A kind of cephalexin-degrading phage and its application

Abstract

The invention relates to a cephalexin-degrading phagocytic bacterium and application thereof. The invention screens a cephalexin-degrading phagocytic bacterium CEF13 which can tolerate high-concentration antibiotics and efficiently degrade cephalexin at high temperature. The concentration of cephalexin is 100-1000 mg / L, the inoculation amount of CEF13 is 1%-20%, the degradation temperature is 20-40 DEG C, and the pH is 5-9. The cephalexin can be effectively degraded. Under the conditions of a temperature of 30 DEG C, a culture medium pH of 7, an inoculation amount of 20%, and a rotation speed of 200 r / min, the degradation rate of 500 mg / L cephalexin by the strain CEF13 reaches 77.33% in 20 hours. The strain has high biological safety, will not endanger human health or pollute the environment during application, and has important application value.

Description

Technical Field

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

[0002] Antibiotics are natural, synthetic or semi-synthetic compounds that can kill or inhibit the growth or metabolic activity of microorganisms and are widely used to treat diseases caused by bacterial infections in humans and animals. Since the first antibiotic was discovered in the late 1920s, the scope of application of antibiotics has become increasingly wide and the amount of use has continued to increase. Due to the excessive use of antibiotics, they continue to accumulate and discharge in the environment and cause serious pollution, leading to the increasing problem of antibiotic resistance (AMR), which has caused serious harm to human health and the ecological environment. Antibiotics have become an emerging pollutant and an important part of pharmaceuticals and personal care products (PPCPs). The drug resistance caused by them has become a serious public health crisis that threatens global social and economic development. my country has included antibiotics in the "List of New Pollutants under Key Control".

[0003] β-lactam antibiotics are the oldest antibiotics discovered, with the largest system of species. The β-lactam ring makes β-lactam antibiotics the most important class of antibiotics. This type of antibiotic has the advantages of strong antibacterial activity, broad antibacterial spectrum, and low toxicity. Cephalexin is the first generation of broad-spectrum β-lactam antibiotics and is widely used to treat human and livestock diseases. Cephalexin has a low utilization rate in organisms, and most of the rest is excreted through urine or feces. With the excessive use of cephalexin, serious pollution has been caused in the environment, causing the accumulation and spread of biological toxicity and drug resistance genes (ARGs), posing a serious threat to human health and ecological security. Therefore, the treatment of cephalexin in the environment has become an urgent problem to be solved. Microbial degradation is a green, efficient and sustainable way to remediate antibiotic pollution. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a phagocytic bacterium capable of degrading cephalexin and its application 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 phage that can degrade cephalexin is Variovorax boronicumulans CEF13, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 13, 2023, with the deposit number CGMCC No.29009.

[0007] The cephalexin-degradable phagocyte is used in the degradation of cephalexin.

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

[0009] Furthermore, during the degradation, the inoculation amount of the cephalexin-degradable phagocytes 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 Variovorax boronicumulans provided by the invention can tolerate high-concentration antibiotics and is a microbial strain that can efficiently degrade cephalexin at high temperature, the concentration of cephalexin is 100-1000 mg / L, the inoculation amount of CEF13 is 1%-20%, the degradation temperature is 20-40°C, and the pH is 5-9, and cephalexin can be effectively degraded. Under the conditions of a temperature of 30°C, a culture medium pH of 7, an inoculation amount of 20%, and a rotation speed of 200 r / min, the degradation rate of 500 mg / L cephalexin by the strain CEF13 reaches 77.33% in 20 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 substrate concentration and peak area;

[0015] Figure 2 This is the colony morphology of strain CEF13 after growing on LB solid medium for 2 days;

[0016] Figure 3 It is a curve diagram showing the effect of temperature on the degradation of CEF13;

[0017] Figure 4 This is a curve diagram showing the effect of pH on the degradation of CEF13;

[0018] Figure 5 This is a curve diagram showing the effect of inoculation amount on the degradation effect of CEF13;

[0019] Figure 6 This is a curve diagram showing the effect of substrate concentration on the degradation of CEF13. DETAILED DESCRIPTION

[0020] 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.

[0021] 1. Screening of cephalexin-degrading phagocytic strains

[0022] 1. Material preparation

[0023] Sample source: coastal sediments from Dashentang, Tianjin

[0024] 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.

[0025] 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.

[0026] 2. Preliminary screening of bacterial strains

[0027] Sediment samples were collected from the coastal area of ​​Dashentang, Tianjin. 5g of sludge was inoculated into 100mL of inorganic salt liquid culture medium. The final concentration of cephalexin in the culture medium was 50mg / L. The culture was continuously cultured in a constant temperature shaking incubator at 30℃ and 200r / min for 5 days. 5mL of culture solution was transferred to an inorganic salt culture medium with a cephalexin concentration of 100mg / L. After another 5 days of culture under the same conditions, the culture was transferred to a culture medium with a cephalexin concentration of 200mg / L. The culture was then cultured for 5 days. 5mL of culture solution was transferred to a culture medium with a cephalexin concentration of 500mg / L. The bacterial solution was then diluted 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5The purified colonies were transferred to a liquid inorganic salt medium with cephalexin as the sole carbon source to screen strains with degradation performance.

[0028] 3. Strain rescreening

[0029] The strain obtained in the above steps is inoculated into LB liquid medium for activation culture, and then inoculated into an inorganic salt liquid medium with cephalexin as the sole carbon source at an inoculum size of 5%, and cultured under shaking conditions at 30°C and 200r / min. The degradation rate of each strain is determined by high performance liquid chromatography, and the strain CEF13 that can efficiently degrade cephalexin is screened. At present, there is no report on the degradation of cephalexin by phagocytic bacteria at home and abroad. Strain CEF13 is a new strain that can degrade cephalexin.

[0030] 4. Cephalexin content determination method

[0031] The bacterial culture solution was centrifuged at 800 rpm for 10 min. The precipitate was discarded, and the supernatant was retained and filtered through a 0.22 um microporous membrane, and then sealed for testing.

[0032] Drawing of the standard curve of cephalexin: Accurately weigh 1000 mg of cephalexin in a 100 mL volumetric flask, dilute to the mark with mobile phase, and prepare a standard solution containing 10 mg of cephalexin per mL. Add mobile phase to dilute and prepare 50, 100, 200, 400, 600, 800, 1000, 1200, and 1400 mg / L of cephalexin standard solution. Draw the standard curve with the mass concentration of cephalexin as the horizontal axis and the peak area as the vertical axis. The curve drawn is as follows: Figure 1 As shown, Y = 12.464X-296.99, R 2 =0.9993, indicating a good linear relationship in the concentration range of 50-1000 mg / L.

[0033] HPLC detection conditions: octadecylsilane bonded silica gel as filler; water-methanol-3.86% sodium acetate solution-4% acetic acid solution (742:240:15:3) as mobile phase; detection wavelength 254nm, column temperature 30°C. Substitute the measured peak area into the standard curve to calculate the substrate concentration and the degradation rate.

[0034] 2. Strain morphology and molecular biological identification

[0035] 1 Observation of colony morphology

[0036] The strain CEF13 was grown on LB solid medium for 2 days, and the colony morphology was as follows Figure 2 As shown, the colonies are yellow with smooth and moist edges.

[0037] 2 Molecular biological identification of strain CEF13

[0038] 2.1 Genome extraction

[0039] The extraction method of strain CEF13 genomic DNA is as follows:

[0040] (1) Collect 1.0×10 9 (1mL bacterial solution OD 600 1-1.5) of bacterial culture, centrifuge at 12000×g for 30s, discard the supernatant, and resuspend the precipitate with 150 μL of Buffer S to which RNase A has been added.

[0041] (2) Add 20 μL of lysozyme storage solution, mix well, and let stand at room temperature for 5 min.

[0042] (3) Add 30 μL 0.25 M EDTA (pH 8.0), mix well, and place on ice for 5 min.

[0043] (4) Add 450 μL Buffer GA, vortex for 15 seconds, and incubate in a 65°C water bath for 10 minutes.

[0044] (5) Add 400 μL of Buffer GB and 1 mL of Buffer DV (precooled at 4°C), mix vigorously, and centrifuge at 12,000 × g for 2 min.

[0045] (6) Discard the upper phase as much as possible and retain the interphase precipitate and lower phase. Add 1 mL of 4°C pre-cooled Buffer DV, mix vigorously, and centrifuge at 12,000 × g for 2 min.

[0046] (7) Discard the upper phase and transfer the lower phase to a filter (the filter is placed in a 2 mL centrifuge tube). Centrifuge at 12000×g for 1 min.

[0047] (8) Discard the filter and add 400 μL of Buffer BV to the filtrate and mix well.

[0048] (9) Place the preparation tube in a 2 mL centrifuge tube, transfer the mixed solution in step (8) into the preparation tube, and centrifuge at 12,000 × g for 1 min.

[0049] (10) Discard the filtrate, place the preparation tube back into the original 2 mL centrifuge tube, add 500 μL Buffer W1, and centrifuge at 12,000 × g for 1 min.

[0050] (11) Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL Buffer W2, and centrifuge at 12,000 × g for 1 min.

[0051] (12) Wash once more with 700 μL Buffer W2 using the same method.

[0052] (13) Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12,000 × g for 1 min.

[0053] (14) Place the preparation tube in another clean 1.5 mL centrifuge tube, add 100-200 μL Eluent or deionized water to the center of the siLica membrane, and let it stand at room temperature for 1 min. Centrifuge at 12,000 × g for 1 min to elute the DNA.

[0054] 2.2 PCR amplification of bacterial genome

[0055] Primer sequence: 27F: 5-AGAGTTTGATCCTGGCTCAG-3

[0056] 1492R:5-GGTTACCTTGTTACGACTT-3

[0057] PCR amplification reaction system

[0058] Add the following ingredients to a 0.2 mL centrifuge tube:

[0059] Table 1 PCR amplification reaction system

[0060]

[0061] Mix by flicking gently, collect the droplets on the tube wall to the bottom of the tube by instant centrifugation, and perform PCR reaction on a PCR amplification instrument. The reaction parameters are as follows:

[0062] Table 2 PCR amplification reaction program

[0063]

[0064] After the reaction was completed, 3uL of PCR product was taken for 1% agarose gel electrophoresis to confirm the PCR amplified fragment. The PCR product was recovered using the AxyPrep DNA gel recovery kit and then sent to Shanghai Paisono Co., Ltd. for sequencing.

[0065] 2.3 Sequencing results

[0066] After sequencing, the length of the 16S rDNA gene sequence of strain CEF13 was 1467bp. Comparison in the NCBI database showed that the 16S rDNA sequence of strain CEF13 had the highest similarity with Variovorax boronicumulans NBRC103145, reaching 99.86%. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 14, 2023, with the deposit number CGMCC NO.29009. Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The obtained 16S rDNA sequence is as follows:

[0067] ACCCAACTGTCGTGGTATCGCCCTCCTTGCGGTTAAGCTAACTACTT

[0068] CTGGCAGAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAGACCC

[0069] GGGAACGTATTCACCGTGACATTCTGATCCACGATTACTAGCGATTCCG

[0070] ACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGACTGGTTT

[0071] TATGGGATTAGCTCCCTCGCGGGTTGGCAACCCTTTGTACCAGCCAT

[0072] TGTATGACGTGTGTAGCCCCACCTATAAGGGCCATGAGGACTTGACGTC

[0073] ATCCCCACCTTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCA

[0074] ACTGAATGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTA

[0075] ACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTG

[0076] TTACGGTTCTCTTTCGAGCACTAAGCCATCTCTGGCGAATTCCGTACAT

[0077] GTCAAAGGTGGGTAAGGTTTTTCGCGTTGCATCGAATTAAACCACATCA

[0078] TCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGC

[0079] GGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTTCGTTACTGA

[0080] GTCAGTGAAGACCCAACAACCAGTTGACATCGTTTAGGGCGTGGACTA

[0081] CCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCATGAGCGTC

[0082] AGTACAGGTCCAGGGGATTGCCTTCGCCATCGGTGTTCCTCCGCATATC

[0083] TACGCATTTCACTGCTACACGCGGAATTCCATCCCCCTCTACCGTACTCT

[0084] AGCTATGCAGTCACAGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCA

[0085] CAACTGTCTTACATAACCGCCTGCGCACGCTTTACGCCCAGTAATTCCG

[0086] ATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAG

[0087] CCGGTGCTTATTCTTACGGTACCGTCATTAGCCTTCTGTATTAGAAAAGA

[0088] CCGTTTCGTTCCGTACAAAAGCAGTTTACAACCCGAAGGCCTTCATCCT

[0089] GCACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCCC

[0090] ACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGG

[0091] CTGGTCGTCCTCTCAGACCAGCTACAGATCGAAGGCTTGGTGAGCCTT

[0092] TACCTCACCAACTACCTAATCTGCCATCGGCCGCTCCATTCGCGCAAGG

[0093] TCTTGCGATCCCCTGCTTTCATCCGTAGATCGTATGCGGTATTAGCACAG

[0094] CTTTCGCTGCGTTATCCCCCACGATTGGGCACGTTCCGATGTATTACTCA

[0095] CCCGTTCGCCACTCGCCGCCAGGATTGCTCCCGCGCTGCCGTTCGACT

[0096] TGCATGTGTAAGGCATGCCGCCAGCGTTCAATCTGAGCCAGGTTTCAA

[0097] ACTCTA

[0098] 3. Degradation characteristics of strain CEF13

[0099] 1 Effect of temperature on CEF13 degradation

[0100] Take the above CEF13 strain, after activation in LB liquid medium, take 10% of the inoculation amount of bacterial liquid, centrifuge at 8000rpm for 2min, discard the supernatant LB medium, resuspend 3 times with sterile PBS solution to remove the residual LB medium, and then inoculate into a sterile inorganic salt medium with a concentration of 500mg / L cephalexin, the culture temperature is set to 20°C, 25°C, 30°C, 35°C and 40°C, the shaking speed is set to 200r / min, the pH is 7, three parallels per group, and a sterile treatment is set as a control for unified culture. Take samples after 20h to determine the residual concentration of cephalexin and calculate the degradation rate.

[0101] At different temperatures, due to the differences in the characteristics of the strains themselves, their degradation performance will also vary. Figure 3It can be seen that under different temperature conditions, the degradation of 500 mg / L cephalexin by the phagocytic bacterium CEF13 is significantly different. Within the temperature range of 20-40°C, cephalexin can be degraded to varying degrees. As the culture temperature increases, the degradation ability of strain CEF13 gradually increases. When the culture temperature reaches 30°C, the degradation ability of the strain is the strongest, reaching 55.75%.

[0102] Effect of 2pH on the degradation of CEF13

[0103] Take the above CEF13 strain, after activation in LB liquid medium, take 10% of the inoculation amount of bacterial liquid, centrifuge at 8000rpm for 2min, discard the supernatant LB medium, resuspend 3 times with sterile PBS solution to remove the residual LB medium, and then inoculate into a sterile inorganic salt medium with a concentration of 500mg / L cephalexin, the medium pH is set to 5, 6, 7, 8 and 9 respectively, the culture temperature is set to 30°C, the shaking speed is set to 200r / min, three parallels per group, and a sterile treatment is set as a control, and uniform culture is performed. Samples are taken after 20h to determine the residual concentration of cephalexin and calculate the degradation rate.

[0104] At different pH levels, the ability of strains to degrade pollutants will vary. Some bacteria can tolerate acid, some can tolerate alkali, and some can only degrade under neutral conditions. Figure 4 It can be seen that the degradation effect of strain CEF13 is poor in a strong acidic environment. As the alkalinity increases, the degradation effect gradually increases, and the degradation rate reaches a maximum of 74.94% at pH 9. Strain CEF13 is better at degrading cephalexin under alkaline conditions.

[0105] 3 Effect of inoculation amount on the degradation effect of CEF13

[0106] Take the above CEF13 strain, after activation in LB liquid medium, take 1%, 2%, 5%, 10% and 20% of the inoculation amount of bacterial solution respectively, centrifuge at 8000rpm for 2min, discard the supernatant LB medium, resuspend 3 times with sterile PBS solution to remove the residual LB medium, and then inoculate into a sterile inorganic salt medium with a concentration of 500mg / L cephalexin, set the culture temperature to 30°C, pH to 7, set the shaking speed to 200r / min, three parallels per group, and set the sterile treatment as a control for unified culture. Take samples after 20h to determine the residual concentration of cephalexin and calculate the degradation rate.

[0107] The inoculum size can affect the initial growth of microorganisms. Too low an inoculum size will result in a longer delay period in bacterial culture, thus affecting the degradation rate. Figure 5It can be seen that with the increase of inoculation amount, the degradation rate of cephalexin by strain CEF13 showed an upward trend. When the inoculation amount was 20%, the degradation effect of strain CEF13 was the best, and the degradation rate of 500 mg / L cephalexin after 20 hours of cultivation could reach 77.33%.

[0108] 4 Effect of substrate concentration on CEF13 degradation

[0109] Take the above CEF13 strain, after activation in LB liquid medium, take 10% of the inoculation amount of bacterial liquid, centrifuge at 8000rpm for 2min, discard the supernatant LB medium, resuspend 3 times with sterile PBS solution to remove the residual LB medium, and then inoculate into sterile inorganic salt medium with initial concentrations of 100, 200, 500 and 1000mg / L cephalexin respectively, set the culture temperature to 30°C, pH to 7, set the shaking speed to 200r / min, three parallels per group, and set the sterile treatment as a control for unified culture. Take samples after 20h to determine the residual concentration of cephalexin, and calculate the degradation rate.

[0110] The degradation effect of strain CEF13 on cephalexin in inorganic salt liquid medium with initial concentrations of 100, 200, 500, and 1000 mg / L is as follows Figure 6 As shown. For strain CEF13, when the concentration is 100-200 mg / L, the degradation rate of cephalexin increases from 62.67% to 69.94%. However, when the concentration is too high, the degradation efficiency of cephalexin by strain CEF13 will gradually decrease. It can be seen that cephalexin is the only carbon source for the growth of the strain, and the concentration has a certain effect on the degradation effect of the strain. When the concentration is 1000 mg / L, CEF13 can still degrade 43.978% of cephalexin, indicating that the bacteria can tolerate high concentrations of cephalexin and can effectively degrade it.

[0111] 4. Preparation of the CEF13 bacterial agent

[0112] 1. Preparation of seed culture solution

[0113] Inoculate phage CEF13 into 10 mL of LB culture medium and culture at 30 °C with shaking for at least 10 h until OD 600 When the value is 0.8±0.05, the culture is terminated and the seed bacterial solution is obtained;

[0114] 2 Fermentation culture

[0115] The seed bacterial liquid obtained in step 1 was inoculated into LB culture medium at a volume ratio of 1:500 for fermentation and culture at 30°C with shaking for 10-14 hours, centrifuged, and the precipitate was diluted with sterile water to obtain a bacterial suspension, so that the OD 600The value is about 0.2, that is, the microbial agent is obtained. The microbial agent obtained in this patent can be used for the remediation of cephalexin pollution in the environment. Under the conditions of temperature of 30°C, medium pH of 7, inoculation amount of 20%, and rotation speed of 200r / min, the degradation rate of strain CEF13 to 500mg / L cephalexin reaches 77.33% in 20h.

[0116] 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 phage capable of degrading cephalexin, characterized in that: For the phagocytic bacteria Variovorax )CEF13, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 14, 2023, with the deposit number CGMCCNo.29009.

2. Use of the cephalexin-degradable phagocyte according to claim 1 in the degradation of cephalexin.

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

4. The use according to claim 2, characterized in that: During the degradation, the inoculation amount of the cephalexin-degradable phagocytes 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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