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CN-122012272-A - Pseudomonas aeruginosa of mucus type and culture method for promoting growth by using IgG

CN122012272ACN 122012272 ACN122012272 ACN 122012272ACN-122012272-A

Abstract

The application relates to the field of microorganism culture, discloses a culture method for promoting the growth of a mucous pseudomonas aeruginosa strain by using IgG, and belongs to the technical field of microorganism culture. The method aims at the technical problems that the growth delay period of the mucous pseudomonas aeruginosa with the genotype of ST270 is overlong and the proliferation is extremely slow in a conventional LB or TSB culture medium, and a specific improvement system is constructed. The preparation method comprises the steps of preparing a basic liquid culture medium, sterilizing, aseptically adding non-heat-inactivated immunoglobulin G (IgG) to a final concentration of 5.0 mu G/mL-5.0 mg/mL, and performing shake culture at 37 ℃ after inoculating a target strain. The application utilizes the modification effect of IgG molecules on the surface mucus layer of the bacteria, effectively overcomes the physical steric hindrance or metabolic inhibition caused by extracellular matrixes, obviously shortens the growth adaptation period, greatly improves the biomass of the bacteria, and successfully realizes the rapid amplification and phenotype recovery of the rare strain difficult to culture.

Inventors

  • WANG XIAOQIONG
  • SU DONGMEI
  • WANG YONGSHENG
  • ZHOU FEI
  • ZHANG YAO
  • HUANG YI
  • YUAN SHIHUI
  • Xie Yanghu
  • LIU XIAOYUE
  • XU CHUCHU
  • YIN XIAONA
  • WANG XI

Assignees

  • 合肥市第二人民医院

Dates

Publication Date
20260512
Application Date
20260113

Claims (10)

  1. 1. A culture method for promoting the growth of a mucous pseudomonas aeruginosa strain by utilizing IgG, which is characterized by comprising the following steps: step S1, preparing a basic liquid culture medium; s2, adding immunoglobulin G (IgG) into the basic liquid culture medium to prepare a liquid culture medium containing the IgG; Step S3, inoculating the mucus type pseudomonas aeruginosa into the liquid culture medium containing the IgG; And S4, shake culturing is carried out on the culture medium inoculated in the step S3 at a proper temperature.
  2. 2. The method for promoting the growth of a strain of Pseudomonas aeruginosa of the mucous type according to claim 1, characterized in that the genotype of the Pseudomonas aeruginosa of the mucous type is ST270 type and the strain exhibits a growth retardation phenotype in conventional LB liquid medium without immunoglobulin G (IgG) added.
  3. 3. The method according to claim 1, wherein in step S2, the final concentration of immunoglobulin G (IgG) in the IgG-containing liquid medium is 5.0 μg/mL to 5.0 mg/mL.
  4. 4. The method for promoting the growth of a mucous-type Pseudomonas aeruginosa strain by using IgG according to claim 3, wherein the final concentration of the immunoglobulin G (IgG) is preferably 5.0 μg/mL-20.0 μg/mL.
  5. 5. The method according to claim 1, wherein in step S1, the basic liquid medium is selected from LB broth, trypticase Soy Broth (TSB) and Muller-Hinton broth (MHB).
  6. 6. The method of claim 1, wherein in step S2, the immunoglobulin G (IgG) is derived from mammalian serum, and the source comprises mouse serum, human serum, fetal bovine serum, sheep serum, or guinea pig serum.
  7. 7. The method according to claim 1, wherein the immunoglobulin G (IgG) is a non-heat-inactivated whole molecule that retains a natural spatial conformation, or a biologically active F (ab') 2 fragment or Fc fragment.
  8. 8. The method for promoting the growth of a mucous type Pseudomonas aeruginosa strain by using IgG according to claim 1, wherein the specific operation of step S2 comprises: Sterilizing the basic liquid culture medium by high-pressure steam and cooling to room temperature; Dissolving the immunoglobulin G (IgG) in a sterile buffer solution and performing filtration sterilization by a filter membrane to prepare a stock solution; The stock solution is added to the cooled basal liquid medium under sterile conditions.
  9. 9. The method according to claim 1, wherein in step S3, the initial bacterial liquid concentration of the inoculation is controlled to be OD 600 to 0.05.
  10. 10. The method for promoting the growth of a mucous-type Pseudomonas aeruginosa strain by using IgG according to claim 1, wherein in the step S4, the shake culture is carried out at a culture temperature of 37+ -1deg.C and a shaking culture rotation speed of 180-220 rpm for 12-48 hours.

Description

Pseudomonas aeruginosa of mucus type and culture method for promoting growth by using IgG Technical Field The invention relates to the technical field of microorganism culture, in particular to a mucous pseudomonas aeruginosa strain and a culture method for promoting growth by using IgG. Background Pseudomonas aeruginosa is a clinically common gram-negative pathogenic bacterium, widely existing in hospital environment, and is one of the main pathogens causing hospital-acquired pneumonia, blood infection and wound infection. Among the numerous popular clones, ST270 type pseudomonas aeruginosa is a high risk clone, has a higher detection rate in intensive care units in east asia in recent years, and often exhibits multiple drug resistance or broad drug resistance characteristics, which brings serious challenges to clinical anti-infection treatment. During chronic respiratory tract infections, pseudomonas aeruginosa often undergoes adaptive mutations that transform into a mucous phenotype. Such strains excessively synthesize and secrete alginate, and form a thick extracellular polysaccharide mucus layer on the surface of the strain. This mucus layer can help bacteria resist clearance of the immune system and killing of antibiotics in living hosts, but in vitro scientific research, this phenotype presents a significant hurdle to the culture and analysis of strains. Because the extracellular alginate layer has higher viscosity and negative charge density, the extracellular alginate layer forms a physical barrier around the thalli, and seriously hinders the diffusion and permeation of nutrient substances in the culture medium into the thalli. When cultured in vitro using conventional laboratory general culture media such as LB broth, the myxotype ST270 strain generally exhibits an extremely long growth lag phase, often requiring 24 hours or more to initiate proliferation, and the biomass eventually achieved is much lower than that of the non-myxotype strain. In addition, the bacterial strain is easy to agglomerate and subside under the conventional culture condition, so that the uniformity of a culture system is poor, and a bacterial sample with consistent growth state is difficult to obtain. Current prior art has focused mainly on improving bacterial growth by increasing nutrient concentration or optimizing carbon-nitrogen source ratios, or on attempting to degrade mucus layers by adding specific enzymes. However, simply increasing the nutrient concentration cannot effectively solve the problem of limited absorption caused by physical barrier of the mucus layer, and too high osmotic pressure may inhibit bacterial metabolism, and enzyme treatment can remove mucus but change the original surface physiological state of bacteria, which is not beneficial to subsequent authenticity research on pathogenic mechanism or drug resistance mechanism. Therefore, a special culture method capable of effectively shortening the lag phase and improving biomass accumulation by adjusting the microenvironment and the surface state on the premise of keeping the mucous type characteristics of the strain is lacking, which limits the in-depth research and monitoring of the high-risk drug-resistant strain. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a mucous type pseudomonas aeruginosa and a culture method for promoting growth by using IgG, which solve the problems of overlong growth lag phase, slow proliferation speed and low final biomass of specific mucous type pseudomonas aeruginosa in the prior art under the conventional in-vitro culture condition. In order to achieve the above purpose, the invention is realized by the following technical scheme: a culture method for promoting growth of a mucous pseudomonas aeruginosa by using IgG comprises the following steps of S1, S2, adding immunoglobulin G (IgG) into a basic liquid culture medium to prepare the liquid culture medium containing the IgG, S3, inoculating the mucous pseudomonas aeruginosa into the liquid culture medium containing the IgG, and S4, carrying out shake culture on the culture medium inoculated in the S3 at a proper temperature. By adopting the technical scheme, the invention utilizes the immunoglobulin G (IgG) as a key growth promoting factor, and establishes an improved culture system aiming at mucous pseudomonas aeruginosa. The mechanism of action is that such mucinous strains are typically covered with a massive extracellular polysaccharide or mucinous matrix on the cell surface, which constitutes a physical barrier or initiates metabolic inhibition signals in a conventional nutritional environment, resulting in the strain entering an extended state of growth arrest. The added immunoglobulin G (IgG) can be specifically combined with or adsorbed on mucus components on the surface of the thalli, the physical and chemical properties of extracellular matrixes of the thalli are changed by the surface modification action of the bio