Enterobacter cloacae sd7-1 strain and application thereof in degrading tetracycline antibiotics

Abstract

The present application belongs to the technical field of microbial strains and environmental microbiology, and discloses a new strain with the function of degrading tetracycline antibiotics. The new strain is Enterobacter hormaechei SD7-1 strain, which was preserved in Guangdong Microbial Culture Collection Center on March 3, 2025, and the preservation number is GDMCC No: 65849. The new strain provided by the present application has a significant degradation effect on tetracycline antibiotics, and the degradation of tetracycline is more than 50% after 24 hours of treatment. Therefore, the Enterobacter hormaechei SD7-1 strain, as a functional strain, has high degradation efficiency and a wide degradation spectrum. The strain can effectively degrade various tetracycline antibiotics in a short time, reduce the environmental half-life discharge of tetracycline, protect the environment, especially the soil and water environment, and provides a potential new microbial resource.

Description

Technical Field

[0001] This invention belongs to the field of microbial strains and environmental microorganisms, specifically relating to a strain of Enterobacter holmieae SD7-1 and its application in degrading tetracycline antibiotics. Background Technology

[0002] Tetracycline antibiotics are a class of broad-spectrum antibacterial drugs whose chemical structure contains a tetracycline system. Representative members include tetracycline, oxytetracycline, chlortetracycline, and doxycycline (Chopra, *Microb. Molecular Biology Reviews*). Since their first discovery in the 1940s, tetracycline has been widely used in clinical medicine and veterinary medicine due to its advantages such as high efficacy, low toxicity, low price, and broad antibacterial spectrum (Nelson, *Annals of the New York Academy of Sciences*). It inhibits protein synthesis by suppressing the 30S subunit of bacterial ribosomes, thus exhibiting inhibitory effects against both Gram-positive and Gram-negative bacteria (Griffin, *Review of Cardiovascular Medicine*).

[0003] Besides its widespread use in the medical field, tetracycline is also extensively 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 feed conversion ratios and growth rates in animals (Sarmah, Chemosphere). However, due to the widespread use of tetracycline, its residues and environmental problems have also become apparent.

[0004] The widespread use and emission of tetracycline has made it one of the most important pollutants in the environment. It has strong chemical stability and can persist in soil and water environments for a long time. Studies have shown that the environmental half-life of tetracycline can be as long as several weeks to several months, which exacerbates its cumulative effects and brings about multiple 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 pathogens through horizontal gene transfer, threatening the clinical effectiveness of antibiotics. (2) Ingestion of tetracycline residues through the food chain may lead to allergic reactions, liver and kidney damage, and gastrointestinal flora imbalance in humans (Hamscher, Analytical Chemistry). In addition, long-term low-dose exposure to tetracycline may interfere with the endocrine system, especially children and adolescents are more susceptible to its adverse effects.

[0005] To address the problem of tetracycline residues, researchers and industry practitioners have explored various methods to reduce tetracycline concentrations in the environment. These methods primarily include physicochemical techniques, such as:

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

[0007] 2. Chemical degradation methods

[0008] One chemical degradation method is photocatalytic oxidation, which uses free radicals generated by semiconductor materials (such as TiO2 and ZnO) under light irradiation to decompose tetracycline molecules. In recent years, the degradation efficiency has been significantly improved by doping with metal ions or modifying photocatalysts supported on carbon-based materials. However, this method may be limited in practical applications by issues related to the recovery of the light source and the catalyst. Another method is advanced oxidation technologies (AOPs), including ozonolysis, Fenton reactions, and electrochemical oxidation, which utilize strong oxidants (such as hydroxyl radicals) to decompose tetracycline. These methods have shown good degradation effects when treating high-concentration tetracycline wastewater, but the need for large amounts of chemical reagents can lead to high operating costs.

[0009] While physical and chemical methods are highly effective at degrading tetracycline antibiotics, their application is significantly limited by drawbacks such as high cost, difficulty in large-scale application, potential for secondary pollution, and even the generation of other non-target toxicities. Biodegradation, which utilizes interactions between organisms, is the most promising approach for tetracycline control in recent years.

[0010] Biodegradation, an environmentally friendly technology that utilizes microorganisms or enzymes to degrade tetracycline, is more conducive to environmental protection. It mainly includes enzyme-catalyzed degradation, where enzymes such as lignin peroxidase and laccase can catalyze the breakdown of tetracycline. Compared to microorganisms, enzymes are more specific and less affected by environmental conditions; however, the production and stability of these enzymes still require further research.

[0011] Microbial degradation, where microorganisms such as the widely used Pseudomonas aeruginosa and Bacillus spp., can degrade tetracycline into non-toxic or low-toxic metabolites under suitable conditions, offers advantages such as low cost and environmental friendliness, largely compensating for the shortcomings of physicochemical techniques. However, ensuring its degradation efficiency remains limited. Therefore, it is necessary to discover more new, highly efficient microbial strains and stable degradation methods to expand the range of options available for microbial degradation and ensure its treatment efficiency. Summary of the Invention

[0012] This invention provides a novel bacterial strain, *Enterobacter hormaechei* SD7-1, capable of degrading tetracycline antibiotics. This strain exhibits high degradation efficiency and a broad degradation spectrum, offering a better solution to the problem of tetracycline antibiotic accumulation in the environment. Through screening, identification, and evaluation of the strain's degradation performance, it was found that this strain can effectively degrade multiple tetracycline antibiotics in a relatively short time, providing a promising new microbial resource for environmental pollution control.

[0013] To address the problems in the prior art, the first objective of this invention is to provide a novel strain capable of degrading tetracycline antibiotics, and to name this strain Enterobacter hormaechei SD7-1.

[0014] A second objective of this invention is to provide the application of the aforementioned Enterobacter hormaechei SD7-1 strain in the treatment of tetracycline antibiotic degradation.

[0015] To achieve the above objectives, the present invention provides the following technical solution:

[0016] This invention provides a strain of Enterobacter hormaechei SD7-1, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 3, 2025, with accession number GDMCC No: 65849.

[0017] Furthermore, the present invention claims protection for the use of the Enterobacter hormaechei SD7-1 strain in the preparation of tetracycline-degrading antibiotic biological agents.

[0018] Furthermore, the tetracycline antibiotic is one or more of tetracycline, chlortetracycline, minocycline, and tigecycline.

[0019] This invention also provides a method for culturing substances that degrade tetracycline antibiotics, comprising the following steps:

[0020] S1. The Enterobacter hormaechei SD7-1 strain described above was cultured on a culture medium;

[0021] S2. Add antibiotics to the cultured bacterial solution from 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: 37°C and 200 rpm for 24 hours.

[0024] This invention also claims protection for the bacterial cells and / or fermentation broth obtained by the method of culturing substances that degrade tetracycline antibiotics.

[0025] Furthermore, this invention protects the use of the aforementioned bacterial cells and / or fermentation broth in the prevention and treatment of tetracycline-degrading antibiotics.

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

[0027] The novel strain provided by this invention exhibits a significant degradation effect on tetracycline antibiotics, achieving a degradation rate of over 98% after 24 hours of treatment. Therefore, Enterobacter horneri SD7-1, as a functional strain, possesses high degradation efficiency and a broad degradation spectrum. This strain can effectively degrade various tetracycline antibiotics in a relatively short time, providing a promising new microbial resource for controlling pollution caused by the widespread use of tetracycline, further reducing the environmental half-life emissions of tetracycline, and protecting the environment, especially soil and water environments. Attached Figure Description

[0028] Figure 1 The results of plate antibacterial experiments for tetracycline, chlortetracycline, minocycline, and tigecycline are presented.

[0029] Figure 2 The results of LC-MS / MS assays for tetracycline degradation by Enterobacter hormaechei SD7-1 strain. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, preparation is carried out according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

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

[0032] Strain source: Pet feces samples were collected from a pet hospital in Liaocheng City, Shandong Province. We isolated and identified bacterial strains from the fecal samples. Feces were swab-dipped into 600 μl of LB broth (Guangdong Huankai Microbial Technology Co., Ltd.) and incubated overnight at 37°C and 180 rpm. Subsequently, the culture was streaked onto LB agar plates containing 50 μg / ml tetracycline (Guangdong Huankai Microbial Technology Co., Ltd.) and incubated overnight at 37°C. Plump colonies on the plates were collected, purified, and preserved.

[0033] 16S rDNA strain identification

[0034] A loopful of the purified bacterial strain was scraped off, and the genome of the strain was extracted according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit (Tiangen Biotech Co., Ltd.). Subsequently, the 16S rDNA gene was amplified by PCR, and the PCR products were electrophoresed on a 1.2% agarose gel. The PCR amplification system (20 μl) using the Tiangen PCR MIX system was as follows: 10 μl 2xPCR MIX, 1 μl upstream primer, 1 μl downstream primer (the upstream and downstream primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), 1 μl template DNA, and 7 μl ddH2O. The 16S rDNA primers were synthesized by Suzhou Genewiz Co., Ltd. The PCR program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 53℃ annealing for 45 s, 72℃ extension for 1 min 30 s, 30 cycles; and a final extension at 72℃ for 10 min.

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

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

[0037] Colony morphology characteristics and physiological and biochemical properties

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

[0039] After identification and analysis, the new strain was determined to be *Enterobacter hormaechei*. Preservation information is as follows:

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

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

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

[0043] Example 2: Determination of Minimum Inhibitory Concentration (MIC)

[0044] The MICs of Enterobacter hormaechei SD7-1 against tetracycline, chlortetracycline, doxycycline, minocycline, and tigecycline (all purchased from Sigma-Aldrich, USA) were determined using the micro-broth dilution method, according to the relevant standards of the Clinical and Laboratory Standards Institute (CLSI: M100-S26). Escherichia coli ATCC25922 was used as the quality control strain.

[0045] The results (Table 1) showed that the EnterobacterhormaecheiSD7-1 strain exhibited different levels of drug sensitivity to tetracycline, chlortetracycline, doxycycline, minocycline, and tigecycline, with values ​​greater than 512 ug / ml, 512 ug / ml, 64 ug / ml, 128 ug / ml, and 4 ug / ml, respectively.

[0046] Table 1. MICs of EnterobacterhormaecheiSD7-1 against tetracycline-like drugs

[0047]

[0048] Note: TC, Tetracycline; CTC, Chlortetracycline; DOX, Doxycycline; MIN, Minocycline; TGC, Tigecycline.

[0049] Example 3: Tetracycline Drug Degradation Experiment

[0050] The E. coli ATCC25922 culture in the logarithmic growth phase was diluted 1:10. 100 μl of the diluted culture was evenly spread onto a fresh MH agar plate using a spreader. Subsequently, three 6 mm diameter wells were punched in the MH agar medium, and the bottom was sealed with sterile MH agar.

[0051] Each tetracycline drug was divided into three test groups, and the tests were conducted in 2 ml EP tubes, with a total EP tube volume of 400 μl.

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

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

[0054] 3. Control group: MH broth + Enterobacter hormaechei SD7-1 strain (without added drugs)

[0055] Three groups of samples were incubated on a 37°C shaker at 220 rpm for 24 hours, followed by centrifugation at 12000 rpm. The supernatant was collected and filtered through a 0.22 μm filter membrane. 20 μL of the filtrate was then added to the corresponding well of MH agar medium coated with ATCC25922. After the filtrate was completely absorbed by the agar, the plates were incubated overnight at 37°C.

[0056] Finally, images were captured using a standard digital camera, and the degradation ability of EnterobacterhormaecheiSD7-1 against tetracycline drugs was determined based on the size of the inhibition zone. Results ( Figure 1 The results showed that the control groups of the four tetracycline drugs all exhibited obvious inhibition zones, while the positive groups showed no obvious inhibition zones or inhibition zones that were significantly smaller than those in the control groups, indicating that strain SD7-1 has a degradation effect on tetracycline drugs. No antibacterial effect was observed in the blank group.

[0057] Example 4

[0058] A single colony of purified Enterobacter hormaechei SD7-1 was picked and inoculated into 4 ml of MH broth, and incubated at 37°C and 200 rpm for 8 hours. 40 μL of the bacterial culture was then transferred to 4 ml of MH broth containing 1.5 μg / ml tetracycline (supplemented with 500 μm magnesium sulfate and 100 μm NADPH; alternatively, commercially available MH broth can be used without these supplements). The broth was incubated at 37°C and 200 rpm in the dark for 24 hours. Simultaneously, MH broth containing only tetracycline (supplemented with 500 μm magnesium sulfate and 100 μm NADPH) was also used. The bacterial culture was then centrifuged at 12,000 rpm for 2 min and filtered through a 0.22 μm filter (Tianjin Jinteng Laboratory Equipment Co., Ltd.). The supernatant after filtration was diluted 10-fold and then analyzed by LC-MS / MS according to the conventional method (Kevin J. Forsberg et al., 2015, Chemistry & Biology). Figure 2 The above experimental group and control group each had 3 replicates.

[0059] The results showed that Enterobacter hormaechei SD7-1 exhibited good degradation activity against tetracycline drugs. Specifically, compared with the control group, the experimental group supplemented with Enterobacter hormaechei SD7-1 achieved a 51.95% degradation rate of tetracycline after 24 hours (p<0.0001).

[0060] The above embodiments of the present invention are merely examples to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A strain of Enterobacter cholerae ( Enterobacter hormaechei SD7-1 strain, characterized in that, The accession number is GDMCC No: 65849.

2. The application of the Enterobacter SD7-1 strain of claim 1 in the preparation of tetracycline-degrading biological agents, characterized in that, The tetracycline antibiotics are one or more of tetracycline, chlortetracycline, minocycline, and tigecycline.

3. A method for degrading tetracycline antibiotics, characterized in that, Includes the following steps: S1. Inoculate the Enterobacter SD7-1 strain of claim 1 onto a culture medium; S2. The bacterial culture obtained in step S1 is used to degrade tetracycline antibiotics, wherein the tetracycline antibiotics are one or more of tetracycline, chlortetracycline, minocycline, and tigecycline.

4. The method for degrading tetracycline antibiotics according to claim 3, characterized in that, The culture medium for step S1 is MH broth.

5. The method for degrading tetracycline antibiotics according to claim 4, characterized in that, Step S1 involves culturing at 37°C and 200 rpm for 8 hours; Step S2 involves culturing at 37°C and 200 rpm for 24 hours.

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