A cefixime-degrading bacterium and its application

Abstract

The present invention relates to a cefixime degrading bacteria and its application. The TY12 of Pseudochrobactrum asaccharolyticum provided by the present invention can tolerate high-concentration antibiotics, the concentration of cefixime is 100-1000mg / L, and the dosage of Pseudochrobactrum asaccharolyticum is 1%-20%; the degradation temperature is 20-40°C, and the pH is 5-9, which can effectively degrade cefixime. Under the conditions of 30°C, 7 pH of culture medium, 10% inoculum amount, and 200r / min rotation speed, the degradation rate of TY12 to 1000mg / L cefixime reaches 83.73% in 24h. The biological safety of the strain is high, human health or environmental pollution will not be endangered during application, and it has important application value.

Description

Technical Field

[0001] The invention relates to the field of cefixime degradation, and in particular to a cefixime-degrading bacterium and an application thereof. Background Art

[0002] Antibiotics, as an important class of pharmaceutical and personal care products (PPCPs), are widely used in medicine and animal husbandry. However, excessive use of antibiotics can lead to their accumulation in the environment, which not only has a toxic effect on organisms in the environment, but also changes the original microbial flora structure, leading to bacterial resistance to antibiotics, causing the spread of drug-resistant genes, and causing serious harm to the ecological environment and human health. In 2019, the World Health Organization (WHO) listed antimicrobial resistance as one of the common health problems. An analysis of antibiotic use in 76 countries from 2010 to 2015 found that global antibiotic consumption increased by 65% ​​in the 15 years studied. Cefixime is a β-lactam antibiotic that has been widely used to treat infections caused by Gram-positive and Gram-negative strains in humans and animals. It has poor solubility in water and low bioavailability, about 40%-50%. A large amount of cefixime was detected in water bodies, ranging from 278-422ng / L. The presence of cefixime in the environment can cause microbial resistance and cause serious damage to the ecosystem. In addition, cefixime can also have toxic effects on other organisms in the environment. Therefore, the removal of residual antibiotics in the environment has become an urgent problem to be solved in the sustainable development of the antibiotic industry and environmental governance.

[0003] Biodegradation is an environmentally friendly, sustainable, non-secondary pollution and highly adaptable pollutant treatment technology. At present, some literature has reported the research on microbial degradation of other β-lactam antibiotics, but there is no report on the degradation of cefixime. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a cefixime-degrading bacterium and an application thereof in view of the above shortcomings.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A cefixime-degrading bacterium, Pseudochrobactrum asaccharolyticum TY12, was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 13, 2023, with the deposit number CGMCC NO.28949.

[0007] The cefixime-degrading bacteria is used in degrading cefixime.

[0008] Furthermore, the concentration of cefixime during degradation is 100-1000 mg / L.

[0009] Furthermore, during the degradation, the inoculation amount of the cefixime-degrading bacteria is 1%-20%.

[0010] Furthermore, the environmental temperature during degradation is 20-40°C.

[0011] Furthermore, the environmental pH during degradation is 5-9.

[0012] The invention has the following beneficial effects: experiments show that the Pseudochrobactrum asaccharolyticum TY12 provided by the invention can tolerate high-concentration antibiotics, the concentration of cefixime is 100-1000 mg / L, the dosage of Pseudochrobactrum asaccharolyticum is 1%-20%, the degradation temperature is 20-40°C, and the pH is 5-9, and cefixime can be effectively degraded. Under the conditions of temperature of 30°C, medium pH of 7, inoculation amount of 10%, and rotation speed of 200 r / min, the degradation rate of 1000 mg / L cefixime by strain TY12 reaches 83.73% in 24 hours. The strain has high biological safety, will not endanger human health or pollute the environment during application, and has important application value.

[0013] The present invention is described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the standard curve of peak area and substrate mass concentration;

[0015] Figure 2 is the growth curve of strain TY12;

[0016] Figure 3 is the colony morphology of strain TY12;

[0017] Figure 4 is the phylogenetic tree of strain TY12;

[0018] Figure 5 is the effect of temperature on the degradation of cefixime by strain TY12;

[0019] Figure 6 is the effect of pH on the degradation of cefixime by strain TY12;

[0020] Figure 7 is the effect of inoculum size on the degradation of cefixime by strain TY12;

[0021] Figure 8 The effect of substrate concentration on the degradation of cefixime by strain TY12. DETAILED DESCRIPTION

[0022] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0023] 1. Screening of degradation strains

[0024] 1. Material preparation

[0025] Sample source: Activated sludge from Yindingzhuang Wastewater Treatment Plant in Baoding City

[0026] LB liquid medium: NaCl 10.0g, peptone 10.0g, yeast powder 5.0g, deionized water to 1L, use NaOH to adjust the pH to 7.0±0.2. On this basis, add 1.5% to 2% agar powder to make LB solid medium, sterilize at 121℃ for 20min.

[0027] Inorganic salt culture medium: composed of 0.5g (NH4) 2SO4, 0.5g KH2PO4, 1.5g K2HPO4, 0.15g MgSO4·7H2O, 0.5g NaCl and 1.0mL trace elements, supplemented to 1L with deionized water, sterilized at 121℃ for 20min. The trace elements used in this patent include 0.3g MnSO4·4H2O, 0.2g ZnSO4·7H2O, 0.02g (NH4) 6Mo7O2·4H2O, 0.1g CuSO4·5H2O, 0.5g CoCl2·6H2O, 0.05g CaCl2·2H2O, 0.5g FeSO4·7H2O.

[0028] 2. Preliminary screening of bacterial strains

[0029] Take 2 mL of fresh sludge from the activated sludge of Yindingzhuang Sewage Treatment Plant in Baoding City, transfer it to 200 mL of LB culture medium in a clean bench, and oscillate it in a constant temperature shaking incubator at 30°C and 200 r / min for 1 day. Set inorganic salt culture medium with mass concentrations of cefixime of 40, 80, 120, 160, and 200 mg / L respectively, and transfer the culture medium to the inorganic salt culture medium in sequence at an inoculation amount of 5% until the mass concentration of cefixime in the culture medium is 200 mg / L, thereby obtaining the final bacterial culture medium for standby use.

[0030] Spreading separation: Take the cultured bacterial solution in the inorganic salt medium and -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5For each dilution concentration, 100 μL of bacterial solution was evenly spread on the solid culture medium and placed in a 30°C constant temperature incubator for 2-3 days. Single colonies with different morphological characteristics were selected from the plate, streaked and purified 2-3 times, and marked for later use.

[0031] 3. Determination method of cefixime content

[0032] Drawing of the cefixime standard curve: Accurately weigh 500 mg of cefixime in a 50 mL volumetric flask, dilute to the mark with mobile phase, and prepare a standard solution containing 10 mg of cefixime per mL. Add mobile phase to dilute and prepare 100, 200, 400, 600, 800, 1000, 1200, and 1400 mg / L cefixime standard solutions, and draw a standard curve with the mass concentration of cefixime as the horizontal axis and the peak area as the vertical axis. The drawn standard curve is as follows: Figure 1 As shown, Y = 22.908X-21.301, R 2 =0.9992.

[0033] The cefixime content was determined by high performance liquid chromatography (HPLC). Chromatographic conditions: octadecylsilane bonded silica gel was used as filler; tetrabutylammonium hydroxide solution (25 mL of 10% tetrabutylammonium hydroxide solution was taken, 1 L of water was added, shaken, and the pH value was adjusted to 7.0 with 1.5 mol / L phosphoric acid solution)-acetonitrile (72:28) was used as mobile phase; the detection wavelength was 254 nm; the column temperature was 40°C; the injection volume was 20 μl.

[0034] Substitute the measured peak area into the standard curve to calculate the substrate concentration and the degradation rate.

[0035] 4. Strain rescreening

[0036] The strain obtained in the above steps was inoculated into LB liquid medium for activation culture, and then inoculated into an inorganic salt medium with cefixime as the sole carbon source at an inoculum size of 5%, and cultured under shaking conditions of 30° C. and 200 r / min. The degradation rate of each strain was determined by high performance liquid chromatography, and a strain TY12 that can efficiently degrade cefixime was screened.

[0037] At present, there is no report on the degradation of cefixime by microorganisms at home and abroad. Strain TY12 is a new strain with the ability to degrade cefixime.

[0038] 2. Growth curve of strain TY12

[0039] Take the activated strain TY12 and inoculate it into LB liquid culture medium. Under appropriate culture conditions, take the bacterial solution regularly and measure the OD value at a wavelength of 600nm. Within a certain range, the bacterial solution concentration and the optical density value are linearly related. Plot the measured OD value and its corresponding culture time to draw the growth curve of the bacteria, as shown in the attached figure. Figure 2 The specific steps are as follows:

[0040] Take the preserved strains, inoculate them into LB liquid culture medium, culture them at 30℃ and 200r / min for 12h, and set aside. The components of LB liquid culture medium are: NaCl5.0g, peptone 10.0g, yeast powder 5.0g, deionized water to 1L, pH adjusted to 7.0±0.2, sterilized at 121℃ for 20min; take 1mL of bacterial solution and transfer it into 200mL of LB liquid culture medium; place it in a constant temperature shaking incubator at 30℃ and 200r / min, take 4mL of bacterial solution at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24h, and immediately put it in the refrigerator for storage, and finally measure the OD value at a wavelength of 600nm to make a growth curve.

[0041] Depend on Figure 2 It can be seen that strain TY12 entered the logarithmic growth phase from 6h.

[0042] 3. Strain morphology and molecular biological identification

[0043] 1 Observation of colony morphology

[0044] Strain TY12 was grown on LB solid medium for 2 days, and the colony morphology was as follows Figure 3 As shown, the colonies are white, round, and have smooth and moist edges.

[0045] 2 Molecular biological identification of strain TY12

[0046] 2.1 Genome extraction

[0047] The genomic DNA of strain TY12 was extracted using a bacterial genomic DNA extraction kit produced by Takara, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for PCR amplification and sequencing.

[0048] 2.2 Sequencing results

[0049] After sequencing, the length of the 16S rDNA gene sequence of strain TY12 was 1410bp. Comparison in the NCBI database showed that the 16S rDNA sequence of strain TY12 had the highest similarity with Pseudochrobactrum asaccharolyticum CCUG 46016, reaching 99.73%. The obtained 16S rDNA sequence is as follows:

[0050] ATGAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTA

[0051] ACACATGCAAGTCGAACGGTCTCTTCGGAGGCAGTGGCAGACGGGTG

[0052] AGTAATGCATGGGAATCTACCGTTCTCTACGGAATAACTCAGGGAAACT

[0053] TGTGCTAATACCGTATACGCCCTTTTGGGGAAAGATTTATCGGAGAATG

[0054] ATGAGCCCATGTTGGATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAG

[0055] GCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACT

[0056] GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGG

[0057] ACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGC

[0058] CCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCG

[0059] GAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGA

[0060] AGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGC

[0061] GGACTTTTAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGC

[0062] CTTTGATACTGGAAGTCTTGAGTATGGAAGAGGTAAGTGGAATTGCGA

[0063] GTGTAGAGGTGAAATTCGTAGATATTCGCAGGAACACCAGTGGCGAAG

[0064] GCGGCTTACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAG

[0065] CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTT

[0066] AGCCGTCGGGGTGTTTACACTTCGGTGGCGCAGCTAACGCATTAAACA

[0067] TTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGAC

[0068] GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG

[0069] CGCAGAACCTTACCAGCTCTTGACATGCCTATGAATCTTAGTGGAGACA

[0070] CTTTGAGCCTTTCGGGGCGTAGGACACAGGTGCTGCATGGCTGTCGTC

[0071] AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

[0072] TCGCCTTTAGTTGCCATCATTTAGTTGGGCACTCTAAAGGGACTGCCAG

[0073] TGATAAGCTGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTT

[0074] ACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGC

[0075] GAGATCGCGAGGTCGAGCTAATCTCCAAAAACCATCTCAGTTCGGATT

[0076] GCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGG

[0077] ATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCC

[0078] GTCACACCATGGGAGTTGGTTTTACCCGAAGGCCGCTGTGCTAACCGCA

[0079] AGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTA

[0080] ACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTT

[0081] 2.3 Phylogenetic tree construction

[0082] The gene sequences of strains with close similarity were selected and a phylogenetic tree was constructed using MEGA 11.0 software. Figure 4 As shown, strain TY12 and strain Pseudochrobactrum asaccharolyticum CCUG 46016 were clustered in the same branch and had the closest phylogenetic relationship.

[0083] The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 13, 2023, with the deposit number CGMCC NO.28949. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0084] At present, there is no report on the degradation of β-lactam antibiotics by Pseudochrobactrum asaccharolyticum at home and abroad. Strain TY12 is a new strain with the function of degrading β-lactam antibiotics.

[0085] IV. Degradation characteristics of strain TY12

[0086] 1 Effect of temperature on TY12 degradation

[0087] Temperature has a significant effect on the growth of microorganisms and the activity of enzymes related to pollutant degradation. 600The LB bacterial solution with a concentration of 1 was centrifuged at 8000rpm for 2min, the supernatant LB medium was discarded, and the culture medium was resuspended with sterile PBS, and repeated three times to ensure that the degradation experiment was carried out with cefixime as the sole carbon source. The inoculum of 10% was inoculated into an inorganic salt medium with an initial cefixime concentration of 500mg / L, pH 7, and placed at 20°C, 25°C, 30°C, 35°C, 40°C, 200r / min shaking table for cultivation, and the cefixime content was measured after 24h, and its degradation rate was calculated.

[0088] Depend on Figure 5 It can be seen that within the range of 20-40℃, the degradation rate of TY12 on cefixime first increased and then decreased with the increase of temperature, reaching a maximum degradation rate of 93.78% at 30℃, and the degradation rates at 20℃ and 40℃ reached 46.98 and 43.81% respectively, which may be due to the fact that temperature affects the growth state of microorganisms and the activity of degradation enzymes. The optimal temperature for strain TY12 is 30℃.

[0089] 2. Effect of pH on the degradation of TY12

[0090] At pH 5, 6, 7, 8, and 9, take OD at 10% inoculation volume. 600 The LB culture medium with a concentration of 1 was centrifuged at 8000 rpm for 2 min, the supernatant LB culture medium was discarded, and the culture medium was resuspended with sterile PBS, and repeated three times to ensure that the degradation experiment was carried out with cefixime as the only carbon source. The bacterial suspension of strain TY12 was inoculated into an inorganic salt culture medium containing 500 mg / L cefixime, placed under the same temperature of 30°C and the same speed of 200 r / min, and the pollutant content was determined after culturing for 24 hours, and its degradation rate was calculated.

[0091] The results are attached Figure 6 As shown in the figure, within the pH range of 5-9, the degradation rate gradually increases with the increase of pH. When the pH is 9, the degradation rate is as high as 89.82%; when the pH is 5, the degradation rate of strain TY12 can also reach 36.87%. Too low pH will affect the activity of the enzyme and the permeability of the cell membrane, which is not conducive to the growth of microorganisms.

[0092] 3 Effect of inoculation size on TY12 degradation

[0093] The OD values ​​were taken at 1%, 2%, 5%, 10% and 20% inoculum respectively. 600LB culture medium with a concentration of 1, centrifuged at 8000rpm for 2min, discarded the supernatant LB culture medium, resuspended with sterile PBS, repeated three times, ensured that the degradation experiment was carried out with cefixime as the only carbon source, inoculated the bacterial suspension into an inorganic salt culture medium containing 500mg / L cefixime, adjusted the incubator temperature to 30℃, the culture medium pH to 7, the rotation speed to 200r / min, and measured the pollutant content after 24h of incubation to calculate its degradation rate. The results are as follows Figure 7 As shown, the degradation rate of cefixime by strain TY12 increased with the increase of inoculum size, and when the inoculum size was 20%, the degradation rate of strain TY12 reached the highest almost 100%.

[0094] 4 Effect of initial concentration on TY12 degradation

[0095] Take OD according to 10% inoculum 600 The LB bacterial solution with a concentration of 1 was centrifuged at 8000rpm for 2min, the supernatant LB medium was discarded, and the culture medium was resuspended with sterile PBS. The experiment was repeated three times to ensure that the degradation experiment was carried out with cefixime as the only carbon source. The incubator temperature was adjusted to 30℃, the medium pH was 7, the rotation speed was 200r / min, and the initial concentrations were set to 100, 200, 500, and 1000mg / L respectively. After culturing for 24h, the pollutant content was determined and its degradation rate was calculated.

[0096] The results are as follows Figure 8 As shown, the degradation rate of strain TY12 for 100 and 200 mg / L cefixime reached 100% in 24 hours, the degradation rate of 500 mg / L cefixime reached 89.69% in 24 hours, and the degradation rate of 1000 mg / L cefixime reached 83.73% in 24 hours. Strain TY12 can efficiently degrade high concentrations of cefixime.

[0097] The above is an example of the best implementation of the present invention, and the parts not described in detail are common knowledge of ordinary technicians in the field. The protection scope of the present invention shall be based on the content of the claims, and any equivalent transformation based on the technical enlightenment of the present invention is also within the protection scope of the present invention.

Claims

1. A cefixime-degrading bacterium, characterized in that: It is Pseudochrobactrum asaccharolyticum TY12, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 13, 2023, with the deposit number CGMCC NO.28949.

2. Use of the cefixime-degrading bacteria according to claim 1 in degrading cefixime.

3. The use according to claim 2, characterized in that: The concentration of cefixime during degradation is 100-1000 mg / L.

4. The use according to claim 2, characterized in that: During the degradation, the inoculation amount of the cefixime-degrading bacteria is 1%-20%.

5. The use according to claim 2, characterized in that: The ambient temperature during degradation is 20-40°C.

6. The use according to claim 2, characterized in that: The environmental pH during degradation is 5-9.

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