudomonas aeruginosa* using IgG includes the following steps: Step S1: Prepare a basic liquid culture medium; Step S2: Add immunoglobulin G (IgG) to the basic liquid culture medium to obtain a liquid culture medium containing IgG; Step S3: Inoculate *Pseudomonas aeruginosa* into the liquid culture medium containing IgG; Step S4: Shake the culture medium after inoculation in Step S3 at a suitable temperature.

[0008] By employing the above-mentioned technical solution, this invention utilizes immunoglobulin G (IgG) as a key growth-promoting factor to establish an improved culture system for myxotropic Pseudomonas aeruginosa. Its mechanism of action lies in the fact that these myxotropic strains typically have a thick layer of extracellular polysaccharides or mucus matrix covering their cell surface. This constitutes a physical barrier or triggers metabolic inhibition signals in a conventional nutrient environment, leading to a prolonged growth arrest state. The added immunoglobulin G (IgG) can specifically bind to or adsorb onto the mucus components on the bacterial surface. This surface modification of the biomolecule alters the physicochemical properties of the bacterial extracellular matrix, effectively removing the steric hindrance effect that restricts the exchange of substances between the bacteria and the external environment, or activating the metabolic activity of the strain through surface binding. Combined with the dissolved oxygen and convection environment provided by shaking culture, this method can significantly shorten the adaptation period (lag phase) of the strain, prompting it to rapidly enter the logarithmic growth phase, thereby obtaining a high-density bacterial biomass in a short time, solving the technical difficulties of culturing and enriching this type of rare strain.

[0009] Preferably, the genotype of *Pseudomonas aeruginosa* is ST270, and this strain exhibits a slow-growth phenotype in conventional LB liquid medium without the addition of immunoglobulin G (IgG).

[0010] By adopting the above technical solution, it is clear that the applicable object of the present invention is the ST270 genotype strain, which is of great clinical significance but extremely difficult to culture, and the precise enrichment of such specific difficult-to-culture microorganisms is achieved.

[0011] Preferably, in step S2, the final concentration of immunoglobulin G (IgG) in the liquid culture medium containing IgG is 5.0 μg / mL to 5.0 mg / mL; more preferably, it is 5.0 μg / mL to 20.0 μg / mL.

[0012] By employing the above technical solution, the effective concentration window for IgG to exert its growth-promoting effect was established. Experimental data show that within this concentration range, IgG can provide sufficient surface binding sites to induce growth, while avoiding the cost waste or potential non-specific interference caused by excessively high concentrations, thus achieving a balance between efficacy and cost.

[0013] Preferably, in step S1, the basic liquid culture medium is selected from LB broth medium, tryptic soy broth medium (TSB), or Muller-Hinton broth medium (MHB).

[0014] By adopting the above technical solution, it has been demonstrated that this growth-promoting strategy does not rely on a single basic nutrient formula, but rather endows conventional general culture media (such as LB, TSB, etc.) with special growth-promoting functions through the specific addition of IgG, which has good universality and promotion value.

[0015] Preferably, in step S2, immunoglobulin G (IgG) is derived from mammalian serum, including mouse serum, human serum, fetal bovine serum, sheep serum, or guinea pig serum.

[0016] By adopting the above technical solution, it was confirmed that the growth-promoting mechanism is not limited to IgG from a specific species, but is based on the universal structural characteristics of IgG molecules, thereby greatly expanding the range of experimental reagents and lowering the implementation threshold.

[0017] Preferably, immunoglobulin G (IgG) is a non-thermally inactivated intact molecule that retains its native spatial conformation, or is a biologically active F(ab')2 fragment or Fc fragment.

[0018] By employing the above technical solution, it was clarified that the growth-promoting effect depends on the intact spatial structure or specific active domains of the IgG molecule, rather than its metabolic utilization as a common nitrogen source nutrient. The non-thermal inactivation treatment method preserves the biological activity of IgG in specifically binding to the bacterial surface mucus layer, which is a key element for achieving rapid proliferation.

[0019] Preferably, step S2 includes the following specific operations: first, autoclaving the basic liquid culture medium and cooling it to room temperature; dissolving immunoglobulin G (IgG) in sterile buffer and filtering it through a filter membrane to prepare a stock solution; and adding the stock solution to the cooled basic liquid culture medium under sterile conditions.

[0020] By adopting the above technical solution and using a stepwise preparation process of "sterilizing the base solution first and then adding IgG under aseptic conditions", the denaturation and inactivation of heat-sensitive protein IgG caused by the high-temperature and high-pressure sterilization process is effectively avoided, ensuring the bioactivity and functional integrity of IgG in the final culture system.

[0021] Preferably, in step S3, the initial bacterial concentration of the inoculated culture is controlled to be an optical density value (OD600) of 0.05 to 0.1.

[0022] By adopting the above technical solution, the inoculum size was standardized, which ensured that the initial bacterial population had sufficient population density to initiate quorum sensing and proliferation, while avoiding interference from excessively high inoculum sizes on growth curve measurements, thus ensuring the reproducibility of culture results.

[0023] Preferably, in step S4, the conditions for shaking culture are: culture temperature 37±1℃, shaker speed 180 to 220 rpm, and culture time 12 to 48 hours.

[0024] By employing the above technical solution, a physical culture environment adapted to IgG induction was provided. 37℃ maintained optimal activity of the bacterial enzyme system, while high-speed shaking at 180-220 rpm provided ample dissolved oxygen and enhanced mass transfer efficiency. This, combined with the surface interaction of IgG, supported the strain to undergo rapid aerobic metabolism and division after inhibition was relieved.

[0025] This invention provides a strain of myxotropic Pseudomonas aeruginosa and a method for promoting its growth using IgG. It has the following beneficial effects: 1. This invention significantly improves the biomass accumulation of *Pseudomonas aeruginosa* by establishing an IgG culture system. In this system, IgG, with its native spatial conformation, effectively reduces the physical steric hindrance effect of the thick mucus layer on nutrient uptake. This eliminates growth inhibition of strain ST270, resulting in a significantly higher final biomass compared to conventional culture conditions, thus solving the technical challenge of obtaining high-density samples of this type of strain in vitro.

[0026] 2. The method provided by this invention exhibits good process stability and subsequent application value. Experimental data show that the culture system can maintain a high bacterial density without drastic cell death within 24 to 48 hours, indicating that the method maintains the physiological activity of the strain while promoting proliferation. Furthermore, this system has a certain degree of compatibility with IgG species, providing sufficient and homogeneous bacterial samples for subsequent transcriptome sequencing, drug sensitivity testing, and pathogenic mechanism studies. Attached Figure Description

[0027] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Please see the appendix Figure 1 This invention provides a strain of myxobolus aeruginosa and a method for culturing it using IgG to promote its growth, comprising the following: Preparation example: Preparation Example 1: Mouse-derived IgG modified LB medium (preferred formulation) This preparation example provides a modified culture medium for promoting the growth of *Pseudomonas aeruginosa* ST270, comprising the following steps: Preparation of basal culture medium: According to the standard LB broth formula, weigh 10.0g of tryptone, 5.0g of yeast extract, and 10.0g of sodium chloride, and dissolve them in 1L of distilled water. After stirring and dissolving, adjust the pH to 7.0-7.2, autoclave at 121℃ for 20 minutes, and cool to room temperature (approximately 25℃). IgG addition: Unheat-inactivated mouse serum-derived IgG lyophilized powder was dissolved in sterile PBS buffer and filtered through a 0.22 μm filter to prepare a stock solution. Under sterile conditions, the IgG stock solution was added to the cooled basal culture medium. Mixing: Gently mix until the final concentration of IgG in the finished culture medium is 5.0 μg / mL.

[0030] Preparation Example 2: Human IgG Modified LB Medium (Alternative Source) This preparation example verifies the applicability of immunoglobulins from different species, and the steps are as follows: Preparation of basal culture medium: Same as in Preparation Example 1; IgG addition: Replace "mouse serum-derived IgG" in Preparation Example 1 with "human serum-derived IgG (polyclonal)". Add to the basal culture medium under aseptic conditions; Mixing: Gently mix until the final concentration of IgG in the finished culture medium is 5.0 μg / mL.

[0031] Preparation Example 3: Mouse-derived IgG modified TSB medium (alternative matrix) This preparation example verifies the applicability of different basal culture media, and the steps are as follows: Preparation of basal culture medium: Weigh 30.0 g of tryptic soy broth (TSB) powder (containing tryptic acid, soy papain digest, sodium chloride, dipotassium hydrogen phosphate, and glucose) and dissolve it in 1 L of distilled water. Stir to dissolve, adjust the pH to 7.3 ± 0.2, sterilize at 121 °C for 15 minutes, and cool. IgG addition: Same as in Preparation Example 1, but with the addition of mouse-derived IgG; Mixing: Gently mix until the final concentration of IgG in the finished culture medium is 5.0 μg / mL.

[0032] Example: Example 1: Rapid culture method for ST270 Pseudomonas aeruginosa (preferred scheme) This embodiment uses the culture medium provided in Preparation Example 1 to culture the difficult-to-culture strain, including the following steps: Strain source: ST270 type mucoid Pseudomonas aeruginosa isolated from bronchoalveolar lavage fluid of patients with bronchiectasis and confirmed by genome sequencing; Inoculation: Pick a single ST270 colony, adjust the initial bacterial suspension concentration, and inoculate it at a ratio of 1:100 into a culture container containing Preparation Example 1 (LB + 5.0 μg / mL mouse IgG). The initial OD is approximately 0.05. Culture conditions: Place the culture container in a constant temperature shaker at 37℃ and shake it at 200 rpm. Results observation: Growth was continuously monitored for up to 48 hours.

[0033] Example 2: Culture method using human IgG (validation of alternative sources) This embodiment aims to verify the growth-promoting effect of human IgG on ST270 strain, including the following steps: Inoculation: ST270 strain was inoculated into a culture vessel containing Preparation Example 2 (LB + 5.0 μg / mL human IgG), with the same inoculation amount as in Example 1; Culture conditions: Same as in Example 1 (37℃, 200 rpm); Results observation: Monitor growth curves and final colony morphology.

[0034] Example 3: Culture method using TSB matrix (alternative matrix validation) This embodiment aims to verify the universality of the method in different nutrient substrates, and includes the following steps: Inoculation: ST270 strain was inoculated into a culture container containing Preparation Example 3 (TSB + 5.0 μg / mL mouse IgG), with the same inoculation amount as in Example 1; Culture conditions: Same as in Example 1 (37℃, 200 rpm); Results observation: Monitor growth.

[0035] Comparative example: Comparative Example 1 (Blank Control): Plain LB liquid medium without any additional IgG was used. Except for the absence of IgG, the strain source, inoculum size, culture temperature (37°C), and shaking speed (200 rpm) were exactly the same as in Example 1.

[0036] Comparative Example 2 (IgG inactivation control): Based on Preparation Example 1, denatured IgG, which had been inactivated by heating in a 56°C water bath for 30 minutes, was used instead of active IgG, with the final concentration remaining at 5.0 μg / mL. The remaining procedures were the same as in Example 1. This comparative example was used to verify whether the growth-promoting effect depended on the active structure of IgG.

[0037] Experimental example: Experiment Example 1: Growth Kinetics and Biomass Detection 1. Experimental Procedure Example 1 (IgG group) and Comparative Example 1 (blank LB group) were cultured under the same conditions. Samples were taken at 0h, 12h, 24h, 36h and 48h of culture to measure the OD value and observe the turbidity and growth status of the bacterial culture.

[0038] 2. Experimental Results Table 1 Comparison of ST270 strain growth under different culture systems 3. Results Analysis Shortened lag period: Comparative Example 1 showed an OD of only 0.15 after 24 hours of culture in conventional LB, exhibiting extremely long growth lag (typically requiring 3-4 days to observe significant growth). In contrast, Example 1 showed significant growth acceleration in approximately 12 hours after the addition of 5 μg / mL IgG, significantly shortening the time required to obtain sufficient sample.

[0039] Biomass enhancement: At the 24-hour time point, the bacterial concentration of Example 1 was more than 10 times that of Comparative Example 1 (1.82 vs 0.15), effectively solving the technical problem of "too low bacterial count to conduct transcriptome or drug resistance studies" for this type of strain.

[0040] Specificity verification: Both Example 2 (human IgG) and Example 3 (TSB matrix) achieved similar growth-promoting effects as Example 1, demonstrating the good universality of the method. The results of Comparative Example 2 (inactivated IgG) were close to those of Comparative Example 1, indicating that the growth-promoting effect of IgG depends on its natural spatial conformation or biological activity, rather than being utilized simply as a nutrient source (nitrogen source).

[0041] In summary, this invention successfully improved the in vitro culture characteristics of ST270 myxomorphic Pseudomonas aeruginosa by adding a trace amount of IgG to the culture medium, achieving rapid proliferation and phenotypic recovery.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for culturing a strain of myxotropic Pseudomonas aeruginosa using IgG to promote its growth, characterized in that, Includes the following steps: Step S1: Prepare the basic liquid culture medium; Step S2: Add immunoglobulin G (IgG) to the basic liquid culture medium to prepare a liquid culture medium containing IgG; Step S3: Inoculate the myxotropic Pseudomonas aeruginosa into the liquid culture medium containing IgG; Step S4: Shake culture the culture medium after inoculation in step S3 at a suitable temperature.

2. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, The myxotrophic Pseudomonas aeruginosa strain has the genotype ST270, and this strain exhibits a slow-growth phenotype in conventional LB liquid medium without the addition of immunoglobulin G (IgG).

3. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, In step S2, the final concentration of immunoglobulin G (IgG) in the liquid culture medium containing IgG is 5.0 μg / mL to 5.0 mg / mL.

4. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 3, characterized in that, The final concentration of immunoglobulin G (IgG) is preferably 5.0 μg / mL to 20.0 μg / mL.

5. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, In step S1, the basic liquid culture medium is selected from LB broth medium, tryptic soy broth medium (TSB), or Muller-Hinton broth medium (MHB).

6. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, In step S2, the immunoglobulin G (IgG) is derived from mammalian serum, including mouse serum, human serum, fetal bovine serum, sheep serum, or guinea pig serum.

7. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, The immunoglobulin G (IgG) is a non-thermally inactivated intact molecule that retains its native spatial conformation, or a biologically active F(ab')2 fragment or Fc fragment.

8. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, The specific operations of step S2 include: First, autoclave the basic liquid culture medium and cool it to room temperature; The immunoglobulin G (IgG) was dissolved in a sterile buffer solution and filtered through a filter membrane to remove bacteria, thus preparing a stock solution. Under aseptic conditions, the stock solution was added to the cooled basal liquid culture medium.

9. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, In step S3, the initial bacterial concentration of the inoculated culture is controlled at OD. 600 Values ​​range from 0.05 to 0.

1.

10. The method for culturing a strain of *Pseudomonas aeruginosa* using IgG to promote its growth according to claim 1, characterized in that, In step S4, the conditions for the shaking culture are: culture temperature 37±1℃, shaking speed 180-220 rpm, and culture time 12-48 hours.

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