Sphingomonas selective culture medium and preparation method thereof
Patent Information
- Application Number
- CN202611552439.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-10-09
- Publication Date
- 2026-11-03
AI Technical Summary
Existing sphingosomal monoclonal isolation techniques suffer from insufficient selectivity of single antibiotics, limited inhibitory effects on non-target bacteria, high false positive and false negative rates due to morphological screening, and complex and costly molecular identification processes, making it difficult to meet the needs of large-scale screening.
A selective culture medium containing streptomycin and polymyxin B was used, with a final concentration of streptomycin of 200 μg/mL and a final concentration of polymyxin B of 64 μg/mL. Through synergistic target complementarity, highly efficient and selective isolation of Sphingosine monocytogenes was achieved.
It achieves highly selective isolation of Sphingosine monocytogenes, significantly reduces false positive and false negative rates, simplifies the molecular identification process, reduces detection costs, and is suitable for large-scale screening.
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Figure CN123027138A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial isolation and culture technology, specifically relating to a method for selectively isolating Sphingosine Monoclonal bacteria from complex environmental samples. Sphingomonas Culture medium and its preparation method. Background Technology
[0002] Sphingosomalidosis (Sphingosomalidosis) Sphingomonas *Sphingosomalidobacteria*, belonging to the class Alphaproteobacteria, is a group of strictly aerobic Gram-negative bacilli widely distributed in diverse ecological environments such as soil, water, sediment, and plant rhizosphere. These bacteria have an outer cell membrane composed of glycosphingolipids (GSL) instead of the lipopolysaccharide (LPS) typically found in Gram-negative bacteria, resulting in lower endotoxin activity and unique membrane permeability. Therefore, they exhibit great application potential in areas such as microbial polysaccharide (e.g., sphingosomalidobacteria) synthesis, degradation of organic pollutants such as polycyclic aromatic hydrocarbons, and plant growth promotion, and are considered important microbial resources. However, *Sphingosomalidobacteria* is usually not a dominant bacterial group in natural environments, and its abundance is relatively low, posing a significant challenge to isolation and purification. Environmental samples have highly complex microbial communities; currently, basal media such as nutrient agar, LB medium, and R2A medium are mainly used with the addition of a single streptomycin as selective pressure for the isolation and screening of *Sphingosomalidobacteria*. This technical route utilizes the natural resistance of the target genus to streptomycin to achieve single selection. However, during isolation, highly competitive bacteria, such as Pseudomonas spp. ( Pseudomonas ), Bacillus spp. Bacillus Agrobacterium spp. Agrobacterium These bacteria easily form dominant colonies, which inhibits the growth of the target strain, Sphingosine monocytogenes. Morphological screening is prone to high false positive and false negative rates, resulting in low isolation efficiency and purity. Molecular identification procedures are cumbersome and costly, making them unsuitable for large-scale screening. Summary of the Invention
[0003] This invention aims to address the technical problems in existing Sphingomonas isolation techniques, such as insufficient selectivity of single antibiotics, limited inhibitory effect on non-target bacteria, poor selectivity of culture media, high false positive and false negative rates due to reliance on morphological screening, complex molecular identification procedures, high detection costs, and difficulty in meeting the needs of rapid screening of large-scale strains. It provides a highly selective culture medium to achieve efficient, highly selective isolation and rapid screening of Sphingomonas.
[0004] Specifically, the present invention provides the following technical solutions: The present invention provides a selective culture medium for Sphingomonas, the culture medium comprising a basal medium, streptomycin and polymyxin B, wherein the final concentration of streptomycin is 200 μg / mL and the final concentration of polymyxin B is 64 μg / mL.
[0005] Furthermore, the basal culture medium includes LB agar medium. In this invention, LB agar medium can be replaced by R2A agar medium, alternative carbon source, nitrogen source, or TSA medium; wherein, R2A culture has a lower nutrient abundance, which is beneficial to the isolation efficiency of slow-growing sphingosine monocytogenes.
[0006] The present invention also provides a selective culture medium for the selective isolation of Sphingomonas spp. from complex environmental samples. Sphingomonas Application in strains.
[0007] Furthermore, the complex environment samples include samples derived from soil, water, sediment, or plant rhizosphere.
[0008] This invention also provides a method for preparing a selective culture medium for Sphingomonas sphingosine monophosphate, comprising the following steps: (1) Prepare and sterilize the basal culture medium; (2) Cool the basal culture medium to 50℃–55℃; (3) Add streptomycin solution and polymyxin B solution filtered through a 0.22 μm filter membrane to the basic culture medium under aseptic conditions, so that the final concentration of streptomycin in the culture medium is 200 μg / mL and the final concentration of polymyxin B is 64 μg / mL. (4) After thorough mixing, pour into a plate and allow to solidify to obtain the selective culture medium.
[0009] Furthermore, the basal culture medium is LB agar medium.
[0010] The present invention also provides a method for isolating Sphingomonas from complex environmental samples, comprising the following steps: (1) Collect samples and perform serial dilution with sterile saline; (2) Prepare the selective culture medium plates described above; (3) Spread the serially diluted samples onto selective culture medium plates; pick yellow or orange-yellow single colonies and purify them to obtain Sphingosine monocytogenes.
[0011] The technical effects achieved by this invention are as follows: (1) Complementary antibacterial spectrum, eliminating selection blind spots Streptomycin: An aminoglycoside antibiotic that exerts its bactericidal effect by specifically binding to the 30S subunit of bacterial ribosomes, interfering with protein translation. Polymyxin B: A polypeptide antibiotic that kills bacteria by electrostatically binding its cationic group to the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria, disrupting membrane integrity, increasing permeability, and causing leakage of intracellular substances. By combining streptomycin and polymyxin B, their different targets are utilized to achieve synergistic antibacterial effects, resulting in complete inhibition of the main interfering bacteria.
[0012] (2) High selectivity and wide range of applications This invention is the first to propose and verify that *Sphingomonas* can grow normally on polymyxin B medium, and constructs a synergistic selective medium of streptomycin and polymyxin B, suitable for screening *Sphingomonas* strains. The combination of streptomycin and polymyxin B can inhibit *Bacillus belyssiensis* (…). B. velezensis ) 、 Lawrence Pseudomonas ( P. laurentiana ) 、 Agrobacterium pusa ( A. pusense ) 、 Escherichia coli ( E. coli This method can promote the growth of various strains, including *Sphingosporium* strains, but has no significant effect on the growth of *Sphingosporium* strains. It offers significant advantages in terms of selectivity and ease of operation, making it suitable for large-scale sample screening. Based on the wide ecological distribution and membrane structure adaptability of *Sphingosporium*, this technique can be used for subsequent isolation and screening in environments such as barren soils, plant phyllodes and endophytic tissues, activated sludge, and biofilms. Attached Figure Description
[0013] Figure 1. Growth inhibition rate of streptomycin against Sphingomonas; A represents Sphingomonas. S. hankookensis, S. yunnanensis, S. aerolata, S. ginsenosidimutans, S. panni Compared with four control strains ( B. velezensis, P. laurentiana, A. pusense, E. coli Growth inhibition comparison of B and B; B is Sphingosine monocytogenes. S. carotinifaciens, S. koreensis, S. paucimobilis, S. faeni, S. aquatilis Growth inhibition compared with four control strains.
[0014] Figure 2. Colony morphology diagram showing the effect of polymyxin B plate screening on the growth of Sphingosomalidone.
[0015] Figure 3 Figure showing the growth inhibition rate of polymyxin B against Sphingomonas and control interfering bacteria; A represents Sphingomonas. S. paucimobilis, S. carotinifaciens, S. panni, S. faeni, S. aquatilis Growth inhibition compared with four control strains; B is Sphingosomalmonella. S. aerolata, S. koreensis, S. ginsenosidimutans, S. yunnanensis, S. hankookensis Growth inhibition compared with four control strains.
[0016] Figure 4Comparison of the growth effects of streptomycin and polymyxin B combined selective culture medium on target bacteria and interfering bacteria. Detailed Implementation
[0017] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.
[0018] Example 1 1. Experimental bacterial strain Ten strains of Sphingosine Monoclonal antibodies from different sources were selected as the target bacterial group, and four typical environmental interference strains were set up as the control group, as detailed in Table 1.
[0019] Table 1 Information on test strains 2. Determination of the growth inhibition rate of streptomycin against Sphingomonas sphingosine mononitrate 2.1 Preparation of bacterial culture The tested strains listed in Table 1 were inoculated onto LB medium for activation. Single colonies were picked and transferred to LB liquid medium and cultured at 28°C and 180 rpm for 24–48 h. The culture was centrifuged at 8000 rpm for 10 min to collect the bacterial cells, resuspended in fresh LB medium, and diluted to OD200. 600 = 0.4, used as the seed culture for the test bacteria.
[0020] 2.2 Micro-broth dilution method The tolerance of each strain to streptomycin was determined using the micro-broth dilution method. Streptomycin stock solution (800 μg / mL) prepared by LB, blank LB solution, and test bacterial seed culture were added to 96-well plates according to the proportions shown in Table 2, resulting in final streptomycin concentrations of 0, 100, 200, 300, and 400 μg / mL in each well. Blank control wells (containing neither test bacteria nor streptomycin) and positive control wells (containing only test bacteria) were also included. Each treatment was performed in triplicate. The 96-well plates were incubated at 28°C for 24–48 h. After incubation, the OD of each well was measured using a microplate reader. 600 Value. The growth inhibition rate is calculated using the following formula: Growth inhibition rate (%) = [OD value] 600 (Positive control) - OD 600 (Test well) / [OD] 600 (Positive control) - OD 600 (Blank control) × 100% Table 2. Sampling Scheme for Micro-broth Dilution Method 3. Determination of the growth inhibition rate of polymyxin B against Sphingomonas. 3.1 Plate screening The Sphingomonas group and control group strains in Table 1 were streaked onto LB agar plates containing 16, 32, and 64 μg / mL polymyxin B, respectively, and incubated at 28°C to observe their growth.
[0021] 3.2 Verification of the micro-broth dilution method The growth inhibition rate of 64 μg / mL polymyxin B on each strain was further determined by micro-broth dilution method.
[0022] 4. Validation of the selectivity of streptomycin and polymyxin B compound culture medium Basic culture medium: LB agar. Selective additives: streptomycin 200 μg / mL; polymyxin B 64 μg / mL.
[0023] Preparation method: Prepare and sterilize LB agar medium according to conventional methods, and cool to 50–55℃; add streptomycin solution and polymyxin B solution that have been filtered through a 0.22 μm filter membrane for sterilization under aseptic conditions, mix thoroughly, pour into plates, and let solidify before use.
[0024] 4.1 Validation of the selectivity of streptomycin and polymyxin B compound culture medium Single colonies of the Sphingosine Monoclonal strain (10 strains) and the control group (4 strains) in Table 1 were picked and inoculated into LB liquid medium. The culture was incubated at 28°C for 24-48 h with shaking. The bacterial suspension was diluted to a certain concentration and plated on LB medium and the above-mentioned compound selective medium. Each group was replicated 3 times and cultured at 28°C for 4 days. The growth of the strains was observed and recorded.
[0025] 5. Experimental Results 5.1 Effect of streptomycin on the growth inhibition rate of Sphingosomalidosis and control interfering bacteria The results are as follows Figure 1 As shown. At a streptomycin concentration of 200 μg / mL, the growth inhibition rate of *Sphingosine monocytogenes* group was less than 20%, demonstrating good tolerance; while in the control group, *Bacillus belye* (… B. velezensis ) and Agrobacterium pusa ( A.pusense ) was significantly inhibited, but Pseudomonas laureate ( P. laurentiana ) and Escherichia coli ( E. coli The inhibition rate of streptomycin was only about 50%, indicating that 200 μg / mL streptomycin was ineffective against Pseudomonas laureate (…). P. laurentiana ) and Escherichia coli ( E. coli The inhibitory effect of ) is insufficient, and it is difficult to achieve complete selection when used alone.
[0026] 5.2 Colony morphology of the effect of polymyxin B plate screening on the growth of Sphingosomalidone. Plate screening indicates ( Figure 2 Sphingosomalidone grew normally at 64 μg / mL polymyxin B, while Pseudomonas laureates ( P. laurentiana ) and Escherichia coli ( E. coli The growth of *Pseudomonas laureate* was inhibited at 64 μg / mL polymyxin B, therefore 64 μg / mL polymyxin B can effectively inhibit *Pseudomonas laureate*. P. laurentiana ) and Escherichia coli ( E. coli ) growth.
[0027] 5.3 Effect of polymyxin B on the growth inhibition rate of Sphingosoma and control interfering bacteria Further verification using the micro-broth dilution method showed that ( Figure 3 At a polymyxin B concentration of 64 μg / mL, the growth inhibition rate of the *Sphingosomalidone* group was significantly lower than that of *Bacillus belyssae*. B. velezensis ), Pseudomonas laureates ( P. laurentiana ) and Escherichia coli ( E. coli Smaller ， And against Agrobacterium pusa ( A.pusense The inhibition rate was close to 50%. ， It exhibits significant tolerance.
[0028] 5.4 Effects of streptomycin and polymyxin B combined selective culture medium on the growth of target bacteria and interfering bacteria The results showed that ( Figure 4 All 10 *Sphingosomalidone* strains grew normally on the combined selective medium, while the 4 control interference strains did not grow on the combined medium. This combined regimen achieved complete inhibition of the target bacteria, with significantly better selectivity than streptomycin-only medium.
[0029] Table 3. Growth of the strains on LB medium and mixed selective medium Note: "+" indicates growth, and "-" indicates no growth.
[0030] In summary, this invention, through systematic screening and validation, established a highly efficient and selective isolation and culture system for *Sphingomonas* based on the synergistic combination of streptomycin and polymyxin B. Further research revealed that *Sphingomonas*, due to its unique GSL outer membrane structure, exhibits tolerance to 64 μg / mL polymyxin B, while LPS-type non-target bacteria are efficiently inhibited. The synergistic effect of streptomycin (200 μg / mL) and polymyxin B (64 μg / mL) is significant: the two antibiotics act on the 30S subunit of bacterial ribosomes (inhibition of protein synthesis) and the outer membrane LPS (membrane structure disruption), respectively, with complementary targets and superimposed antibacterial spectrum, effectively covering the selection blind spot of streptomycin alone. The selective culture medium formulation and construction method provided by this invention are suitable for the efficient screening, isolation, and purification of *Sphingomonas* strains.
Claims
1. A selective culture medium for Sphingomonas, characterized in that, The culture medium comprises basal medium, streptomycin, and polymyxin B, wherein the final concentration of streptomycin is 200 μg / mL and the final concentration of polymyxin B is 64 μg / mL.
2. The selective culture medium according to claim 1, characterized in that, The basal culture medium includes LB agar medium.
3. The selective culture medium according to claim 1 or 2 for the selective isolation of Sphingomonas spp. from complex environmental samples ( Sphingomonas Application in strains.
4. The application according to claim 3, characterized in that, The complex environment samples include samples from soil, water, sediment, or plant rhizosphere.
5. A method for preparing a selective culture medium for Sphingomonas sphingosine monophosphate, characterized in that, Includes the following steps: (1) Prepare and sterilize the basal culture medium; (2) Cool the basal culture medium to 50℃–55℃; (3) Add streptomycin solution and polymyxin B solution filtered through a 0.22 μm filter membrane to the basic culture medium under aseptic conditions, so that the final concentration of streptomycin in the culture medium is 200 μg / mL and the final concentration of polymyxin B is 64 μg / mL. (4) After thorough mixing, pour into a plate and allow to solidify to obtain the selective culture medium.
6. The preparation method according to claim 5, characterized in that, The basic culture medium is LB agar medium.
7. A method for isolating Sphingosine Monoclonal bacteria from complex environmental samples, characterized in that, Includes the following steps: (1) Collect samples and perform serial dilution with sterile saline; (2) Prepare the selective culture medium plate according to claim 1 or 2; (3) Spread the serially diluted samples onto selective culture medium plates; pick yellow or orange-yellow single colonies and purify them to obtain Sphingosine monocytogenes.