Enterobacter hormaechei SD7-1 strain and application thereof in degradation of tetracycline antibiotics

Abstract

The invention belongs to the technical field of microbial strains and environmental microbiology, and discloses a novel strain capable of degrading tetracycline antibiotics. The new bacterial strain is an Enterobacter hormaechei SD7-1 bacterial strain, and is preserved in the Guangdong Microbial Culture Collection Center on March 3, 2025, and the preservation number is GDMCC No: 65849. The invention further discloses a preparation method of the new bacterial strain. The new strain provided by the invention has a remarkable degradation effect on tetracycline antibiotics, and the degradation rate of tetracycline after 24-hour treatment reaches 50% or above. Therefore, as a functional strain, the enterobacter hormaechei SD7-1 strain has high degradation efficiency and a wide degradation spectrum, can effectively degrade various tetracycline antibiotics in a short time, and has the advantages of treating pollution caused by wide use of tetracycline, further reducing environmental half-life emission of tetracycline, protecting the environment, and having good application prospects. The invention provides a novel microbial resource with potential in soil and water environments, especially in soil and water environments.

Description

Technical Field

[0001] The present invention belongs to the field of microbial strains and environmental microorganisms, and particularly relates to an Enterobacter hormaechei SD7-1 strain and its application in degrading tetracycline antibiotics. Background Art

[0002] Tetracycline antibiotics are a class of broad-spectrum antibacterial drugs with a tetracyclic system in their chemical structure, and their representative members include tetracycline, oxytetracycline, chlortetracycline, and doxycycline (Chopra, I Microb Molecular Biology Reviews). Since being first discovered in the 1940s, tetracycline has been widely used in clinical medicine and veterinary medicine due to its advantages such as high efficiency, low toxicity, low price, and broad antibacterial spectrum (Nelson, Annals of the New York Academy of Sciences). It inhibits the 30S subunit of the bacterial ribosome, thereby blocking protein synthesis, and has an inhibitory effect on both Gram-positive and Gram-negative bacteria (Griffin, Review of Cardiovascular Medicine).

[0003] In addition to its extensive use in the medical field, tetracycline is also widely used in animal husbandry and aquaculture to prevent animal diseases and promote growth. In agriculture, tetracycline is often used as a feed additive, greatly improving the feed conversion rate and growth rate of animals (Sarmah, Chemosphere). However, due to the extensive use of tetracycline, its residues and environmental problems have emerged.

[0004] The extensive use and discharge of tetracycline have made it one of the important pollutants in the environment. It has strong chemical stability and can exist in soil and water environments for a long time. Research shows that the environmental half-life of tetracycline can be as long as several weeks to several months, which exacerbates its cumulative effect and brings various potential hazards. The main hazards of tetracycline are as follows: (1) Residues in the environment may promote the emergence and spread of drug-resistant strains. Long-term exposure to antibiotics will exert selective pressure on the microbial community, causing drug-resistant genes to spread rapidly in the environment (Aminov, Environmental Microbiology). These drug-resistant genes may spread to pathogenic bacteria through horizontal gene transfer, threatening the effectiveness of antibiotics in clinical practice. (2) Intake of tetracycline residues through the food chain may cause allergic reactions, liver and kidney function damage, and gastrointestinal flora imbalance in the human body (Hamscher, Analytical Chemistry). In addition, long-term low-dose exposure to the tetracycline environment may interfere with the endocrine system, especially children and adolescents are more vulnerable to its adverse effects.

[0005] To address the issue of tetracycline residues, researchers and industry practitioners have explored various means to reduce the concentration of tetracycline in the environment. These methods mainly include physicochemical technologies, such as:

[0006] 1. Physical degradation methods, such as adsorption, utilize adsorbent materials like activated carbon, modified clay, or novel nanomaterials (such as graphene oxide) to remove tetracycline from water or soil. These adsorbent materials have a high specific surface area and porous structure, enabling effective capture of tetracycline molecules. However, the adsorption process is merely physical migration and cannot completely degrade tetracycline, requiring subsequent treatment to avoid secondary pollution. The second is membrane separation technology. For example, nanofiltration membrane and reverse osmosis membrane technologies can effectively remove tetracycline and its metabolites through a physical barrier. This method is highly efficient, but membrane fouling and high operating costs are its main bottlenecks.

[0007] 2. Chemical degradation methods

[0008] One of the chemical degradation methods is photocatalytic oxidation, which uses free radicals generated by semiconductor materials (such as TiO2, ZnO) under light irradiation to decompose tetracycline molecules. In recent years, the degradation efficiency has been significantly improved by modified photocatalysts doped with metal ions or supported on carbon-based materials. However, this method may be limited by light sources and catalyst recovery problems in practical applications. The second is advanced oxidation processes (AOPs), including ozonation, Fenton reaction, and electrochemical oxidation, which use strong oxidants (such as hydroxyl radicals) to decompose tetracycline. These methods show good degradation effects when treating high-concentration tetracycline wastewater, but due to the need for a large amount of chemical reagents, the operating costs may be relatively high.

[0009] Although physical and chemical methods have a strong degradation effect on tetracycline antibiotics, their high costs, difficulty in large-scale application, easy generation of secondary pollution, and even the production of other non-target toxicities have greatly limited the application of these methods. Biodegradation methods, which utilize biological interactions for degradation, are the most promising means for the treatment of tetracyclines in recent years.

[0010] As an environmentally friendly technology that uses microorganisms or enzymes to degrade tetracycline, biodegradation methods are more environmentally friendly and mainly include: enzyme-catalyzed degradation. Some enzymes such as lignin peroxidase and laccase can catalyze the decomposition of tetracycline. Compared with microorganisms, enzymes have stronger specificity and are less affected by environmental conditions, but the production and stability of enzymes still need further research.

[0011] Microbial degradation: Microorganisms such as the widely used Pseudomonas spp. and Bacillus spp. can degrade tetracycline into non-toxic or low-toxic metabolites under suitable conditions. Microbial degradation has the advantages of low cost and environmental friendliness, which greatly makes up for the deficiencies of physical and chemical technologies to a certain extent. However, its efficiency is still limited. Therefore, it is necessary to discover more new highly efficient microbial strains and stabilized degradation methods to expand the scope of options for microbial degradation and ensure the treatment efficiency of microorganisms. Summary of the Invention

[0012] The present invention provides a new strain Enterobacter hormaechei SD7-1 that can degrade tetracycline drugs, which has high degradation efficiency and a wide degradation spectrum, and can better solve the problem of the accumulation of tetracycline antibiotics in the environment. Through the screening, identification and evaluation of the degradation performance of this strain, it is found that this strain can effectively degrade a variety of tetracycline antibiotics in a short time, providing a potential new microbial resource for environmental pollution control.

[0013] To solve the above problems in the prior art, the first object of the present invention is to provide a new strain with the ability to degrade tetracycline antibiotics, and this strain is named Enterobacter hormaechei SD7-1 strain.

[0014] The second object of the present invention is to provide the application of the above Enterobacter hormaechei SD7-1 strain in the treatment and degradation of tetracycline antibiotics.

[0015] To achieve the above object, the present invention provides the following technical solutions:

[0016] The present invention provides a strain Enterobacter hormaechei SD7-1, which was deposited with the Guangdong Provincial Microbial Culture Collection Center on March 3, 2025, and the deposit number is: GDMCC No: 65849.

[0017] Furthermore, the present invention claims the application of the Enterobacter hormaechei SD7-1 strain in the preparation of a biological agent for degrading tetracycline antibiotics.

[0018] Still further, the tetracycline antibiotics are one or more of tetracycline, chlortetracycline, minocycline, tigecycline.

[0019] The present invention also provides a method for culturing substances with the ability to degrade tetracycline antibiotics, comprising the following steps:

[0020] S1. Culturing the Enterobacter hormaechei SD7-1 strain on a culture medium;

[0021] S2. Adding antibiotics to the cultured bacterial liquid obtained in step S1 for degradation.

[0022] Preferably, the culture medium in step S1 is MH broth.

[0023] Preferably, step S1 is cultured at 37°C and 200 rpm for 8 hours; the degradation culture conditions for step S2 are: cultured at 37°C and 200 rpm for 24 hours.

[0024] The present invention also claims the bacterial cells and / or fermentation broth obtained by the method for culturing substances with the ability to degrade tetracycline antibiotics.

[0025] Furthermore, the present invention protects the application of the bacterial cells and / or fermentation broth in the prevention and treatment of degradation of tetracycline antibiotics.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The new strain provided by the present invention has a significant degradation effect on tetracycline antibiotics, and the degradation of tetracycline reaches more than 98% after 24 hours of treatment. Therefore, as a functional strain, the Enterobacter hormaechei SD7-1 strain has a high degradation efficiency and a wide degradation spectrum. This strain can effectively degrade a variety of tetracycline antibiotics in a short time, and provides a potential new microbial resource for controlling the pollution caused by the widespread use of tetracycline, further reducing the environmental half-life emission of tetracycline, and protecting the environment, especially the soil and water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Results of the plate antibacterial experiment for tetracycline, chlortetracycline, minocycline and tigecycline;

[0029] Figure 2 LC-MS / MS determination results of the degradation of tetracycline by the Enterobacter hormaechei SD7-1 strain. DETAILED DESCRIPTION OF THE INVENTION

[0030] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are prepared according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0031] Example 1 Isolation, purification and preservation of Enterobacter hormaechei SD7-1 strain:

[0032] Source of the strain: Pet fecal samples were collected from a pet hospital in Liaocheng City, Shandong Province. We carried out the isolation and identification of strains on the fecal samples. A cotton swab was dipped in feces and added to 600 μl of LB broth (Guangdong Huankai Microbial Technology Co., Ltd.), and cultured overnight at 37°C and 180 rpm. Subsequently, the above-mentioned culture solution was streaked on an LB agar plate containing 50 μg / ml tetracycline (Guangdong Huankai Microbial Technology Co., Ltd.) and cultured overnight at 37°C. Take the plump colonies on the petri dish, purify and preserve the bacteria.

[0033] 16S rDNA strain identification

[0034] Scrape a loop of the purified strain, and extract the genomic DNA of the strain with reference to the steps of the Tiangen Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). Subsequently, PCR amplification was performed on the 16S rDNA gene, and the PCR product was electrophoresed on a 1.2% agarose gel. The PCR amplification PCR system (20 μl) using the Tiangen PCRMIX system is as follows: 10 μl of 2xPCR MIX, 1 μl of upstream primer, 1 μl of downstream primer (the upstream primer and the downstream primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively), 1 μl of template DNA, and 7 μl of ddH2O. Among them, the 16S rDNA primers were synthesized by Genewiz Co., Ltd., Suzhou. The PCR program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 53°C for 45 s, extension at 72°C for 1 min 30 s, for 30 cycles; extension at 72°C for another 10 min.

[0035] According to the results of agarose gel electrophoresis, the PCR positive samples were sent to Genewiz Co., Ltd., Suzhou for sequencing, and nucleotide sequence alignment and analysis were performed using the NCBI database:

[0036] (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), the strain was identified at the molecular level as Enterobacter hormaechei (the 16S rDNA gene is shown in SEQ ID NO: 3).

[0037] Colony morphological characteristics and physiological and biochemical properties

[0038] Enterobacter hormaechei strain SD7-1 is a Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive straight rod bacterium that is transparent on MacConkey agar plates.

[0039] After identification and analysis, the new strain was Enterobacter hormaechei. The preservation information is as follows:

[0040] It was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms (GDMCC) on March 3, 2025;

[0041] The deposit number is GDMCC No: 65849;

[0042] The deposit address is: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0043] Example 2 Determination of the minimum inhibitory concentration (MIC)

[0044] The microbroth dilution method was used to determine the MIC of Enterobacter hormaechei SD7-1 against tetracycline, chlortetracycline, doxycycline, minocycline, and tigecycline (all purchased from Sigma-Aldrich, USA) according to the relevant standards of the Clinical and Laboratory Standards Institute of the United States (CLSI: M100-S26). Escherichia coli ATCC 25922 was used as the quality control strain.

[0045] The results showed (Table 1) that Enterobacter hormaechei SD7-1 strain showed different levels of drug sensitivity to tetracycline, chlortetracycline, doxycycline, minocycline, and tigecycline, which were greater than 512 μg / ml, 512 μg / ml, 64 μg / ml, 128 μg / ml, and 4 μg / ml, respectively.

[0046] Table 1 MICs of Enterobacter hormaechei SD7-1 against tetracycline-like drugs

[0047]

[0048] Note: TC, tetracycline; CTC, chlortetracycline; DOX, doxycycline; MIN, minocycline; TGC, tigecycline.

[0049] Example 3 Degradation Experiment of Tetracycline Drugs

[0050] The bacterial solution of E. coli ATCC25922 cultured to the logarithmic phase was diluted 1:10. 100 μl of the diluted bacterial solution was evenly spread on a fresh MH agar plate with a spreading rod. Subsequently, 3 holes with a diameter of 6 mm were punched on the MH agar medium with a puncher and sealed with sterile MH agar at the bottom.

[0051] Each tetracycline drug was divided into three test groups and carried out in 2-ml EP tubes with a total volume of 400 μl in the EP tubes:

[0052] 1. Control group: Tetracycline drug + MH broth

[0053] 2. Positive group: Tetracycline drug + MH broth + Enterobacter hormaechei SD7-1 strain

[0054] 3. Blank group: MH broth + Enterobacter hormaechei SD7-1 strain (without drug)

[0055] The three groups of samples were incubated at 37 °C on a constant temperature shaker at 220 rpm for 24 hours, and then centrifuged at 12,000 rpm. The supernatant was taken and filtered through a 0.22-μm filter membrane. 20 μL of the filtered solution was pipetted and dropped into the corresponding holes of the MH agar medium coated with ATCC25922. After the filtered solution was completely absorbed by the agar, it was inverted and cultured overnight in a 37 °C incubator.

[0056] Finally, an image was taken using a standard digital camera, and the degradation ability of Enterobacter hormaechei SD7-1 to tetracycline drugs was judged according to the size of the inhibition zone. The results ( Figure 1 ) showed that obvious inhibition zones appeared in the control groups of all four tetracycline drugs, while no obvious inhibition zones or the inhibition zones were significantly smaller than those in the control groups in the positive group, indicating that the strain SD7-1 had a degradation effect on tetracycline drugs. No antibacterial effect was seen in the blank group.

[0057] Example 4

[0058] Pick a single colony of purified Enterobacter hormaechei SD7-1 and inoculate it into 4 ml of MH broth. Incubate at 37 °C and 200 rpm for 8 hours. Pipette 40 μl of the above bacterial culture solution into MH broth with a final volume of 4 ml containing 1.5 μg / ml tetracycline (supplemented with 500 μM magnesium sulfate and 100 μM NADPH. Without the above supplements, regular commercially available MH broth can also be used), and incubate at 37 °C, 200 rpm and in the dark for 24 hours. At the same time, use MH broth (supplemented with 500 μM magnesium sulfate and 100 μM NADPH) with only tetracycline-class (tetracycline) drugs added. Subsequently, centrifuge the bacterial culture solution at 12,000 rpm for 2 min and filter it with a 0.22-μm pore-size filter head (Tianjin Jinteng Laboratory Equipment Co., Ltd.). Take the filtered supernatant, dilute it 10-fold, and perform LC-MS / MS determination according to the traditional method (Kevin J. Forsberg et al., 2015, Chemistry & Biology), see Figure 2 Set up 3 parallels for each of the above experimental group and control group.

[0059] The results show that Enterobacter hormaechei SD7-1 exhibits good degradation activity against tetracycline-class drugs. Among them, compared with the control group, the degradation rate of tetracycline by the experimental group supplemented with Enterobacter hormaechei SD7-1 reached 51.95% in 24 hours (p < 0.0001).

[0060] The above embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, rather than limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. An Enterobacter hormaechei SD7-1 strain, characterized in that, It was deposited at the Guangdong Microbial Culture Collection Center on March 3, 2025, with the deposit number: GDMCC No: 65849.

2. Use of the Enterobacter hormaechei SD7-1 strain according to claim 1 in the preparation of a biological agent for degrading tetracycline antibiotics.

3. The application according to claim 2, characterized in that, The tetracycline antibiotics are one or more of tetracycline, chlortetracycline, minocycline, and tigecycline.

4. A method for culturing a substance with the ability to degrade tetracycline antibiotics, characterized in that, It includes the following steps: S1. Culturing the Enterobacter hormaechei SD7-1 strain according to claim 1 on a culture medium; S2. Adding antibiotics to the cultured bacterial liquid in step S1 for degradation.

5. The method for culturing a substance with the ability to degrade tetracycline antibiotics according to claim 4, characterized in that, The culture medium in step S1 is MH broth.

6. The method for culturing a substance having the ability to degrade tetracycline antibiotics according to claim 4 or 5, characterized in that Step S1 is cultured at 37 °C and 200 rpm for 8 hours; the degradation culture conditions in step S2 are: cultured at 37 °C and 200 rpm for 24 hours.

7. The bacterial cells and / or fermentation broth obtained by the method for culturing a substance with the ability to degrade tetracycline antibiotics according to claim 4.

8. Use of the bacterial cells and / or fermentation broth according to claim 7 in the prevention and treatment of degradation of tetracycline antibiotics.

Images