Steroid hormone degrading bacterium as well as screening method and application thereof

Abstract

The invention discloses a steroid hormone degrading bacterium as well as a screening method and application thereof, and relates to the field of biological medicines, the steroid hormone degrading bacterium comprises klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) with the preservation number of CCTCC (China Center for Type Culture Collection) NO: M2025478. The steroid hormone degrading bacterium provided by the invention has the capability of efficiently degrading dexamethasone, is beneficial to degrading dexamethasone in the environment, is conveniently obtained by adopting a screening method, and provides a sample for research on degradation of steroid hormones.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to a steroid hormone-degrading bacterium, a screening method thereof, and an application thereof. Background Art

[0002] In the treatment of allergic rhinitis, intranasal glucocorticoids are often used, and steroid hormones such as dexamethasone are used during the treatment process. With the increase in the use of steroid hormones, their residual amounts in the environment also increase.

[0003] However, the steroid hormones remaining in the environment have adverse effects on aquatic plants, mammals, and even humans. Moreover, the current research on how to degrade steroid hormones and the degradation principle is limited. Therefore, it is necessary to study a method for degrading steroid hormones. Summary of the Invention

[0004] In view of the deficiencies of the above-mentioned related technologies, this application provides a steroid hormone-degrading bacterium, a screening method thereof, and an application thereof. This application provides a steroid hormone-degrading bacterium, which has the ability to efficiently degrade dexamethasone, is beneficial to the degradation of dexamethasone in the environment, and can be conveniently obtained by the screening method, providing a sample for the research on degrading steroid hormones.

[0005] In the first aspect, a steroid hormone-degrading bacterium provided by this application adopts the following technical solution: A steroid hormone-degrading bacterium includes Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae), deposit number: CCTCC NO: M2025478.

[0006] In the second aspect, a screening method for a steroid hormone-degrading bacterium provided by this application adopts the following technical solution: A screening method for a steroid hormone-degrading bacterium includes the following steps: collecting a specimen from the nasal cavity of a patient resistant to allergic rhinitis hormones, placing the specimen in a selective medium for selective culture, taking the culture solution for culture and separating out single colonies to obtain the steroid hormone-degrading bacterium.

[0007] Preferably, the selective medium includes steroid hormones and an inorganic salt medium.

[0008] Preferably, the mass concentration of the steroid hormone is 0.5 - 1.5 g / L.

[0009] Preferably, the steroid hormone includes dexamethasone.

[0010] Preferably, the inorganic salt medium includes one or more of magnesium sulfate heptahydrate, calcium chloride dihydrate, disodium hydrogen phosphate, ammonium chloride, sodium dihydrogen phosphate, and sodium chloride.

[0011] Preferably, the selective medium comprises 1.9 - 2.1 mmol / L magnesium sulfate heptahydrate, 0.08 - 0.12 mmol / L calcium chloride dihydrate, 45 - 50 mmol / L disodium hydrogen phosphate, 18 - 20 mmol / L ammonium chloride, 21 - 23 mmol / L sodium dihydrogen phosphate, and 8.5 - 9.5 mmol / L sodium chloride.

[0012] Preferably, the selective medium comprises 2 mmol / L magnesium sulfate heptahydrate, 0.1 mmol / L calcium chloride dihydrate, 48 mmol / L disodium hydrogen phosphate, 19 mmol / L ammonium chloride, 22 mmol / L sodium dihydrogen phosphate, and 9 mmol / L sodium chloride.

[0013] Preferably, the selective culture comprises the following steps: culturing the selective medium containing the specimen in the dark with shaking at 36.5 - 37.5 °C and 150 - 250 rpm, sampling every 3 - 5 days and transferring it to a new selective medium for culture, repeating 3 - 5 times.

[0014] Preferably, the selective culture comprises the following steps: culturing the selective medium containing the specimen in the dark with shaking at 37 °C and 200 rpm, sampling every 4 days and transferring it to a new selective medium for culture, repeating 4 times.

[0015] Preferably, the sampling ratio is 5 - 15% by volume.

[0016] Preferably, the sampling ratio is 10% by volume.

[0017] In a third aspect, the present application provides an application of a steroid hormone-degrading bacterium in degrading steroid hormones.

[0018] Preferably, the above steroid hormone-degrading bacterium is used for degrading steroid hormones in one of medical wastewater, industrial sewage, urban sewage, and aquaculture wastewater.

[0019] Preferably, the steroid hormone is dexamethasone.

[0020] In summary, the present application includes at least one of the following beneficial technical effects: 1. The Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) of the present application can degrade dexamethasone; 2. When the Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) of the present application degrades dexamethasone with a concentration of 0.5 g / L within one week, the degradation rate reaches 85%; 3. The Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) of the present application degrades dexamethasone, providing a new method for the degradation of dexamethasone in medical wastewater, industrial sewage and other environments. Description of the Drawings

[0021] Figure 1 is the whole-genome sequencing result of Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae).

[0022] Figure 2 Degradation result of dexamethasone in Application Example 1. Detailed Description of the Invention

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the technical solutions of the present invention in combination with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Example 1 Example 1 of the present application provides a method for screening steroid hormone-degrading bacteria, and the specific steps are as follows: (1) Specimen collection: Let the back of the head of a patient with allergic rhinitis resistant to hormones lean against the treatment chair. The collector holds a cotton swab and slowly inserts it into the nasal cavity along the nostril. After reaching the posterior nasopharyngeal wall, the cotton swab is gently taken out to obtain a cotton swab with the specimen collected, and it is properly stored in an environment of -80°C.

[0025] (2) Isolation of steroid hormone-degrading bacteria strains: Take a 100 mL conical flask, add 30 mL of selective medium, put the tip cotton ball of the cotton swab with the specimen collected into the conical flask, place the conical flask in a shaker and shake it in the dark at 37°C and 200 rpm. Every 4 days, take 3 mL of the sample from the conical flask and transfer it to a conical flask containing 27 mL of new selective medium for continued cultivation, and repeat 4 times. Then, in a laminar flow hood, dip the sample solution with an inoculation loop and spread it on a Columbia blood agar culture dish, and place it in a 37°C bacterial incubator for overnight cultivation. The next day, pick out the single colonies on the Columbia blood agar culture dish and store them at -20°C to obtain steroid hormone-degrading bacteria.

[0026] Application Example 1 Application Example 1 of the present application provides an application of steroid hormone-degrading bacteria, and the specific steps are as follows: Prepare LB medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl with ultrapure water and autoclave it. Drop the PBS suspension of the steroid hormone-degrading bacterium screened in Example 1 into a 50 mL conical flask, add 10 mL of sterile LB medium, and culture it in a shaker at 37 °C and 200 rpm to obtain LB bacterial liquid. Take 3 mL of the cultured LB bacterial liquid and add it to 30 mL of degrading medium containing 0.5 g / L dexamethasone. Place the conical flask containing the degrading medium in a shaker and shake it in the dark at 37 °C and 200 rpm to obtain a degrading solution.

[0027] Test detection (1)Identify the strain of the steroid hormone-degrading bacterium screened in Example 1 by 16sRNA sequencing.

[0028] (2)Perform whole-genome sequencing on the steroid hormone-degrading bacterium screened in Example 1, and the results are shown in Figure 1 the figure.

[0029] (3)Collect 500 μL of the degrading solution from the conical flask in Application Example 1 every day and store it in a -20 °C refrigerator for one week. Thaw the degrading solutions collected for seven days at room temperature and extract them with ethyl acetate respectively. Then, centrifuge them at 12000 rpm at 4 °C for 10 min, carefully aspirate the upper organic solution into a new test tube, and finally drop 2 μL of dexamethasone and other hormone standards and the extractive solution onto a thin-layer chromatography silica gel plate in sequence. Visualize the band intensity of dexamethasone with a UV analyzer (labeled as Day1, Day2, Day3, Day4, Day5, Day6, and Day7 in the order of collection time), as shown in Figure 2 Figure (a), and analyze the content of dexamethasone in each degrading solution from the ethyl acetate / petroleum ether chromatography solution, as shown in Figure 2 Figure (b).

[0030] Result analysis The following combines Figure 1-2 the provided experimental results to elaborate on this application in detail.

[0031] The steroid hormone-degrading bacterium screened in Example 1 is a single strain with a raised shape, milky white color, and smooth surface. After 16sRNA sequencing, it shows that the 16s rRNA sequence of this bacterium has a similarity of 99.740% with Klebsiella pneumoniae. This bacterium is named Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae), which was deposited at the China Center for Type Culture Collection (CCTCC) on March 17, 2025. Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Deposit number: CCTCC NO: M2025478.

[0032] Reference Figure 1 , the whole genome sequencing of Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) was carried out. The results showed that Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) contained 1 chromosome and 1 plasmid, the total gene length was 4,755,756 bp, the gene spacer length was 672,878 bp, there were 5,026 coding genes, and there were 86 and 25 tRNAs and rRNAs respectively in non-coding genes.

[0033] Reference Figure 2 , from Figure 2 (a), it can be seen that the visualized patterns gradually change from deep to light in the order of collection time. From Figure 2 (b), it can be seen that the degradation rate of Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) to dexamethasone reached 85% on the 7th day, indicating that the Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) of the present application has a high degradation rate and degradation speed for 0.5 g / L dexamethasone.

[0034] In summary, the Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) of the present application has an efficient degradation effect on steroid hormones, and has a good degradation effect when applied to the degradation of steroid hormones in one of medical wastewater, industrial sewage, urban sewage and aquaculture wastewater. Moreover, in view of the degradation effect of Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae) on steroid hormones, in the medical field, the drug resistance of patients using steroid hormones (such as patients with allergic rhinitis, allergic asthma and other hormone therapy-related diseases) can be evaluated by detecting Klebsiella pneumoniae (KP2022 Klebsiella pneumoniae).

[0035] This specific embodiment is only an interpretation of the present application, and it is not a limitation of the present application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A steroid hormone-degrading bacterium, characterized in that: Including Klebsiella pneumoniae (KP2022 Klebsiellapneumoniae), deposit number: CCTCC NO: M2025478.

2. The method for screening steroid hormone-degrading bacteria according to claim 1, characterized in that: The following steps are involved: A specimen is collected from the nasal cavity of a patient with allergic rhinitis and hormone resistance, the specimen is placed in a selective culture medium for selective culture, the culture fluid is taken for culture and a single colony is isolated to obtain the steroid hormone degrading bacteria.

3. The method for screening steroid hormone-degrading bacteria according to claim 2, characterized in that: The selective culture medium comprises a steroid hormone and an inorganic salt medium.

4. The method for screening steroid hormone-degrading bacteria according to claim 3, characterized in that: The mass concentration of the steroid hormone is 0.5-1.5 g / L.

5. The method for screening steroid hormone-degrading bacteria according to claim 3, characterized in that: The steroid hormone is dexamethasone.

6. The method for screening steroid hormone-degrading bacteria according to claim 3, characterized in that: The inorganic salt medium includes one or more of magnesium sulfate heptahydrate, calcium chloride dihydrate, disodium hydrogen phosphate, ammonium chloride, sodium dihydrogen phosphate and sodium chloride.

7. The method for screening steroid hormone-degrading bacteria according to claim 2, characterized in that: The selective culture medium includes 1.9-2.1 mmol / L magnesium sulfate heptahydrate, 0.08-0.12 mmol / L calcium chloride dihydrate, 45-50 mmol / L disodium hydrogen phosphate, 18-20 mmol / L ammonium chloride, 21-23 mmol / L sodium dihydrogen phosphate and 8.5-9.5 mmol / L sodium chloride.

8. The method for screening steroid hormone-degrading bacteria according to claim 2, characterized in that: The selective culture comprises the following steps: culturing the selective culture medium containing the specimen under the conditions of 36.5-37.5° C. and 150-250 rpm in the dark with shaking, taking samples and transferring them to new selective culture medium for culture every 3-5 days, and repeating for 3-5 times.

9. The method for screening steroid hormone-degrading bacteria according to claim 8, characterized in that: The sampling ratio is 5-15% volume fraction.

10. Use of the steroid hormone-degrading bacteria according to claim 1 in degrading steroid hormones.

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