Fluoroquinolone antibiotic efficient degrading bacterium and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a fluoroquinolone antibiotic efficient degrading bacterium and application thereof. The fluoroquinolone antibiotic degrading bacterium is Kocuria sp. X03, the preservation number of the Kocuria sp. X03 is CCTCC NO: M 2025522, the invention provides the fluoroquinolone antibiotic degrading bacterium Kocuria sp. X03 separated from soil, the Kocuria sp. X03 can efficiently degrade fluoroquinolone antibiotics, an antibiotic degrading strain library is enriched, biotransformation mechanism research and bioremediation application are promoted, and the application prospect is wide. The strain can be used for degrading and eliminating livestock and poultry manure and residual antibiotics in farmland soil, and has certain research and application values.

Description

Technical Field

[0001] This specification relates to the field of microbial technology, and particularly to a highly efficient fluoroquinolone antibiotic-degrading bacterium and its application. Background Art

[0002] In the global antibiotic market, the consumption of fluoroquinolone antibiotics (FQs) accounts for 17% of the total, and they are widely used in many fields such as clinical medicine and aquaculture. They are one of the main types of antibiotics in various surface waters and soils. After being absorbed by the animal intestine, more than half of the FQs will be excreted from the body in the form of the original form or metabolites through feces and urine, resulting in a large amount of active ingredients entering the natural environment, and generating a series of more toxic metabolic and degradation intermediate products, inducing microorganisms to gradually develop resistance to them, affecting their normal growth or reproduction, causing the enrichment of drug-resistant flora and the generation of antibiotic resistance genes (ARGs). After entering the food chain, they ultimately pose a potential threat to human health.

[0003] Currently, bacteria that can degrade FQs screened from the environment include Ochrobactrum, Thermus, Paraclostridium, Bacillus, Lactobacillus, Micrococcus, Rhodococcus, Staphylococcus, Pseudomonas, and Enterobacter, etc. However, there are not many research examples on using highly drug-resistant strains to degrade fluoroquinolone antibiotics at present. And due to obstacles such as high-concentration mixed pollutants and limited availability of nutrients, the efficiency of inoculated microorganisms in the natural environment is poor, which is one of the main challenges in the effective degradation of new antibiotic pollutants by microorganisms in the natural environment.

[0004] The pollution of fluoroquinolone antibiotics poses a potential threat to human health. Microbial degradation is an effective method for removing antibiotics, and people are looking forward to microorganisms with strong drug resistance, high efficiency, and ecological safety characteristics. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the present application provides a highly efficient fluoroquinolone antibiotic-degrading bacterium and its application, in order to solve the above problems.

[0006] To achieve the above object, the present invention specifically adopts the following technical solutions.

[0007] In the first aspect of the present invention, a fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 is protected. It is Kocuria sp., and the deposit number is CCTCC NO: M 2025522.

[0008] In the second aspect of the present invention, a biological preparation is protected, which includes the aforementioned fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, its bacterial suspension, and culture solution.

[0009] In the third aspect of the present invention, the use of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 or the biological preparation in degrading fluoroquinolone antibiotics is protected.

[0010] In the fourth aspect of the present invention, a method for degrading fluoroquinolone antibiotics is protected. The aforementioned fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 or the aforementioned biological preparation is used to degrade the material to be degraded containing fluoroquinolone antibiotics.

[0011] The fluoroquinolone antibiotic-degrading bacterium and its application of the present invention have the following beneficial effects compared with the prior art:

[0012] 1. In order to screen out strains with the ability to efficiently degrade FQs, the commonly used, cheap, and broad-spectrum enrofloxacin (ENR) was selected as the target pollutant. A strain that can efficiently degrade ENR was screened from the farmland soil where pig manure is commonly applied through enrichment and domestication. It was identified as a bacterium belonging to Kocuria, and was named Kocuria sp. X03.

[0013] 2. The Kocuria sp. X03 isolated in the present invention has high degradation efficiency, the ability to simultaneously degrade multiple FQs drugs, and the toxicity of the biotransformation product is much lower than that of the parent compound.

[0014] 3. The present invention enriches the antibiotic-degrading bacterial species library, and at the same time promotes the research on biotransformation mechanisms and bioremediation applications. It has certain research and application values, and can further be used to degrade and eliminate residual antibiotics in livestock and poultry manure and farmland soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It shows the morphological diagram of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 of the present invention;

[0016] Figure 2 It shows the 16S rDNA sequence of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 of the present invention, which is the sequence shown in SEQ ID No. 1;

[0017] Figure 3 Shown is the growth and degradation curve of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 of the present invention. Detailed implementation mode

[0018] The present invention discloses a fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, and also discloses the application of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, which has a high efficiency in degrading fluoroquinolone antibiotics, has the ability to simultaneously degrade multiple fluoroquinolone antibiotic drugs, and the toxicity of the biotransformation product is much lower than that of the parent compound. The present invention provides more basis and conditions for the field of biodegradation of fluoroquinolone antibiotics and has high application value in industry.

[0019] To achieve the object of the present invention, the present invention provides a fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, which is Kocuria sp., and the preservation number is CCTCC NO: M 2025522.

[0020] The preservation information of the strain of this application is as follows:

[0021] Strain name: Fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03;

[0022] Preservation number: CCTCC NO: M 2025522;

[0023] Preservation date: March 18, 2025;

[0024] Name of the preservation unit: China Center for Type Culture Collection;

[0025] Abbreviation of the preservation unit: CCTCC;

[0026] Address of the preservation unit: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province

[0027] The fluoroquinolone antibiotic-degrading bacterium of this application is screened from farmland soil.

[0028] This strain was screened by the inventor from the farmland soil in Shanghai and was registered and preserved in the China Center for Type Culture Collection (abbreviation CCTCC) on March 18, 2025.

[0029] The biological morphology of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 is round, yellow, smooth, with a viscous surface and easy to pick up.

[0030] Among them, the 16S rDNA sequence of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 contains the sequence shown in SEQ ID No. 1. When the sequence was aligned with gene sequences on the NCBI website, it was found that the closest relative was Kocuria marina.

[0031]

[0032] The fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 provided by the present invention was screened from farmland soil where pig manure was frequently applied. Through 16S rDNA sequencing and morphological observation, it conforms to the genus Kocuria and can efficiently degrade fluoroquinolone antibiotics.

[0033] The degradation rate of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 provided by the present invention for fluoroquinolone antibiotics can reach more than 85%. Preferably, the fluoroquinolone antibiotics can be enrofloxacin (ENR), norfloxacin, ofloxacin, and moxifloxacin. Specifically, the fluoroquinolone antibiotic can be enrofloxacin.

[0034] The degradation rate of the present invention was determined by the following method: The strain Kocuria sp. X03 was inoculated into the basal medium containing 0.5 mg / L of enrofloxacin at an inoculation amount of 5%, and cultured with shaking at 30 °C and 150 rpm, and the residual degradation rate of enrofloxacin within 168 hours was measured.

[0035] The present invention also provides a biological preparation, comprising the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described above, its bacterial suspension, and culture solution, and the biological preparation is used for degrading fluoroquinolone antibiotics.

[0036] The present invention also provides the use of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described above or the biological preparation as described above in degrading fluoroquinolone antibiotics.

[0037] The present invention also provides a method for degrading fluoroquinolone antibiotics, using the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described above or the biological preparation as described above to degrade the material to be degraded containing fluoroquinolone antibiotics.

[0038] Furthermore, for the aforementioned fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 or biological preparation, based on the mass of the highly efficient fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, the inoculation amount is 4% - 6%, preferably 5%.

[0039] In the present invention, the mass concentration of fluoroquinolone antibiotics in the aforementioned material to be degraded is 0.5 - 1 mg / L, preferably 0.5 mg / L or 1 mg / L.

[0040] In a preferred embodiment, the aforementioned fluoroquinolone antibiotics can be enrofloxacin, norfloxacin, ofloxacin, and moxifloxacin. Specifically, the fluoroquinolone antibiotic can be enrofloxacin.

[0041] In the present invention, the temperature of the aforementioned degradation is 20 - 40 °C, preferably 30 °C; the pH value of the aforementioned degradation is preferably 6.5 - 7.5, preferably 7.0.

[0042] The culture media used in the following examples are as follows:

[0043] Basal medium: It is used for the preliminary screening and enrichment of ENR-degrading bacteria and the study of ENR degradation performance. The main components (per liter) are: 1.0 g of CH3COONa, 1.5 g of (NH4)2SO4, 1.5 g of K2HPO4, 0.5 g of KH2PO4, 0.2 g of MgSO4·7H2O, 5.0 g of NaCl, 1 mL of trace elements, pH = 7.0. When used for enriching degrading bacteria, a certain concentration of antibiotics is added to the medium, and when used for antibiotic degradation experiments, the corresponding volume of antibiotic stock solution is added.

[0044] LB medium: It is used for the purification and isolation of degrading bacteria. The main components (per liter) are: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1 mL of trace element solution, pH = 7.0. For solid medium, 20 g of agar is added. For the LB medium containing antibiotics, the corresponding volume of antibiotic stock solution should be added when it is cooled to about 60 °C.

[0045] Trace element solution: It is used to provide trace elements for the culture medium. The main components (per liter) are: 50 g of EDTA-2Na, 22 g of ZnSO4·7H2O, 11.4 g of H3BO3, 5.06 g of MnCl2·4H2O, 1.61 g of CoCl2·6H2O, (NH4)6Mo7O 24 ·4H2O 1.1 g, 5.0 g of FeSO4·7H2O, 0.50 g of CaCl2, pH = 7.0.

[0046] Example 1 - Enrichment, isolation, purification and re-screening of fluoroquinolone antibiotic-degrading bacteria

[0047] Place 1 g of soil sample in 100 mL of basal medium containing 0.5 mg / L of ENR, and culture it in a shaker at 30 °C and 150 rpm in the dark with oscillation. Every 2 days, suck 5 mL of the pre-acclimated culture into 100 mL of fresh basal medium containing ENR, and gradually increase the ENR concentration in the medium. Repeat the above steps until the ENR concentration reaches 5 mg / L. Use a pipette to suck 5 mL of the supernatant into a 50 mL test tube, add physiological saline to 50 mL, and repeat this step after shaking evenly until it is diluted 10 -8 times. From 10 -4 、10 -6 、10 -8100 μL was taken from the dilution solution of [multiple] times and spread on the LB solid medium with an ENR concentration of 1 mg / L. Three parallel samples were made for each dilution multiple, and they were cultured in the dark at 30 °C for 48 h. Single colonies with different shapes, sizes and colors were selected from the medium, and streaking was repeated on the fresh LB solid medium. After purification culture, the strains with regular colony morphology and faster growth were subcultured by streaking until the colony morphology on the medium was completely consistent, and pure strains were obtained.

[0048] Although many bacteria are resistant to specific antibiotics, they cannot effectively degrade antibiotics. The ENR degradation performance of the screened resistant bacteria was detected to re-screen the strains that can effectively degrade ENR. The pure strain was inoculated into 100 mL of LB medium containing 1 mg / L ENR and cultured in the dark in a shaker at 30 °C and 150 rpm for 24 h. Then, it was inoculated into 100 mL of basic medium containing 1 mg / L ENR at an inoculation amount of 5% and cultured in the dark in a shaker at 30 °C and 150 rpm for 7 d. The residual amount of ENR was detected by HPLC, and the degradation rate was calculated. Each experiment was repeated 3 times, and the strain with the highest ENR degradation rate was obtained and named X03. It was stored in a -80 °C refrigerator by the glycerol method and the slant streaking method respectively. Before use, the freezing tube was taken out and quickly thawed in a 37 °C constant temperature water bath. 1 mL of the thawed cell suspension was taken out and inoculated into the basic medium, and it was activated and expanded under the conditions of 30 °C and 150 rpm.

[0049] Example 2 - Identification of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03

[0050] After the strain X03 was spread and cultured on the LB solid medium for 48 h, the colonies were regular round, yellow, smooth, sticky on the surface and easy to pick up ( Figure 1 ). Optical microscopy examination found that the size of a single colony was about 2 - 3 mm, the color of the center was slightly darker, and the color of the edge was lighter. Gram staining was positive. Molecular biological identification was carried out on the strain X03, and a DNA fragment about 1431 bp in length was amplified by PCR of 16S rRNA ( Figure 2 ). The gene sequence was aligned in Genbank, and the homology of the strain with Kocuria marina was as high as 99.44%, confirming that the degrading bacterium was Kocuria marina, and it was further named Kocuriasp. X03.

[0051] Example 3 - Growth curve and degradation effect of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03

[0052] The fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 was inoculated into the basal medium containing 0.5 mg / L ENR at an inoculation amount of 5%, and cultured with shaking at 30 °C and 150 rpm. Samples were taken at 0, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144, and 168 h respectively. The OD value of the bacterial solution was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer to determine the growth curve of the strain. At the same time, the residual concentration of ENR was measured, and the degradation rate was calculated. The growth and degradation curve of the strain Kocuria sp. X03 is as Figure 3 shown. The strain began to grow rapidly after 8 h of inoculation and entered the logarithmic growth phase. At this time, the ENR concentration also began to decrease rapidly. At 48 h, the strain entered the stationary phase, and the biomass reached the maximum value. Within 96 h, the degradation rate of ENR was 79.9%. After that, the ENR concentration decreased slowly, and the bacteria entered the decline phase, and the OD 600 value of the culture solution decreased slowly. This is mainly because after 96 h, the bacteria no longer grew or the reproduction rate was lower than the death rate of the bacteria, which further indicated that the degradation of ENR mainly occurred during the growth stage of the biomass of Kocuria sp. X03. After 168 h, the degradation rate of ENR reached 87.6%.

[0053] The fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 of the present application was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit number: CCTCC NO: M 2025522) on March 18, 2025, and it was isolated and screened from the soil.

[0054] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are proposed in this specification, so such modifications, improvements, and corrections still fall within the spirit and scope of the exemplary embodiments of this specification.

[0055] At the same time, this specification uses specific terms to describe the embodiments of this specification. Such as "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that the "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification is not necessarily the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0056] In some embodiments, numbers are used to describe components and the quantity of attributes. It should be understood that such numbers used in the description of embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the stated numbers allow for a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may vary according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the method of retaining the general number of digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, such numerical settings are made as precise as possible within the feasible range.

[0057] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered to be consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, which is Kocuria, and its preservation number is CCTCC NO: M 2025522.

2. The Kocuria sp. X03, a fluoroquinolone antibiotic-degrading bacterium as described in claim 1, is characterized in that, The 16S rDNA sequence of the highly efficient fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 contains the sequence shown in SEQ ID No.

1.

3. The Kocuria sp. X03 for degrading fluoroquinolone antibiotics as described in claim 1, characterized in that, The biological morphology of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 is round, yellow, smooth, with a viscous surface and is easily picked up.

4. The fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described in claim 1, characterized in that, The degradation rate of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 for fluoroquinolone antibiotics reaches more than 85%. The fluoroquinolone antibiotics are selected from one or more of enrofloxacin, norfloxacin, ofloxacin, and moxifloxacin, preferably enrofloxacin.

5. A biological agent, characterized in that, Comprising the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described in any one of claims 1-3; and / or its bacterial suspension; and / or its culture.

6. Use of the fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described in any one of claims 1-3 or the biological preparation as described in claim 4 in degrading fluoroquinolone antibiotics.

7. The use according to claim 6, characterized in that, The fluoroquinolone antibiotics are selected from one or more of enrofloxacin, norfloxacin, ofloxacin, and moxifloxacin, preferably enrofloxacin.

8. A method for degrading fluoroquinolone antibiotics, characterized in that, Degrading the material to be degraded containing fluoroquinolone antibiotics by using the highly efficient fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03 as described in any one of claims 1-3 or the biological preparation as described in claim 4.

9. The method according to claim 8, wherein Based on the mass of the highly efficient fluoroquinolone antibiotic-degrading bacterium Kocuria sp. X03, the inoculation amount is 5%.

10. The method according to claim 8, characterized in that The temperature of the degradation is 20-40 °C; and / or the pH value of the degradation is 6-8.

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