Pandora bacteria, bacterial agent and use thereof in degradation of quinolone antibiotics

Abstract

The present invention belongs to the field of microbial degradation technology, and specifically relates to a Pandora bacteria, a bacterial agent and its application in the degradation of quinolone antibiotics. Pandoraea sp.) was named AYGW‑1 and deposited with the China Center for Type Culture Collection on March 10, 2025, with the accession number CCTCC M 2025415. This Pandoracone AYGW‑1 is capable of degrading quinolone antibiotics, providing an effective biodegradation solution for the treatment of quinolone antibiotic pollution in the environment, especially in water. It also offers a safe and environmentally friendly solution to the problem of quinolone antibiotic residues.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation, and particularly relates to a Pandora bacteria, a bacterial agent and application thereof in the degradation of quinolone antibiotics. Background Art

[0002] Quinolone antibiotics are a widely used class of antimicrobial drugs, widely used in fields such as medicine and animal husbandry due to their broad antimicrobial spectrum and strong antibacterial activity. However, their widespread use has led to a continuous increase in residual quinolone antibiotics in the environment. These antibiotics enter water bodies, soil, and other environmental media through various pathways, posing a potential threat to ecosystems and human health.

[0003] Currently, treatment methods for quinolone antibiotic contamination primarily include physical and chemical methods and biodegradation. While physical and chemical methods, such as adsorption and redox, can rapidly reduce antibiotic concentrations, they pose challenges such as high costs and secondary pollution. Biodegradation, on the other hand, offers advantages such as low cost and environmental friendliness. However, traditional microbial degradation of quinolone antibiotics is inefficient and requires strict environmental conditions.

[0004] In recent years, research on microbial degradation of antibiotics has gradually attracted attention. Some studies have reported that specific microbial strains have the ability to degrade quinolone antibiotics. For example, patent CN117467583A discloses the ability of Achromobacterium Achromobacter sp.) JK7 has a strong ability to degrade quinolone antibiotics such as levofloxacin, moxifloxacin and enoxacin; Patent CN114107065A discloses that Geotrichum sp. 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 dealing with antibiotic pollution, but further exploration of more efficient and adaptable strains is still needed. Summary of the Invention

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

[0007] A second object of the present invention is to provide a use of the Pandora bacteria described above in degrading quinolone antibiotics.

[0008] A third object of the present invention is to provide a use of the Pandora bacteria described above in the preparation of a bacterial agent for degrading quinolone antibiotics.

[0009] Preferably, the quinolone antibiotic is ciprofloxacin.

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

[0011] A fourth object of the present invention is to provide a bacterial agent for degrading quinolone antibiotics, wherein the bacterial agent comprises the Pandora candida AYGW-1 described above.

[0012] Preferably, the bacterial agent is a liquid seed bacterial agent, and the preparation method is as follows: the Pandora bacteria AYGW-1 is inoculated into LB liquid culture medium, cultured in a constant temperature shaker at 30°C and 180-220r for 20-24 hours, centrifuged to collect the bacteria, and resuspended with MSM culture medium to obtain the desired liquid seed bacterial agent.

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

[0014] Preferably, the viable bacteria count of Pandora bacteria AYGW-1 in the liquid seed inoculum is (5.0-8.0)×10 7 CFU / mL.

[0015] The beneficial effects of this application are:

[0016] 1. This application discloses a strain capable of degrading quinolone antibiotics, which was obtained by enriching, acclimating, isolating and purifying the lake bottom sludge in the center of Chaohu Lake, Anhui Province, China (N 31°36′7.56″, E 117°21′7.20″). The strain was identified as Pandora bacteria ( Pandoraea sp.), named AYGW-1. This bacterium has the ability to degrade quinolone antibiotics, which could provide a basis for the removal of quinolone antibiotics in the environment.

[0017] 2. The efficiency of the strain used in this application in degrading quinolone antibiotics was optimized through single-factor experiments at the laboratory level. At an initial ciprofloxacin concentration of 5 mg / L, the strain achieved a degradation rate of over 82.05% after 10 days. In laboratory experiments, degradation of wastewater containing quinolone antibiotics was conducted. At ciprofloxacin concentrations of 2 mg / L, 5 mg / L, and 10 mg / L, the degradation rates after 10 days were 76.37%, 68.82%, and 71.74%, respectively. This indicates that Pandora's AYGW-1 can be used to degrade quinolone antibiotics or to prepare bacterial agents for degrading quinolone antibiotics.

[0018] 3. The Pandora bacteria AYGW-1 provided in this application enriches the bacterial 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 problem of quinolone antibiotic residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The phylogenetic tree of Pandora bacteria AYGW-1 provided in this application.

[0020] Figure 2 The plate streak image (A) and scanning electron microscope image (B) of Pandora bacteria AYGW-1 provided in this application.

[0021] Figure 3 Figure 4 shows the degradation of quinolone antibiotic ciprofloxacin by Pandora bacteria AYGW-1 at different concentrations, where Figure A shows the results at CIP 2 mg / L, Figure B shows the results at CIP 5 mg / L, and Figure C shows the results at CIP 10 mg / L.

[0022] Figure 4 This is the degradation diagram of the quinolone antibiotic ciprofloxacin by Pandora bacteria AYGW-1 when different concentrations of carbon sources are added, where Figure A is sodium acetate carbon source and Figure B is glucose carbon source.

[0023] Figure 5 These are the degradation diagrams of Pandora's bacteria AYGW-1 on wastewater containing different concentrations of the quinolone antibiotic ciprofloxacin, where Figure A shows the results of CIP 2 mg / L, Figure B shows the results of CIP 5 mg / L, and Figure C shows the results of CIP 10 mg / L. DETAILED DESCRIPTION

[0024] The present invention will be further described below by way of examples.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the experiments can be purchased from the market or prepared by existing methods.

[0026] Example 1

[0027] Isolation and purification of quinolone antibiotic-degrading strain AYGW-1

[0028] 1. Source of sediment samples:

[0029] The samples were collected from the central sludge of Chaohu Lake in Hefei City, Anhui Province, at the latitude and longitude of N 31°36′7.56″, E 117°21′7.20″. The samples were collected into sterilized sample bags and stored at 4°C until use.

[0030] 2. Enrichment and domestication:

[0031] 10.0 g of the collected sludge sample was weighed and added to 90 mL of sterile mineral salt medium (MSM), placed in a shaker at 30°C and 200 rpm for 24 h, taken out and allowed to stand for 15 min, 5.0 mL of the supernatant was taken and added to 15 mL of MSM liquid culture medium containing the third-generation quinolone antibiotic ciprofloxacin (CIP) at a concentration of 1 mg / L, and placed in a shaker at 30°C and 200 rpm for acclimation culture. After acclimation for seven days, the acclimation liquid was obtained, and the acclimation liquid was transferred to 20 mL of MSM liquid culture 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 at a ratio of 2%, and placed in a constant temperature shaker at 30°C and 200 rpm for incubation in the dark until the culture medium became turbid. During the acclimation process, samples were taken regularly to detect the degradation rate of CIP by high-performance liquid chromatography (HPLC) to evaluate the degradation capacity of the bacterial community;

[0032] The main components of mineral salt medium (MSM) are: 50 mM HEPES, major elements (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 / LNiCl2, 0.1 g / L Na2SeO4, 1 mM MgSO4.

[0033] 3. Separation and purification:

[0034] After acclimation, dilute the bacterial solution to 10⁻¹ with sterile physiological saline (NS). Spread 100 μL of the dilution onto a pressure plate (LB agar plate containing 5.0 mg / L and 10.0 mg / L CIP) and incubate inverted at 37°C until colonies form. Aseptically select individual colonies and repeatedly streak them onto the pressure plate to purify them to obtain single colonies with essentially uniform morphological characteristics. Identify the purified strains based on their morphology, color, transparency, and other characteristics.

[0035] 4. Screening of quinolone antibiotic-degrading bacteria:

[0036] Different colonies on the culture medium were picked out and separated repeatedly by streaking until a single colony was obtained under a microscope. The corresponding single colony was inoculated into an enrichment medium containing 10 mg / L CIP. After shaking culture for 24 hours, 100 μL of the supernatant was aspirated and diluted to 10 -3 , 10 -5 , 10 -7 The strain was then plated onto LB solid medium with an antibiotic concentration of 10 mg / L. After 48 hours, colonies that grew were selected and placed in enriched medium containing 10 mg / L antibiotic for further acclimation. This cycle was repeated for 6 cycles to obtain the corresponding antibiotic-degrading strain. The purified strain was transferred to sterile LB liquid medium and cultured until turbid. The bacterial solution was collected by centrifugation, washed with 0.01 M PBS, and resuspended in MSM medium. For the experimental group, 1 mL of bacterial solution (OD 600 =1.0). For the control group, equal amounts of bacterial suspension were sterilized by autoclaving at 121°C for 20 min. Both groups were inoculated into 100 mL of MSM medium (containing 2 mg / L CIP) and cultured in a shaker at 30°C and 200 rpm. Samples were taken regularly, and residual CIP concentrations were determined using HPLC. A strain with the ability to degrade quinolone antibiotics was identified, and the strain with the strongest degradation ability was named AYGW-1.

[0037] In this example, the isolated and purified strains were stored according to the glycerol method, identified by 16S rDNA, compared with NCBI and BLAST, and a phylogenetic tree was prepared using the software MEGA11.0. Figure 1 It can be seen that strain AYGW-1 is most closely related to Pandoraea pnomenusa strain I3-10 and Pandoraea apnomenusa strain MAQ10, and the genetic similarity among the three reaches 99%.

[0038] AYGW-1 was identified as Pandora candida by 16S rDNA. Pandoraea sp.), the 16S rRNA gene sequence of AYGW-1 strain is shown in the sequence listing (SEQ ID NO: 1), and its plate streaking pattern and scanning electron micrograph are shown in Figure 2 .

[0039] Example 2

[0040] Optimization of degradation conditions for AYGW-1

[0041] Preparation of strain inoculum: The strain stored in a -80°C refrigerator was quickly streaked on a stress medium (LB agar plate containing 5.0 mg / L CIP) for activation, and then placed in a 30°C constant temperature incubator for 18 h~24 h. A single colony on the stress medium was selected and inoculated into 15 mL LB medium containing 10.0 mg / L CIP. The culture was shaken in a constant temperature shaker at 30°C and 200 rpm for about 20 h~24 h. 30 μL or 60 μL of the expansion culture solution was inoculated into a glass test tube containing 4 mL LB medium, and the culture was shaken in a constant temperature shaker at 37°C and 200 rpm. The OD was tested every 30 min during the culture. 600 Until the bacterial solution OD 600 = 0.4±0.03, the bacterial solution at this time is the inoculum solution, and all subsequent experimental verifications are carried out using the inoculum solution prepared by this method.

[0042] 1. Effects of different carbon sources and their concentrations on degradation performance:

[0043] The experiment was carried out using different amounts of sodium acetate (AcOH) and glucose (Glu). The MSM culture medium 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 was prepared and sterilized at 121°C for 20 min. The culture medium was dispensed into 100 mL sterile conical flasks in a clean bench, and 20 mL of culture medium was added to each conical flask. The CIP concentration in the bottle was set to 5.0 mg / L, and the inoculation volume was 2%. Each concentration was set to be treated with the inoculated strain solution and the sterilized strain solution, and 3 replicates were set for each treatment.

[0044] 2. Effect of substrate concentration on degradation performance:

[0045] The sterilized MSM culture medium (pH = 7.00) was dispensed into 100 mL sterile conical flasks in a clean bench. 20 mL of culture medium was added to each conical flask. The substrate concentrations were set to 2.0 mg / L, 5.0 mg / L, and 10.0 mg / L, respectively, and the inoculation volume was 2%. Each concentration was treated with the inoculated strain solution and the sterilized strain solution, and 3 replicates were set for each treatment.

[0046] After the treatment, the experimental group and the control group were placed in a 30 ℃ constant temperature shaker at 200 rpm for 24 hours. After that, samples were taken and the CIP residual concentration in the culture medium was determined by HPLC. The pH of the culture medium after the culture was also determined. Figure 3 、 Figure 4 .

[0047] Without adding any carbon source, only CIP is used as the sole carbon source for degradation. Due to the stable chemical structure of ciprofloxacin, high concentrations of CIP may have toxic effects on microorganisms, inhibiting their growth and metabolism, resulting in a low degradation rate. 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. 2 mg / L ( Figure 3 Medium A), 5mg / L ( Figure 3 Medium B), 10mg / L ( Figure 3 (C) After 10 days of degradation experiment, the degradation rates of CIP were 28.44%, 27.86% and 21.60%.

[0048] During the co-metabolism process, microorganisms do not need to be exposed to pollutants in advance. As long as they are provided with nutrient substrates (such as glucose, sodium acetate, etc.), they can achieve the degradation of pollutants. 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 shown in Figure 2. Figure 4 As shown in the figure, compared with glucose ( Figure 4 B), sodium acetate ( Figure 4 Middle A) showed an advantage in promoting CIP degradation by strain AYGW-1. After adding 1.0 g / L AcOH, the degradation rate increased to 82.05% in 10 days. Therefore, sodium acetate was selected as the co-metabolic carbon source for CIP degradation by the strain.

[0049] In summary, the optimized conditions for the degradation of quinolone antibiotics by Pandora bacteria AYGW-1 were: substrate concentration of 5 mg / L ciprofloxacin, initial pH = 7.0, and addition of 1.0 g / L AcOH. The degradation rate of antibiotics reached a maximum of 82.05%, showing a good degradation effect.

[0050] Example 3

[0051] Degradation of quinolone antibiotics in sewage by AYGW-1

[0052] Water from Chaohu Lake was collected as the experimental wastewater sample. The collected water was first filtered through gauze to remove large suspended particles and impurities. A portion of the lake water was sterilized at high temperature and high pressure (121°C, 15 minutes) as a control group to eliminate the possibility of interference from other microorganisms in the lake water. Another portion of lake water was not sterilized and used to simulate degradation experiments in a natural environment.

[0053] Pretreated Chaohu Lake water was spiked with 2 mg / L, 5 mg / L, and 10 mg / L CIP standard solutions to prepare wastewater with varying concentration gradients. The initial pH was adjusted to 7.0, and 1.0 g / L AcOH was added as a supplemental carbon source. Three parallel groups were set up for each concentration, along with a blank control group without ciprofloxacin.

[0054] The activated AYGW-1 bacterial solution was inoculated into the polluted lake water at a 2% inoculum volume to make the bacterial solution concentration reach 10 6 The sterilized lake water was also inoculated with AYGW-1 bacterial solution as a control experiment.

[0055] The inoculated wastewater was placed in a thermostatic shaker at 30°C and 200 rpm for degradation. Samples were taken regularly and the residual CIP concentration in the culture medium was determined by HPLC.

[0056] As can be seen from the figure, compared with the control group, the degradation effect of the experimental group is obvious, 2 mg / L ( Figure 5 Medium A), 5mg / L ( Figure 5 Medium B), 10mg / L ( Figure 5 After 10 days of degradation experiment, the degradation rates of CIP in (C) were 76.37%, 68.82% and 71.74%, respectively.

[0057] This indicates that strain AYGW-1 has the ability to enhance the degradation of quinolone antibiotics in wastewater, providing an effective biodegradation solution reference for the treatment of quinolone antibiotic pollution in the environment, especially in water bodies.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A Pandora bacterium ( Pandoraeasp. ), characterized in that, Pandora's bacteria was named AYGW-1 and deposited in the China Center for Type Culture Collection on March 10, 2025, with the deposit number CCTCC M 2025415.

2. Use of Pandora bacteria as claimed in claim 1 in degrading quinolone antibiotics, wherein the quinolone antibiotic is ciprofloxacin.

3. A use of the Pandora bacteria according to claim 1 in the preparation of a bacterial agent for degrading quinolone antibiotics, wherein the quinolone antibiotic is ciprofloxacin.

4. The use according to claim 2 or 3, characterized in that The concentration of the quinolone antibiotics 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 includes the Pandora bacteria AYGW-1 as claimed in claim 1.

6. A bacterial agent for degrading quinolone antibiotics as claimed in claim 5, characterized in that The bacterial agent is a liquid seed bacterial agent, and the preparation method is as follows: Pandora bacteria AYGW-1 is inoculated into LB liquid culture medium, cultured in a constant temperature shaker at 30°C and 180-220 r for 20-24 hours, centrifuged to collect the bacterial cells, and resuspended with MSM culture medium to obtain the required liquid seed bacterial agent.

7. A bacterial agent for degrading quinolone antibiotics as claimed in claim 6, characterized in that The centrifugation conditions are: relative centrifugal force 4000-6000 g, time 3-5 min.

8. A bacterial agent for degrading quinolone antibiotics according to claim 6, characterized in that: The number of viable bacteria of Pandora bacteria AYGW-1 in the liquid seed inoculum is (5.0-8.0)×10 7 CFU / mL.

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