Pandoraea sp., fungicide and application of pandoraea sp. In degradation of quinolone antibiotics

Abstract

The invention belongs to the technical field of microbial degradation, and particularly relates to pandoraea sp., a microbial agent and application of the pandoraea sp. In degradation of quinolone antibiotics. The Pandoraea sp. Is named as AYGW-1, and is preserved in the China Center for Type Culture Collection on March 10, 2025, and the preservation number is CCTCC M 2025415. The Pandoraea sp. Is named as Pandoraea sp. The Pandoraea sp. AYGW-1 has the function of degrading the quinolone antibiotics, provides an effective biodegradation scheme for treatment of pollution of the quinolone antibiotics in the environment, especially in a water body, and also provides a safe and environment-friendly solution for the residue problem of quinolone antibiotic drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation, and specifically relates to a Pandoraea bacterium, a bacterial agent, and their application in degrading quinolone antibiotics. Background Art

[0002] Quinolone antibiotics are a class of widely used antibacterial drugs. Due to their broad antibacterial spectrum, strong antibacterial activity and other advantages, they are widely used in the fields of medicine and animal husbandry. However, their extensive use has led to an increasing amount of quinolone antibiotics remaining in the environment. These antibiotics enter environmental media such as water bodies and soil through various channels, posing a potential threat to the ecosystem and human health.

[0003] Currently, the treatment methods for quinolone antibiotic pollution mainly include physical and chemical methods and biodegradation methods. Physical and chemical methods such as adsorption, oxidation-reduction, etc., although they can quickly reduce the antibiotic concentration, have problems such as high cost and secondary pollution. Biodegradation methods, on the other hand, have the advantages of low cost and environmental friendliness, but the efficiency of traditional microorganisms in degrading quinolone antibiotics is relatively low, and they have high requirements for environmental conditions.

[0004] In recent years, the research on microbial degradation of antibiotics has gradually received attention. Some studies have reported that specific microbial strains have the ability to degrade quinolone antibiotics. For example, patent CN117467583A discloses that Achromobacter sp. JK7 has strong degradation ability for quinolone antibiotics such as levofloxacin, moxifloxacin and enoxacin; patent CN114107065A discloses that Geotrichum sp. fungi have the potential to degrade quinolone antibiotics under low temperature conditions. Achromobacter sp.) fungi have the potential to degrade quinolone antibiotics under low temperature conditions. Geotrichum sp.) fungi have the potential to degrade quinolone antibiotics under low temperature conditions.

[0005] It can be seen that microbial degradation is a feasible method for treating antibiotic pollution, but there is still a need to further explore more efficient and more adaptable strains. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, one of the purposes of the present invention is to provide a Pandoraea bacterium ( Pandoraea sp.), named AYGW-1, which was deposited at the China Center for Type Culture Collection on March 10, 2025. The deposit address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 2025415.

[0007] Another purpose of the present invention is to provide the application of the above-mentioned Pandoraea bacterium in degrading quinolone antibiotics.

[0008] A third object of the present invention is to provide an application of Pandoraea sp. as described above in the preparation of a microbial agent for degrading quinolone antibiotics.

[0009] Preferably, the quinolone antibiotic is ciprofloxacin.

[0010] Preferably, the concentration of the quinolone antibiotic is 10 mg / L or less.

[0011] A fourth object of the present invention is to provide a microbial agent for degrading quinolone antibiotics, which comprises Pandoraea sp. AYGW-1 as described above.

[0012] Preferably, the microbial agent is a liquid seed microbial agent. The preparation method is to inoculate Pandoraea sp. AYGW-1 into an LB liquid medium and culture it in a constant temperature shaker at 30 °C and 180-220 r for 20-24 h, then centrifuge to collect the bacterial cells and resuspend them with an MSM medium to obtain the required liquid seed microbial agent.

[0013] Preferably, the centrifugation conditions are a relative centrifugal force of 4000-6000 g and a time of 3-5 min.

[0014] Preferably, in the liquid seed microbial agent, the viable count of Pandoraea sp. AYGW-1 is (5.0-8.0)×10 7 CFU / mL.

[0015] The beneficial effects of the present application are as follows: 1. In the present application, a strain with the ability to degrade quinolone antibiotics was enriched, domesticated, isolated and purified from the bottom sludge of the middle of Chaohu Lake in Anhui Province, China (N 31°36′7.56″, E 117°21′7.20″). It was identified as Pandoraea sp. by 16S rDNA and named AYGW-1. This strain has the function of degrading quinolone antibiotics and can provide a basis for the removal of quinolone antibiotics in the environment. Pandoraea sp.), named AYGW-1. This strain has the function of degrading quinolone antibiotics and can provide a basis for the removal of quinolone antibiotics in the environment.

[0016] 2. The efficiency of the strain used in the present application to degrade quinolone antibiotics was optimized for its influencing factors through single-factor experiments at the laboratory level. Under the condition that the initial concentration of ciprofloxacin was 5 mg / L, the degradation rate of the strain was more than 82.05% after 10 d. At the laboratory level, a degradation experiment was carried out on sewage containing quinolone antibiotics. Under the conditions of ciprofloxacin at 2 mg / L, 5 mg / L, and 10 mg / L, after 10 d of degradation experiments, the degradation rates were 76.37%, 68.82%, and 71.74% respectively. It shows that Pandoraea sp. AYGW-1 can be used for degrading quinolone antibiotics or in the preparation of a microbial agent for degrading quinolone antibiotics.

[0017] 3. The Pandoraea sp. AYGW-1 provided by this application enriches the strain resource library of quinolone antibiotic-degrading bacteria, provides an effective biodegradation solution for the treatment of quinolone antibiotic pollution in the environment, especially in water bodies, and also provides a safe and environmentally friendly solution to the residue problem of quinolone antibiotic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the phylogenetic tree of the Pandoraea sp. AYGW-1 provided by this application.

[0019] Figure 2 It is the streak plate diagram (A in the figure) and scanning electron microscope image (B in the figure) of the Pandoraea sp. AYGW-1 provided by this application.

[0020] Figure 3 It is the degradation diagram of the quinolone antibiotic ciprofloxacin by the Pandoraea sp. AYGW-1 at different concentrations. Among them, Figure A is the result of CIP 2 mg / L, Figure B is the result of CIP 5 mg / L, and Figure C is the result of CIP 10 mg / L.

[0021] Figure 4 It is the degradation diagram of the quinolone antibiotic ciprofloxacin by the Pandoraea sp. AYGW-1 with different concentrations of carbon sources added. Among them, Figure A is the sodium acetate carbon source, and Figure B is the glucose carbon source.

[0022] Figure 5 It is the degradation diagram of the sewage containing different concentrations of the quinolone antibiotic ciprofloxacin by the Pandoraea sp. AYGW-1. Among them, Figure A is the result of CIP 2 mg / L, Figure B is the result of CIP 5 mg / L, and Figure C is the result of CIP 10 mg / L. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The present invention will be further described below by way of examples.

[0024] Unless otherwise specified, all kinds of raw materials, reagents, instruments and equipment used in the experiments can be obtained through market purchase or can be prepared by existing methods.

[0025] Example 1 Isolation and purification of the quinolone antibiotic-degrading strain AYGW-1 1. Source of sediment samples: The samples were taken from the sludge in the center of Chaohu Lake, Hefei City, Anhui Province. The latitude and longitude are: N 31°36′7.56″, E 117°21′7.20″. The samples were collected into a sterilized sample bag and stored at 4°C for future use.

[0026] 2. Enrichment and domestication: Weigh 10.0 g of the collected sludge sample and add it to 90 mL of sterile mineral salt medium (MSM). Place it in a shaker at 30 °C and 200 rpm for 24 h. Then take it out and let it stand for 15 min. Take 5.0 mL of the supernatant and add it to 15 mL of MSM liquid medium containing 1 mg / L of the third-generation quinolone antibiotic ciprofloxacin (CIP). Place it in a shaker at 30 °C and 200 rpm for acclimation culture. After seven days of acclimation, the acclimated liquid is obtained. Then, transfer the acclimated liquid to 20 mL of MSM liquid medium containing CIP at concentrations of 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 40.0 mg / L, and 50.0 mg / L in sequence at a ratio of 2%. Place it in a constant temperature shaker at 30 °C and 200 rpm and culture it in the dark until the medium becomes turbid. During the acclimation process, samples are taken regularly to detect the degradation rate of CIP by high-performance liquid chromatography (HPLC) to evaluate the degradation ability of the bacterial community; The main components of the mineral salt medium (MSM) are: 50 mM HEPES, macroelements (10×): 4.6 g / L NaCl, 2.25 g / L K2HPO4, 2.25 g / L KH2PO4, 2.25 g / L (NH4)2SO4, trace elements (100×): 1.5 g / L NTA, 0.1 g / L MnCl2•4H2O, 0.3 g / L FeSO4•7H2O, 0.17 g / L CoCl2•6H2O, 0.1 g / L ZnCl2, 0.04 g / L CuSO4•5H2O, 0.005 g / L AlK(SO4)2•12H2O, 0.005 g / L H3BO3, 0.09 g / L Na2MoO4, 0.12 g / L NiCl2, 0.1 g / L Na2SeO4, 1 mM MgSO4.

[0027] 3. Isolation and purification: After the acclimation is completed, dilute the bacterial liquid with sterile normal saline (NS) to 10-6 times. Take 100 μL of the diluted liquid and spread it on a pressure plate (LB agar plate containing 5.0 mg / L and 10.0 mg / L CIP). Invert it and culture it in a constant temperature incubator at 37 °C until colonies form. Pick single colonies under sterile conditions and purify them by repeated streaking on the pressure plate to obtain single colonies with basically the same colony morphological characteristics. Select the purified strains according to the morphological, color, transparency and other characteristics of the strains to distinguish them.

[0028] 4. Screening of quinolone antibiotic-degrading bacteria: Separate different colonies on the selective medium and streak them repeatedly until colonies with a single morphology are obtained under the microscope. Pick the corresponding single colonies and inoculate them into the enrichment medium containing 10 mg / L CIP. After shaking culture for 24 h, take 100 μL of the supernatant, dilute it to 10 -3 、10 -5 、10 -7 times and spread them on the LB solid medium with an antibiotic concentration of 10 mg / L. After 48 h, pick the grown colonies and place them in the enrichment medium with 10 mg / L antibiotic for further domestication. Repeat this cycle 6 times to obtain the corresponding antibiotic-degrading strains. The above-mentioned purified and preserved strains were transferred to a sterile LB liquid medium and cultured until turbid. The bacterial solution was centrifuged and collected, washed with 0.01 M PBS, and then resuspended in the MSM medium. In the experimental group, directly take 1 mL of the bacterial solution (OD 600 = 1.0), and in the control group, an equal amount of the bacterial solution was sterilized by autoclaving at 121 °C for 20 min. Both groups were inoculated into 100 mL of the MSM medium (containing 2 mg / L CIP) and cultured on a shaker at 30 °C and 200 rpm. Samples were taken at regular intervals, and the residual CIP concentration was detected by the HPLC method. Strains with the ability to degrade quinolone antibiotics were obtained, and the strain with the strongest degradation ability was named AYGW-1.

[0029] In this example, the isolated and purified strains were stored according to the glycerol method. Through 16S rDNA identification, NCBI and BLAST alignment, and using the software MEGA 11.0 to construct a phylogenetic tree, the phylogenetic tree is as shown in Figure 1 . It can be seen that the strain AYGW-1 has the closest genetic relationship with Pandoraea pnomenusa strain I3-10 and Pandoraea pnomenusa strain MAQ10, and the gene similarity among the three reaches 99%.

[0030] Identified by 16S rDNA, AYGW-1 is Pandoraea ( Pandoraea sp.), The 16S rRNA gene sequence of the AYGW-1 strain is shown in the sequence listing (SEQ ID NO: 1), and its plate streaking map and scanning electron microscopy map are shown in Figure 2 .

[0031] Example 2 Optimization of the degradation conditions of AYGW-1 Preparation of strain inoculum: The strain stored in an -80 °C refrigerator was quickly streaked and activated on a pressure medium (LB agar plate containing 5.0 mg / L CIP), placed in an incubator at 30 °C for 18 h - 24 h. Single colonies on the pressure medium were selected and inoculated into 15 mL of LB medium containing 10.0 mg / L CIP, and then cultured in a constant temperature shaker at 30 °C and 200 rpm for about 20 h - 24 h. 30 μL or 60 μL of the amplified culture solution was inoculated into a glass test tube containing 4 mL of LB medium and cultured in a constant temperature shaker at 37 °C and 200 rpm. OD was measured every 30 min during this period. 600 until the OD of the bacterial solution in the test tube 600 = 0.4 ± 0.03. At this time, the bacterial solution was the inoculum, and all subsequent experimental verifications were carried out using the inoculum prepared by this method.

[0032] 1. Effects of different carbon sources and their concentrations on degradation performance: Experiments were carried out using different amounts of added sodium acetate (AcOH) and glucose (Glu). MSM media with sodium acetate and glucose concentrations of 0.0 g / L, 0.1 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, and 5.0 g / L were prepared, sterilized at 121 °C for 20 min, and then dispensed into 100 mL sterile conical flasks in a laminar flow hood. 20 mL of the medium was added to each conical flask, the CIP concentration in the flask was set to 5.0 mg / L, and the inoculation amount was 2%. For each concentration, an inoculated strain solution and an inoculated sterilized strain solution treatment were set, and each treatment was set with 3 replicates.

[0033] 2. Effects of substrate concentration on degradation performance: The sterilized MSM medium (pH = 7.00) was dispensed into 100 mL sterile conical flasks in a laminar flow hood. 20 mL of the medium was added to each conical flask, and the substrate concentrations were set to 2.0 mg / L, 5.0 mg / L, and 10.0 mg / L respectively. The inoculation amount was 2%. For each concentration, an inoculated strain solution and an inoculated sterilized strain solution treatment were set, and each treatment was set with 3 replicates.

[0034] After the above experimental groups and control groups were processed, they were all placed in a constant temperature shaker at 30 °C and shaken at 200 rpm for 24 h, then sampled and analyzed by HPLC to determine the residual concentration of CIP in the medium, and the pH of the medium after cultivation was also measured. The results are shown in Figure 3 and Figure 4 .

[0035] Without adding any carbon source, and using only CIP as the sole carbon source for degradation, due to the stable chemical structure of ciprofloxacin, high concentrations of CIP may have a toxic effect on microorganisms, inhibiting their growth and metabolism, thus resulting in a low degradation rate. From Figure 3 it can be seen that the effect of different initial concentrations of CIP on the degradation efficiency of strain AYGW-1 is not very obvious. For CIP at 2 mg / L ( Figure 3 A in Figure 3 ), 5 mg / L ( Figure 3 B in

[0036] ), and 10 mg / L ( Figure 3 C in Figure 4 ), after 10 days of degradation experiments, the degradation rates were 28.44%, 27.86%, and 21.60%, respectively. Figure 4 In the co-metabolism process, microorganisms do not need to be pre-exposed to pollutants. As long as a nutrient substrate (such as glucose, sodium acetate, etc.) is given, the degradation of pollutants can be achieved. A suitable carbon source is very important for the biodegradation of antibiotics. Glucose (Glu) and sodium acetate (AcOH) are common external carbon sources in the co-metabolism process of microorganisms. Therefore, the effects of these two carbon sources on the degradation of CIP by strain AYGW-1 were compared. The results are as Figure 4 shown. It can be seen from the figure that compared with glucose ( Figure 4 B in Figure 4 ), sodium acetate ( Figure 4 A in

[0037] ) has more advantages in promoting strain AYGW-1 to increase the CIP degradation rate. After adding 1.0 g / L AcOH, the degradation rate increased to 82.05% after 10 days. Therefore, sodium acetate was selected as the co-metabolic carbon source for the strain to degrade CIP.

[0038] Example 3 Degradation of quinolone antibiotics in sewage by AYGW-1 Lake Chaohu water was collected as an experimental sewage sample. The collected lake water was first filtered through a gauze to remove large suspended particles and impurities. A part of the lake water was subjected to high-temperature and high-pressure sterilization treatment (121 °C, 15 min) as a control group to exclude the interference of other microorganisms in the lake water on the experimental results. Another part of the lake water was not sterilized and was used to simulate the degradation experiment under natural conditions.

[0039] Take the pre-treated Chaohu Lake water and add 2 mg / L, 5 mg / L, and 10 mg / L of CIP standard solution respectively to prepare sewage with different concentration gradients. Adjust the initial pH to 7.0 and add 1.0 g / L of AcOH as a supplementary carbon source. Set up 3 parallel groups for each concentration, and at the same time set up a blank control group without ciprofloxacin.

[0040] Inoculate the activated AYGW-1 bacterial solution into the polluted lake water at an inoculation amount of 2%, so that the bacterial solution concentration reaches about 10 6 CFU / mL. For the sterilized lake water, inoculate the AYGW-1 bacterial solution in the same way as a control experiment.

[0041] Place the inoculated sewage in a constant temperature shaker, set the temperature to 30 °C and the rotation speed to 200 r / min for the degradation reaction. Regularly take samples and measure the residual concentration of CIP in the culture medium on a machine (HPLC).

[0042] It can be seen from the figure that compared with the control group, the degradation effect of the experimental group is obvious. After 10 days of degradation experiment, the degradation rates of CIP at 2 mg / L ( Figure 5 A in the figure), 5 mg / L ( Figure 5 B in the figure), and 10 mg / L ( Figure 5 C in the figure) are 76.37%, 68.82%, and 71.74% respectively.

[0043] It shows that the strain AYGW-1 has the ability to enhance the degradation of quinolone antibiotics in sewage, providing an effective reference for the biodegradation scheme for the treatment of quinolone antibiotic pollution in the environment, especially in water bodies.

[0044] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Pandoraea bacterium ( Pandoraea sp . ), characterized in that The Pandoraea was named AYGW-1 and was deposited in the China Center for Type Culture Collection on March 10, 2025, with the deposit number of CCTCC M 2025415.

2. Use of the Pandoraea according to claim 1 in degrading quinolone antibiotics, wherein the quinolone antibiotic is ciprofloxacin.

3. Use of the Pandoraea according to claim 1 in preparing a bacterial agent for degrading quinolone antibiotics, wherein the quinolone antibiotic is ciprofloxacin.

4. The application according to claim 2 or 3, characterized in that The concentration of the quinolone antibiotic is 10 mg / L or less.

5. A bacterial agent for degrading quinolone antibiotics, characterized in that, The quinolone antibiotic is ciprofloxacin, and the bacterial agent comprises the Pandoraea AYGW-1 according to claim 1.

6. The bacterial agent for degrading quinolone antibiotics according to claim 5, wherein The bacterial agent is a liquid seed bacterial agent, and the preparation method is as follows: inoculate the Pandoraea AYGW-1 into an LB liquid medium, culture it in a constant temperature shaker at 30°C and 180 - 220 r for 20 - 24 h, then centrifuge to collect the bacterial cells, and resuspend them with an MSM medium to obtain the required liquid seed bacterial agent.

7. The microbial agent for degrading quinolone antibiotics according to claim 6, characterized in that, The centrifugation conditions are: relative centrifugal force of 4000 - 6000 g and time of 3 - 5 min.

8. The bacterial agent for degrading quinolone antibiotics according to claim 6, characterized in that, In the liquid seed bacterium agent, the viable count of Pandoraea sp. AYGW-1 is (5.0 - 8.0)×10 7 CFU / mL.

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