Marine source beta-lactam antibiotic biodegradation bacteria and application thereof

Abstract

The application discloses a marine source beta-lactam antibiotic biodegradation bacterium and application thereof. The marine source beta-lactam antibiotic biodegradation bacterium is isolated and identified from coastal seawater and has the ability of degrading beta-lactam antibiotics. Research shows that the DT01 strain has certain degradation effect on beta-lactam antibiotics, such as penicillin potassium, amoxicillin and ceftriaxone sodium, wherein the DT01 has excellent degradation capacity on penicillin potassium; and the DT01 strain has good temperature and pH adaptability and penicillin potassium resistance, can grow rapidly in a harsh environment and can be better used for the treatment of antibiotic environmental pollution, thereby providing a new effective repair scheme for beta-lactam antibiotic pollution and making contribution to the biological treatment of antibiotic pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms and antibiotic degradation, in particular to a marine source beta-lactam antibiotic biodegradation bacterium and application thereof. BACKGROUND

[0002] Antibiotics are important protectors of human and animal life health. However, their extensive use has also caused adverse effects on humans, animals and the environment. Since these antibiotics cannot be effectively absorbed or degraded by the human and animal body, some of them are released into the environment in the form of excrement after use. Studies have shown that 23.4% of pharmaceutical antibiotics and 55.8% of veterinary antibiotics still exist in the environment. These antibacterial agents accumulate in wastewater or other environments, selectively affecting susceptible environmental bacteria, leading to community structure imbalance, loss of function and spread of antibiotic resistance, which can result in antibiotics being useless to both humans and animals. As the most popular antibiotics, beta-lactam drugs are widely used in human and veterinary medicine, resulting in a large amount of antibiotics being discharged into the environment in the form of metabolites or even original state, causing pollution, which is called antibiotic pollution. Antibiotics not only cause pathogenic microorganisms to develop resistance, making the effective dose of antibiotics to kill bacteria increase, but also, when discharged into the environment for a long time, can cause the enhancement of drug resistance of susceptible bacteria. Moreover, drug resistance genes can spread and evolve in the environment, posing a potential threat to the ecological environment and human health.

[0003] Therefore, it is crucial to protect susceptible members of environmental bacteria by degrading antibiotics to reduce their concentration, alleviate antibiotic pollution in the environment, and eliminate the need for susceptible bacteria to acquire their own resistance genes. As part of the ecosystem, environmental degradation bacteria have the advantages of low cost and environmental protection in removing antibiotics. The marine environment is the most diverse in terms of species diversity in the ecosystem, with a rich variety of bacterial populations. Especially in coastal waters, these bacteria are important drivers of biogeochemical cycles and organic matter decomposition. A large number of bacteria have been isolated from the ocean, which can degrade various types of hydrocarbons and adapt to different environments. However, previous studies on bacteria from the ocean did not involve the biodegradation of antibiotics, although some research results revealed their potential role. Therefore, it is necessary to develop more beta-lactam antibiotic-degrading microorganisms based on marine source microbial resources, which is of great significance for the environmental management of antibiotic pollution. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the shortcomings of existing beta-lactam antibiotic-degrading bacterial resources in the environment, and to provide a marine source beta-lactam antibiotic biodegradation bacterium and application thereof.

[0005] The first object of the present application is to provide a Klebsiella variicola strain (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] The 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:

[0012] 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 collection number GDMCC No: 66214.

[0013] 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.

[0014] Therefore, the present invention provides the use of the DT01 strain or its bacterial solution in the degradation of β-lactam antibiotics.

[0015] The present invention provides use of the DT01 strain or its bacterial liquid in preparing a β-lactam antibiotic degradation product.

[0016] The application provides application of a DT01 strain or a bacterial liquid thereof in antibiotic environmental pollution treatment.

[0017] Preferably, the antibiotic environmental pollution refers to beta-lactam antibiotic environmental pollution.

[0018] Preferably, the beta-lactam antibiotic is one or more of penicillin potassium, amoxicillin and ceftriaxone sodium.

[0019] The application provides a degrading agent containing the DT01 strain or the bacterial liquid thereof.

[0020] The application provides a product containing the degrading agent.

[0021] The application provides a method for degrading beta-lactam antibiotics, which adopts the DT01 strain or the degrading agent.

[0022] The application also provides a method for treating beta-lactam antibiotic contaminated environment, which adopts the DT01 strain or the degrading agent to treat the contaminated environment.

[0023] The application has the following beneficial effects:

[0024] The application separates and identifies a marine beta-lactam antibiotic biodegradation bacterium from coastal seawater, and the bacterium has the ability to degrade beta-lactam antibiotics. Research shows that the DT01 strain has a certain degradation effect on beta-lactam antibiotics, such as penicillin potassium, amoxicillin and ceftriaxone sodium, wherein the DT01 has excellent degradation ability for penicillin potassium; and the DT01 strain has good temperature and pH adaptability and penicillin potassium resistance, can grow rapidly in harsh environment, and can be better used for antibiotic environmental pollution treatment, thereby providing a new effective repair scheme for beta-lactam antibiotic pollution, and making contributions to biological treatment of antibiotic pollution. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a colony morphology diagram of the DT01.

[0026] Figure 2 It is a phylogenetic tree diagram based on 16S rRNA sequencing.

[0027] Figure 3 It is a degradation result diagram of the DT01 for penicillin potassium (A) and ceftriaxone sodium (B).

[0028] Figure 4 It is a result diagram of effects of different temperatures (A), initial pH (B) and initial drug concentrations (C) on growth of the DT01. DETAILED DESCRIPTION

[0029] The present application is further described in conjunction with the accompanying drawings and specific examples, which do not limit the present application in any manner. Unless otherwise specified, the reagents, methods and apparatus employed in the present application are those conventional in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] The penicillin potassium, amoxicillin and ceftriaxone sodium used in the examples are purchased from Shanghai Maikelin Company. The MSM medium is purchased from Beijing Coolabor Company. The LB broth medium is purchased from Beijing Luqiao Technology Co., Ltd.

[0032] Example 1 Isolation and identification of bacteria

[0033] 1. Sampling

[0034] Coastal seawater samples were obtained in Zhanjiang, South China (110.41924 E, 21.20237 N). The samples were stored in portable ice boxes after sampling and transported to the laboratory within 2 hours for pretreatment.

[0035] 2. Isolation of degrading bacteria

[0036] In order 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 medium containing 100 mg / L of amoxicillin and shaken at 30°C and 180 rpm overnight. 1 mL of bacterial solution was centrifuged at 4°C and 12000 rpm for 30 minutes, the supernatant was discarded, and the bacterial solution was resuspended in MSM medium containing 100 mg / L of amoxicillin and adjusted to a concentration of OD 600 nm=1.0, and inoculated in 50 mL of the above MSM medium at an inoculation amount of 1% (v / v). After 5 days of culture at 30°C and 180 rpm, 100 μL of bacterial solution was spread on MSM agar solid medium containing 100 mg / L of amoxicillin, and incubated at 30°C for 48 h. Single colonies were picked and cultured in LB broth medium, and 20% (v / v) of glycerol was added after overnight shaking, and the mixture was stored at -20°C.

[0037] 3. Identification of degrading bacteria

[0038] Morphological identification: The colony morphology was observed by plate streaking method, and the activated strain was subjected to Gram staining, and the morphology of the bacterial body and spores was observed under an optical microscope. The colony morphology is shown in Figure 1 Fig. 1, which presents smooth and wet white round colonies on LB plate medium, and belongs to Gram-negative bacteria.

[0039] 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.

[0040] Example 2 Degradation performance determination of DT01 strain

[0041] 1. Rapid degradation test

[0042] 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.

[0043] 2. Analysis Method

[0044] The quantitative detection of antibiotics was performed using a high-performance liquid chromatography system (HPLC, SHIMADZU LC-16P, Japan) equipped with a ShimNex CS C18 chromatographic column (4.6 x 250 mm, 5 μm, SHIMADZU, Japan). The mobile phase A and the mobile phase B were ultrapure water + 0.1% formic acid and acetonitrile + 0.1% formic acid, respectively. The detailed conditions are shown in Table 1. Before injection, all samples must be filtered with a 0.22 μm filter membrane. The detection conditions of potassium penicillin were mobile phase A:B = 40%:60%, flow rate 1 mL / min, detection wavelength 210 nm, injection volume 20 μL, column temperature same as ambient temperature, and detection time 13 min. The detection conditions of amoxicillin were mobile phase A:B = 80%:20%, flow rate 1 mL / min, detection wavelength 210 nm, injection volume 20 μL, column temperature same as ambient temperature, and detection time 10 min. The detection conditions of ceftriaxone sodium were mobile phase A:B = 70%:30%, flow rate 1 mL / min, detection wavelength 254 nm, injection volume 10 μL, column temperature same as ambient temperature, and detection time 6 min.

[0045] Table 1 Analysis conditions

[0046]

[0047] 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:

[0048] Biodegradation rate (%) = (C CK -C t ) / C0x 100%;

[0049] In the formula, C CK is the antibiotic concentration of the negative control at 12 h (mg / L), C t is the antibiotic concentration after biodegradation (mg / L), and C0is the antibiotic concentration in the culture medium at 0 h.

[0050] The results showed that the DT01 strain had excellent degradation capacity for potassium penicillin, degrading 77.9% of the potassium penicillin within 12 h; it also had a certain degradation effect on ceftriaxone sodium, degrading 52.76% of the ceftriaxone sodium within 12 h; while the DT01 strain only degraded 20.56% of amoxicillin.

[0051] 3. Preparation of degradation curve

[0052] 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.

[0053] 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 ).

[0054] Example 3 Effects of different environmental factors on the growth of DT01

[0055] 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.

[0056] 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.

[0057] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

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, characterized in that: The β-lactam antibiotics are one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

3. Use of the DT01 strain or its bacterial solution according to claim 1 in preparing a β-lactam antibiotic degradation product, characterized in that: The β-lactam antibiotics are one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

4. The use of the DT01 strain or its bacterial solution according to claim 1 in the treatment of antibiotic environmental pollution, characterized in that: The antibiotic environmental pollution refers to β-lactam antibiotic environmental pollution; the β-lactam antibiotics are one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

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

6. A product, characterized in that Containing the degradation agent according to claim 5.

7. A method for degrading β-lactam antibiotics, characterized in that: The DT01 strain according to claim 1 or the degradation agent according to claim 5 is used for treatment; the β-lactam antibiotic is one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

8. 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 5 is used to treat the polluted environment; the polluted environment refers to β-lactam antibiotic pollution; the β-lactam antibiotic is one or more of penicillin potassium, amoxicillin, and ceftriaxone sodium.

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