Marine source beta-lactam antibiotic biodegradation bacterium and application thereof

Abstract

The invention discloses a marine-derived beta-lactam antibiotic biodegradation bacterium and an application of the marine-derived beta-lactam antibiotic biodegradation bacterium. The marine-derived beta-lactam antibiotic biodegradation bacterium is separated and identified from coastal seawater, and has the capacity of degrading beta-lactam antibiotics. Researches show that the DT01 strain has a certain degradation effect on beta-lactam antibiotics such as benzylpenicillin potassium, amoxicillin and ceftriaxone sodium, and the DT01 strain has an excellent degradation capability on the benzylpenicillin potassium; and the DT01 strain has good temperature and pH adaptability and penicillin potassium tolerance, can rapidly grow in a severe environment, can be better used for treatment of antibiotic environmental pollution, provides a new effective repair scheme for beta-lactam antibiotic pollution, and makes a contribution to biological treatment of antibiotic pollution.

Description

Technical Field

[0001] The present application relates to the technical field of microorganisms and antibiotic degradation, and in particular to a marine-derived β-lactam antibiotic biodegrading bacterium and its application. Background Art

[0002] Antibiotics are crucial protectors of human and animal health. However, their widespread use also has adverse effects on humans, animals, and the environment. Because these antibiotics are not effectively absorbed or degraded by humans and animals, some are released into the environment through excretion after use. Studies have shown that 23.4% of pharmaceutical antibiotics and 55.8% of veterinary antibiotics remain in the environment. These antimicrobials accumulate in wastewater and other environmental environments, selectively affecting susceptible environmental bacteria, leading to imbalanced community structure, loss of function, and the spread of antibiotic resistance, potentially rendering antibiotics useless for both humans and animals. β-lactams, the most popular antibiotic class, are widely used in both human and veterinary medicine. This leads to the release of large quantities of antibiotics into the environment as metabolites or even in their original form, a contamination known as antibiotic pollution. Antibiotics not only contribute to the development of resistance in pathogens, increasing the effective dose of antibiotics required to kill bacteria, but also, long-term exposure to the environment can lead to the development of resistance in susceptible bacteria. Furthermore, resistance genes can spread and evolve in the environment, posing a potential threat to the ecological environment and human health.

[0003] Therefore, protecting susceptible members of environmental bacteria is crucial. This can reduce antibiotic concentrations by degrading antibiotics, mitigate antibiotic pollution in the environment, and eliminate the need for susceptible bacteria to acquire their own resistance genes. As part of the ecosystem, environmental degrading bacteria offer the advantages of low cost and environmental friendliness in removing antibiotics. The marine environment is the most species-rich ecosystem, harboring a rich and diverse bacterial community. Especially in coastal waters, these bacteria are important drivers of biogeochemical cycles and the decomposition of organic matter. Numerous bacteria have been isolated from the ocean, capable of degrading various types of hydrocarbons and adapting to diverse environments. However, previous studies of marine-derived bacteria have not addressed the biodegradation of antibiotics, although some findings have revealed their potential role. Therefore, developing more marine-derived microbial strains capable of degrading β-lactam antibiotics in the environment is essential, based on marine-derived microbial resources and is of great significance for the environmental management of antibiotic pollution. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortage of bacterial species resources of β-lactam antibiotics in the existing degradation environment and provide a marine-derived β-lactam antibiotic biodegrading bacterium and its application.

[0005] The first object of the present invention is to provide a strain of Klebsiella mutans (Klebsiella variicola ) DT01 strain.

[0006] The second object of the present invention is to provide the application of Klebsiella mutans DT01 strain.

[0007] The third object of the present invention is to provide a degradation agent.

[0008] A fourth object of the present invention is to provide a product.

[0009] A fifth object of the present invention is to provide a method for degrading β-lactam antibiotics.

[0010] The sixth object of the present invention is to provide a method for treating an environment polluted by β-lactam antibiotics.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a strain of Klebsiella mutans ( Klebsiella variicola ) DT01 strain, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 25, 2025, with the deposit number GDMCC No: 66214.

[0012] The present invention isolated and identified a bacterial strain DT01 with the ability to degrade antibiotics from coastal seawater. The genome of the strain was studied and it was found that the colony morphology of the DT01 strain was smooth and moist white round. Molecular identification showed that the DT01 strain was similar to Klebsiella variicola The origin of the bacteria was high and it was identified as Klebsiella mutans ( Klebsiella variicola ) strain DT01. Studies have shown that the DT01 strain can degrade three commonly used β-lactam antibiotics: penicillin potassium, amoxicillin, or ceftriaxone sodium. DT01 has excellent degradation capacity for penicillin potassium, degrading 77.9% of penicillin potassium, 52.76% of ceftriaxone sodium, and 20.56% of amoxicillin within 12 hours. Furthermore, the DT01 strain exhibits excellent temperature and pH adaptability and tolerance to penicillin potassium, allowing it to grow rapidly in harsh environments. This strain has the potential to degrade penicillin potassium in these environments, making it well-suited for the remediation of antibiotic environmental pollution and providing a new and effective remediation solution for β-lactam antibiotic contamination.

[0013] Therefore, the present invention provides the use of the DT01 strain or its bacterial solution in the degradation of β-lactam antibiotics.

[0014] The present invention provides use of the DT01 strain or its bacterial liquid in preparing a β-lactam antibiotic degradation product.

[0015] The present invention provides application of the DT01 strain or its bacterial solution in the treatment of antibiotic environmental pollution.

[0016] Preferably, the antibiotic environmental pollution refers to β-lactam antibiotic environmental pollution.

[0017] Preferably, the β-lactam antibiotic is one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

[0018] The present invention provides a degradation agent containing the DT01 strain or its bacterial liquid.

[0019] The present invention provides a product containing the above-mentioned degradation agent.

[0020] The present invention provides a method for degrading beta-lactam antibiotics, which uses the DT01 strain or the above-mentioned degradation agent for treatment.

[0021] The present invention also provides a method for treating an environment polluted by β-lactam antibiotics, which uses the DT01 strain or the above-mentioned degradation agent to treat the polluted environment.

[0022] The present invention has the following beneficial effects: This study isolated and identified a marine-derived β-lactam antibiotic biodegrading bacterium from coastal seawater, which has the ability to degrade β-lactam antibiotics. Studies have shown that the DT01 strain has a certain degradation effect on β-lactam antibiotics: penicillin potassium, amoxicillin, and ceftriaxone sodium, with DT01 having excellent degradation ability for penicillin potassium. Furthermore, the DT01 strain has good temperature and pH adaptability, as well as tolerance to penicillin potassium, allowing it to grow rapidly in harsh environments. This makes it suitable for the remediation of antibiotic environmental pollution, providing a new and effective remediation solution for β-lactam antibiotic pollution and contributing to the bioremediation of antibiotic pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the colony morphology of DT01.

[0024] Figure 2 This is a phylogenetic tree based on 16S rRNA sequencing.

[0025] Figure 3 The degradation results of DT01 on penicillin potassium (A) and ceftriaxone sodium (B) are shown.

[0026] Figure 4 The results show the effects of different temperatures (A), initial pH (B) and initial drug concentrations (C) on the growth of DT01. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0029] Penicillin potassium, amoxicillin, and ceftriaxone sodium used in the examples were purchased from Shanghai Macklin Co., Ltd. MSM culture medium was purchased from Beijing Coolabor Co., Ltd. LB broth medium was purchased from Beijing Luqiao Technology Co., Ltd.

[0030] Example 1 Isolation and identification of bacteria 1. Sampling Coastal seawater samples were obtained in Zhanjiang, South China (110.41924 E, 21.20237 N). After sampling, the samples were stored in portable ice boxes and transported to the laboratory for pretreatment within 2 h.

[0031] 2. Isolation of degradation bacteria To remove non-resistant bacteria and select bacteria that degrade β-lactam antibiotics, 1 mL of seawater was placed in 50 mL of LB broth containing 100 mg / L amoxicillin and incubated overnight at 30°C and 180 rpm. 1 mL of bacterial suspension was centrifuged at 4°C and 12,000 rpm for 30 minutes, the supernatant was discarded, and the suspension was resuspended in MSM medium containing 100 mg / L amoxicillin and the bacterial concentration was adjusted until the OD value reached 0. 600 With nm = 1.0, inoculate 50 mL of the aforementioned MSM medium at a 1% (v / v) inoculum. After culturing at 30°C and 180 rpm for 5 days, spread 100 μL of the bacterial solution onto MSM agar solid medium containing 100 mg / L amoxicillin and incubate at 30°C for 48 hours. Pick a single colony and culture it in LB broth. After shaking overnight, add 20% (v / v) glycerol, mix thoroughly, and store at -20°C.

[0032] 3. Identification of degrading bacteria Morphological identification: observe the colony morphology by streaking the plate, take the activated strain for Gram staining, and observe the bacterial and spore morphology by optical microscope. Figure 1 As shown, smooth, moist, white, round colonies appear on LB plate culture medium, which are Gram-negative bacteria.

[0033] Molecular identification: Single colonies were picked and the DNA of the strains was extracted using the boiling method. The DNA was sent to Shanghai Sangon Biotechnology Service Co., Ltd. for 16s rRNA sequencing and a phylogenetic tree was drawn. The BLAST comparison results of 16s rRNA sequencing are as follows: Figure 2 As shown, the strain Klebsiella variicola There is 97.48% homology, combined with morphological identification, the taxonomic status of the strain was identified as Klebsiella variegata ( Klebsiella variicola ), and named it DT01 strain, and deposited it in Guangdong Provincial Microbiological Culture Collection Center on April 25, 2025, with the deposit number GDMCC No: 66214, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0034] Example 2 Degradation performance determination of DT01 strain 1. Rapid degradation test To test the degradation ability of the DT01 strain on penicillin potassium, amoxicillin, and ceftriaxone sodium, the DT01 strain was placed in LB medium and shaken at 30°C and 180 rpm overnight. 3 mL of the bacterial solution was centrifuged at 4°C and 8000 rpm for 30 min and resuspended in MSM medium containing 100 mg / L penicillin potassium. The bacterial solution concentration was adjusted to OD 600 nm = 1.0, and a 1% inoculum was added to 3 mL of MSM culture medium containing 100 mg / L penicillin potassium. A control group without bacteria was established, and three replicates were performed for both the experimental and control groups. The cells were placed in a shaker and degraded at 30°C, 180 rpm for 12 hours. The penicillin potassium concentration in the MSM culture medium was then measured. The degradation effect of DT01 on amoxicillin and ceftriaxone sodium was tested using the same method.

[0035] 2. Analysis Method Quantitative determination of antibiotics was performed using a high-performance liquid chromatography system (HPLC, SHIMADZU LC-16P, Japan) equipped with a ShimNex CS C18 column (4.6 × 250 mm, 5 μm, SHIMADZU, Japan). Mobile phases A and B consisted of ultrapure water + 0.1% formic acid and acetonitrile + 0.1% formic acid, respectively. Detailed operating conditions are shown in Table 1. All samples were filtered through a 0.22 μm filter membrane before injection. The detection conditions for penicillin potassium were a mobile phase A:B ratio of 40%:60%, a flow rate of 1 mL / min, a detection wavelength of 210 nm, an injection volume of 20 μL per sample, column temperature maintained at ambient temperature, and a detection time of 13 min. The detection conditions for amoxicillin were a mobile phase A:B ratio of 80%:20%, a flow rate of 1 mL / min, a detection wavelength of 210 nm, an injection volume of 20 μL per sample, column temperature maintained at ambient temperature, and a detection time of 10 min. The detection conditions for ceftriaxone sodium were as follows: mobile phase A:B = 70%:30%, flow rate of 1 mL / min, detection wavelength of 254 nm, injection volume of 10 μL each time, column temperature the same as ambient temperature, and detection time of 6 min.

[0036] Table 1 Analysis conditions

[0037] The antibiotic concentrations of the experimental group and the blank control group at 0 h and 12 h were measured by liquid chromatography, and the degradation capacity of the strain was calculated according to the following formula: Biodegradation rate (%) = (C CK -C t ) / C0× 100%; Where: C CK is the negative control antibiotic concentration at 12 h (mg / L), C t is the antibiotic concentration after biodegradation (mg / L), and C0 is the antibiotic concentration in the culture medium at 0 h.

[0038] The results showed that the DT01 strain had excellent degradation ability for penicillin potassium, degrading 77.9% of penicillin potassium within 12 hours; it also had a certain degradation effect on ceftriaxone sodium, degrading 52.76% of ceftriaxone sodium within 12 hours; while the DT01 strain only degraded 20.56% of amoxicillin.

[0039] 3. Drawing of degradation curve The DT01 strain was incubated in LB medium and shaken at 30°C and 180 rpm overnight. A 50 mL aliquot of the bacterial suspension was centrifuged at 8000 rpm for 30 min at 4°C and resuspended in MSM medium containing 100 mg / L penicillin potassium. The bacterial suspension was adjusted to an OD600nm of 1.0 and added to 50 mL of MSM medium containing 100 mg / L penicillin potassium at a 1% inoculum. A control group without bacterial inoculation was established, and three replicates were performed for both the experimental and control groups. The cells were shaken at 30°C and 180 rpm for 120 h. The penicillin potassium concentration was determined every 12 h by high-performance liquid chromatography, and degradation curves were plotted. The degradation of ceftriaxone sodium by DT01 was tested using the same method.

[0040] Draw a time-degradation curve as Figure 3 As shown in the results, DT01 can degrade 75.56% of penicillin potassium within 120 h ( Figure 3 A) and 24.74% of ceftriaxone sodium ( Figure 3 B in ).

[0041] Example 3 Effects of different environmental factors on the growth of DT01 The DT01 strain was placed in LB medium and shaken at 30°C and 180 rpm for 12 h. After taking out, the OD 600 The bacterial solution was inoculated into LB medium at a ratio of 1% (v / v) and the OD was measured every 2 h. 600 Plot the growth curves of DT01 at different culture temperatures (20, 25, 30, 35, 40°C), initial pH values (5, 6, 7, 8, 9), and initial drug concentrations (0, 50, 100, 200, 300 mg / L). Select the temperature, pH, and drug concentration suitable for bacterial growth.

[0042] The results of the test are as follows Figure 4 As shown, the DT01 strain grew well at 25℃, 30℃, and 35℃, but grew slowly at 40℃ and 20℃, indicating good temperature adaptability ( Figure 4 A in the figure); the best growth effect was achieved when the initial pH was 7, but it also grew well at pH 5-9, showing good pH adaptability ( Figure 4 B); DT01 has excellent tolerance to penicillin potassium. The growth of the experimental group with the addition of antibiotics was inhibited within 4 hours, but after 10 hours of culture, there was almost no effect on bacterial growth ( Figure 4 C in the figure). This shows that the DT01 strain is a penicillin potassium-degrading bacterium with good temperature and pH adaptability, can grow rapidly in harsh environments, and has the potential to degrade penicillin potassium in harsh environments.

[0043] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Klebsiella mutans ( Klebsiella variicola ) DT01 strain, characterized in that This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 25, 2025, with the collection number GDMCC No: 66214.

2. Use of the DT01 strain or its bacterial solution according to claim 1 in the degradation of β-lactam antibiotics.

3. Use of the DT01 strain or its bacterial solution according to claim 1 in the preparation of β-lactam antibiotic degradation products.

4. Use of the DT01 strain or its bacterial solution according to claim 1 in the treatment of environmental pollution caused by antibiotics.

5. The application according to claim 4, characterized in that: The antibiotic environmental pollution refers to β-lactam antibiotic environmental pollution.

6. The use according to any one of claims 2 to 3 or claim 5, characterized in that: The β-lactam antibiotics are one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

7. A degradation agent, characterized in that Containing the DT01 strain according to claim 1 or its bacterial liquid.

8. A product, characterized in that Containing the degradation agent according to claim 7.

9. A method for degrading β-lactam antibiotics, characterized in that: The treatment is carried out using the DT01 strain according to claim 1 or the degradation agent according to claim 7.

10. A method for treating environmental pollution caused by β-lactam antibiotics, characterized in that: The DT01 strain according to claim 1 or the degradation agent according to claim 7 is used to treat the polluted environment.

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