Rausella ornithinolyticus SD8-1 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 novel bacterial strain is a strain of Raoultella ornithinolyticus SD8-1, the novel bacterial strain is preserved in the Guangdong Microbial Culture Collection Center on January 22, 2025, and the preservation number is GDMCC No: 65848. The invention further discloses a preparation method of the novel bacterial strain. The new strain provided by the invention has a remarkable degradation effect on tetracycline antibiotics, and the tetracycline degradation rate reaches 98% or above after 24-hour treatment. Therefore, the strain can effectively degrade various tetracycline antibiotics in a short time, and an efficient and environment-friendly solution is provided for the problem of residues of tetracycline antibiotic drugs.

Description

Technical Field

[0001] The invention belongs to the field of microbial strains and environmental microorganisms, and particularly relates to a Ralstonia ornithine-soluble SD8-1 strain and an application thereof in degrading tetracycline antibiotics. Background Art

[0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics, including tetracycline, oxytetracycline, chlortetracycline, doxycycline, minocycline, tigecycline and eravacycline. Due to their broad antibacterial spectrum, low cost and ease of use, tetracycline antibiotics are widely used in the treatment of infectious diseases in humans and animals and in animal growth promotion. However, due to the low absorption rate of tetracycline drugs, most of the unabsorbed drugs will be discharged into the environment in the form of prototype or active metabolites (Zheng Maojia et al., 2018, Tianjin Agricultural Sciences). According to relevant studies, the residual problem of tetracycline antibiotics in the environment is becoming increasingly serious. The average residues of chlortetracycline, tetracycline and oxytetracycline in the surface soil of farmland treated with livestock and poultry manure were 38, 13 and 12 times that of farmland without livestock and poultry manure, respectively, indicating that livestock and poultry manure is an important source of tetracycline in the environment (ZHANG, 2008, J Ecology and Rural Environ).

[0003] The residues of tetracycline antibiotics not only pollute the environment and affect soil and water quality, but also provide a breeding ground for the spread of drug-resistant bacteria and drug-resistant genes, which seriously threatens the ecological environment and public health. (Knapp CW et al., Environmental Science & Technology, 2010). Therefore, how to effectively degrade tetracycline antibiotics in the environment has become an important issue that needs to be solved in the field of environmental protection and public health.

[0004] As a green and environmentally friendly method, biodegradation has gradually become an important means to solve this problem due to its high efficiency and sustainability. Biodegradation methods mainly include plant degradation and microbial degradation, among which microbial degradation shows good prospects in the degradation of tetracycline antibiotics. Studies have shown that certain specific strains can effectively degrade tetracycline drugs in a relatively short period of time. For example, the degradation rate of tetracycline by Escherichia coli LHM10-1 reached 83.91% after 16 hours (CN110055192A), Chryseobacterium DDW4-2 degraded 48.4% of tetracycline within 16 hours (CN111088192A), and Elizabethkingia anopheles strain 279-2 degraded 64.2% of eravacycline within 16 hours (CN111893068A).

[0005] Although there are currently some effective microbial degradation strains, the degradation efficiency of tetracycline antibiotics still has certain limitations. Therefore, it is urgent to expand the existing bacterial library resources and develop more efficient strains to address the increasingly serious environmental pollution problem. By exploring and screening new strain resources, more effective solutions can be provided for the treatment of tetracycline antibiotic pollution. Summary of the Invention

[0006] The present invention provides a new strain, Raoultella ornithinolytica SD8-1, which can degrade tetracycline antibiotics. It 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.

[0007] To solve the above problems in the prior art, the first object of the present invention is to provide a new strain capable of degrading tetracycline antibiotics, and this strain is named Raoultella ornithinolytica SD8-1 strain.

[0008] The second object of the present invention is to provide the application of the above Raoultella ornithinolytica SD8-1 strain in the treatment and degradation of tetracycline antibiotics.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a Raoultella ornithinolytica SD8-1 strain, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 22, 2025, with the deposit number: GDMCC No: 65848.

[0011] Furthermore, the present invention claims the application of the Raoultella ornithinolytica SD8-1 strain in the preparation of a biological agent for degrading tetracycline antibiotics.

[0012] Furthermore, the tetracycline antibiotics are one or more of tetracycline, chlortetracycline, minocycline, and tigecycline.

[0013] The present invention also provides a method for culturing a substance with the ability to degrade tetracycline antibiotics, including the following steps:

[0014] S1. Culture the Raoultella ornithinolytica SD8-1 strain on the medium and incubate it at 37 °C and 200 rpm for 8 hours.

[0015] S2. Add antibiotics to the cultured bacterial liquid in step S1 for degradation.

[0016] Preferably, the medium in step S1 is MH broth; the degradation culture conditions in step S2 are: incubate at 37 °C and 200 rpm for 24 hours.

[0017] The present invention also claims the bacterial cells and / or fermentation broth obtained by the method for culturing the substance capable of degrading tetracycline antibiotics.

[0018] Furthermore, the present invention protects the application of the bacterial cells and / or fermentation broth in the prevention and treatment of tetracycline antibiotics degradation.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention provides a new strain Raoultella ornithinolytica SD8-1 capable of degrading tetracycline drugs, which has high degradation efficiency and a wide degradation spectrum. The new strain provided by the present invention has a significant degradation effect on tetracycline antibiotics, and the degradation rate of tetracycline reaches more than 98% after 24 hours of treatment. It provides a more effective solution for expanding the existing bacterial library resources, developing more efficient strains and treating tetracycline antibiotic pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Results of the plate antibacterial experiment for tetracycline, chlortetracycline, minocycline and tigecycline;

[0022] Figure 2 LC-MS / MS determination results of the degradation of tetracycline by the Raoultella ornithinolytica SD8-1 strain. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention. Those not specified in the embodiments are prepared according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased commercially.

[0024] Isolation, purification and preservation of Raoultella ornithinolytica SD8-1 in Example 1:

[0025] Source of the strain: A sewage sample was collected from a pig farm in Liaocheng City, Shandong Province. We carried out the work of isolating and identifying the strains from this sewage sample. 50 μl of the sewage was taken and added to 600 μl of LB broth (Guangdong Huankai Microbial Sci-Tech Co., Ltd.), and cultured overnight at 37 °C and 180 rpm. Subsequently, the above culture solution was streaked on an LB agar plate containing 50 μg / ml tetracycline (Guangdong Huankai Microbial Sci-Tech Co., Ltd.) and cultured overnight at 37 °C. The plump colonies on the petri dish were taken, purified and preserved.

[0026] 16S rDNA strain identification

[0027] A loop of the purified strain was scraped, and the genomic DNA of the strain was extracted with reference to the steps of the Tiangen Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). Subsequently, the 16S rDNA gene was amplified by PCR, and the PCR product was electrophoresed on a 1.2% agarose gel. The PCR amplification system (20 μl) using the Tiangen PCR MIX system was as follows: 10 μl of 2x PCR MIX, 1 μl of the upstream primer, 1 μl of the 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 the 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; and then extension at 72 °C for 10 min.

[0028] 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 analysis was carried out using the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). At the molecular level, the strain was identified as Raoultella ornithinolytica (the 16S rDNA gene is shown in SEQ ID NO: 3).

[0029] Colony morphological characteristics and physiological and biochemical characteristics

[0030] Raoultella ornithinolytica SD8-1 strain is transparent on MacConkey agar plates and is a capsulated Gram-negative, oxidase-negative, catalase-positive, aerobic, non-motile rod-shaped bacterium; the optimal growth temperature is 35-38°C.

[0031] After identification and analysis, the new strain was found to be Raoultella ornithinolytica. The deposit information is as follows:

[0032] Deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on January 22, 2025;

[0033] The deposit number is GDMCC No: 65848;

[0034] The storage address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0035] Example 2 Determination of Minimum Inhibitory Concentration (MIC)

[0036] The MICs of Raoultella ornithinolytica SD8-1 against tetracycline, chlortetracycline, doxycycline, minocycline and tigecycline (all purchased from Sigma-Aldrich, USA) were determined by the broth microdilution method in accordance with the relevant standards of the Clinical and Laboratory Standards Institute (CLSI: M100-S26). Escherichia coli ATCC25922 was used as the quality control strain.

[0037] The results showed (Table 1) that the Raoultella ornithinolytica SD8-1 strain showed different levels of drug sensitivity to tetracycline, chlortetracycline, doxycycline, minocycline and tigecycline, which were >512ug / ml, 512ug / ml, 32ug / ml, 64ug / ml and 8ug / ml respectively.

[0038] Table 1 MICs of Raoultella ornithinolytica SD8-1 against tetracycline-like drugs

[0039]

[0040] Note: TC, tetracycline; CTC, chlortetracycline; DOX, doxycycline; MIN, minocycline; TGC, tigecycline.

[0041] Example 3 Tetracycline drug degradation experiment

[0042] The bacterial solution of E. coli ATCC 25922 cultured to the logarithmic phase was diluted 1:10, and 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.

[0043] 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:

[0044] 1. Control group: Tetracycline drug + MH broth

[0045] 2. Positive group: Tetracycline drug + MH broth + Raoultella ornithinolytica SD8-1 strain

[0046] 3. Blank group: MH broth + Raoultella ornithinolytica SD8-1 strain (without adding drugs)

[0047] 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 filtrate was aspirated and dropped into the corresponding holes of the MH agar medium coated with ATCC 25922. After the filtrate was completely absorbed by the agar, it was inverted and cultured overnight in a 37 °C incubator.

[0048] Finally, images were taken using a standard digital camera, and the degradation ability of Raoultella ornithinolytica SD8-1 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 SD8-1 had a degradation effect on tetracycline drugs. No antibacterial effect was seen in the blank group.

[0049] Example 4

[0050] Pick a single colony of purified Raoultella ornithinolytica SD8-1 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, conventional 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 only tetracycline drugs (supplemented with 500 μM magnesium sulfate and 100 μM NADPH). Subsequently, centrifuge the bacterial culture solution at 12,000 rpm for 2 min and filter it through a filter head with a pore size of 0.22 μm (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), as Figure 2 shown. Set 3 parallels for each of the above experimental group and control group.

[0051] The results show that Raoultella ornithinolytica SD8-1 exhibits good degradation activity against tetracycline drugs. Among them, compared with the control group, the degradation rate of tetracycline by the experimental group supplemented with Raoultella ornithinolytica SD8-1 reached 98.71% after 24 hours (p < 0.0001).

[0052] The above embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit 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. A Raoultella ornithinolytica SD8-1 strain, characterized in that, It was deposited at the Guangdong Provincial Culture Collection of Microorganisms on January 22, 2025, with the deposit number: GDMCC No: 65848.

2. Use of the Raoultella ornithinolytica SD8-1 strain according to claim 1 in the preparation of a biological agent for degrading tetracycline antibiotics.

3. The application according to claim 2, wherein 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. Cultivate the Raoultella ornithinolytica SD8-1 strain according to claim 1 on a culture medium and culture it at 37 °C and 200 rpm for 8 hours. S2. Add the antibiotic 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; the degradation culture conditions in step S2 are: culture at 37 °C and 200 rpm for 24 hours.

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

7. Use of the bacterial cells and / or fermentation broth according to claim 6 in the prevention and treatment of degradation of tetracycline antibiotics.

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