CN113637481A - Application of chitosan and derivatives thereof in reducing antibiotic resistance gene pollution in soil - Google Patents
Application of chitosan and derivatives thereof in reducing antibiotic resistance gene pollution in soil Download PDFInfo
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- CN113637481A CN113637481A CN202110836992.1A CN202110836992A CN113637481A CN 113637481 A CN113637481 A CN 113637481A CN 202110836992 A CN202110836992 A CN 202110836992A CN 113637481 A CN113637481 A CN 113637481A
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Abstract
The invention discloses application of chitosan and derivatives thereof in reducing antibiotic resistance gene pollution in soil, according to the actual condition of the soil, chitosan and derivatives thereof in a proper proportion are directly added into the soil, the operation is simple and rapid, the abundance of various antibiotic resistance genes in the soil can be obviously reduced, a new and effective way is provided for controlling the ARGs pollution in the soil, and the application prospect is wide.
Description
Technical Field
The invention relates to the technical field of environmental engineering, in particular to application of chitosan and derivatives thereof in reducing antibiotic resistance gene pollution in soil.
Background
The potential risk of Antibiotic Resistance Genes (ARGs) in the environment to the ecological environment and human health processes has been recognized as an emerging environmental pollutant. A great deal of research finds that in some clinical pathogenic bacteria, ARGs are derived from non-pathogenic bacteria in the environment, and are transmitted and spread by horizontal gene transfer by virtue of movable genetic elements such as plasmids, transposons and the like.
Soil is an important storage bank of the ARGs in the environment, and exogenous ARGs brought by human activities are main sources of the ARGs in the soil, for example, antibiotics are frequently used in animal husbandry as feed additives and widely used for promoting animal growth and treating diseases, but the antibiotics cannot be completely absorbed after entering animal bodies and can be discharged out of the animal bodies along with excrement, so that the excrement of the livestock and poultry often contains the antibiotics and the ARGs. In order to improve crop yield, livestock manure is applied to agricultural soil in large quantities as an organic fertilizer, resulting in continuous residue of antibiotics in the soil, directly resulting in increased diversity and abundance of ARGs in the soil. In addition, sludge and effluent from sewage treatment plants contain hundreds of different types of ARGs that enter agricultural soils with sludge application and sewage irrigation. The global pattern of the ARGs in the soil of the global multi-land farmland has been researched by a plurality of researches, the detection rate of the ARGs is high, the types are diversified, and the abundance is high, particularly, the pollution situations of the ARGs such as sulfonamide resistance genes (sul1 and the like), tetracycline resistance genes (tetM, tetW and the like) are serious, and the hot spot areas polluted by the ARGs are mainly agricultural planting areas with active human activities.
Bacterial communities in soil are one of the evolutionary origins of ARGs, which can be migratory transmitted between environmental bacteria and clinical pathogens by horizontal gene transfer. In addition, various ARGs can be detected at different parts of vegetables planted in agricultural soil, potential harm can be caused to human bodies through the migration of food chains, the use effect of clinical medicines is seriously reduced by the resistance of generated antibiotics, the medicine selection range is narrowed, and the human health is threatened.
The prior art has few strategies for controlling such pollution of ARGs, for example, aerobic composting or anaerobic digestion of mainly manure or sludge applied to the soil in order to reduce ARGs from the source, but only by adding soil amendments to ARGs that have entered the soil or have accumulated for many generations. Currently, a few types of soil conditioners are found, and not all soil conditioners have a positive effect of reducing the pollution of ARGs.
Disclosure of Invention
Aiming at the problems in the prior art, the chitosan and the derivatives thereof are used as soil conditioners and act on the soil polluted by antibiotic resistance genes, so that the pollution of the antibiotic resistance genes in the soil can be greatly reduced.
In the invention, the chitosan and the derivatives thereof are applied to the reduction of the antibiotic resistance gene pollution in soil.
Specifically, the chitosan and the derivatives thereof are applied to reducing the abundance of antibiotic resistance genes in soil. The gene abundance refers to the copy number of each type of antibiotic resistance gene in the genome, and the higher the abundance is, the more the number of the gene is. Abundance includes absolute and relative abundances, where relative abundance is used to describe the percentage of individual base factors to the total base factor.
Optionally, the chitosan derivative comprises carboxylated chitosan, hydroxylated chitosan, chitosan salts, and chitosan oligosaccharide.
Optionally, the carboxylated chitosan is carboxymethyl chitosan or carboxyethyl chitosan; the hydroxylated chitosan is hydroxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, and methyl glycol chitosan; the chitosan salt is chitosan lactate, chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan nitrate, chitosan sulfate, chitosan acetate, chitosan citrate, chitosan succinate, chitosan glutamate, chitosan tartrate and chitosan acetylsalicylate; the chitosan oligosaccharide is an oligosaccharide product obtained by degrading chitosan.
Optionally, the antibiotic resistance gene is diaminopyrimidines, fosfomycins, nucleosides, macrolides-lincomycins-streptogramins B (MLSB), glycopeptides, tetracyclines, chloramphenicol, aminoglycosides, fluoroquinolones, rifamycins, aminocoumarins, sulfonamides, and peptides.
Optionally, the application includes: adding chitosan and its derivatives into the soil to be treated.
Optionally, the addition amount of the chitosan and the derivatives thereof is 20-50 g/kg of dry soil weight.
Optionally, the addition amount of the chitosan and the derivatives thereof is 30g/kg of dry soil weight.
Optionally, the chitosan and the derivatives thereof are added to the soil in powder form.
Optionally, the deacetylation degree of the chitosan and the derivatives thereof is more than or equal to 95%, the viscosity is less than 200mPa & s, and the molecular weight is 10000-50000 Da.
The chitosan and the derivatives thereof used in the invention are purchased from Xia chemical technology (Shandong) limited company, wherein the chitosan is prepared by processing Alaska deep sea snow crab by the processes of decalcification, deproteinization, decoloration, deacetylation and the like, and is white-like to yellow brown powder.
The invention provides a new application of chitosan and derivatives thereof, which has good application in reducing the pollution of antibiotic resistance genes in soil, can obviously reduce the abundance of various antibiotic resistance genes, and provides an ideal path for controlling the ARGs pollution in soil.
Drawings
FIG. 1 is a graph showing the effect of chitosan (added at a ratio of 30g/kg dry weight of soil) on various types of antibiotic resistance genes;
FIG. 2 is a graph showing the rate of decrease of various types of antibiotic resistance genes after chitosan treatment.
FIG. 3 is a graph showing the rate of decrease of various types of antibiotic resistance genes after carboxymethyl chitosan treatment.
Detailed Description
The technical solutions of the present invention will be further described with reference to the following embodiments, but the present invention is not limited thereto.
Example 1
The invention verifies and evaluates the application effect of chitosan in controlling the antibiotic gene pollution in soil through a laboratory simulation test, and the specific operation steps are as follows:
harvesting of soil
Soil used in the test is collected from a large-scale vegetable planting facility base with years of livestock manure application history in Jiaxing city of Zhejiang province in 2020 and 9 months, and 0-15 cm of surface soil is collected after the surface layer withered and fallen substances are removed by adopting a five-point sampling method. A total of 40kg of soil was collected and quickly transferred to a laboratory, where plant residues and stones were removed, air dried, and then sieved through a 2mm sieve to obtain sample soil. Chicken manure was added to the sample soil at 30g/kg dry weight of soil and mixed well to simulate a common soil improvement that may introduce multiple ARGs into the soil. Adjusting the water content of the soil to 60% of the maximum water holding capacity, placing the soil in an artificial climate box, and culturing for 7 days at the temperature of 25 ℃ and the humidity of 70% for later use.
Microcosm test
Weighing 500.0g (dry weight) of the soil to be used in a white porcelain dish, uniformly scattering chitosan (Xiyaichou chemical technology (Shandong) Co., Ltd.) in the soil and continuously stirring to ensure that the content of the chitosan in the soil reaches 30g/kg of the dry weight of the soil, then spraying a proper amount of sterile water, adjusting the water content of the soil to 60 percent of the maximum water holding capacity, and taking a group without adding the chitosan as a control. Dividing the soil into 3 parts (each part is 150.0g in dry weight, the rest soil is retained in a refrigerator at-20 ℃), transferring the soil into a numbered polyethylene basin (the inner diameter is 10cm, the height is 9cm, the bottom diameter is 6.5cm), covering the basin mouth with tinfoil paper, pricking 5 holes with 1mm, placing the basin mouth in an artificial climate box, carrying out dark culture for 90 days under the conditions of 25 ℃ and 70% humidity, and storing the obtained soil sample at-20 ℃. And monitoring the water content of the soil by adopting a soil weighing method every two days in the dark culture process, and adding a proper amount of sterile water to keep the water content of the soil constant.
Extraction and quality detection of total DNA of soil
0.5g of a soil sample was taken and FastDNA was usedTExtracting total DNA of Soil by using an M Spin Kit for Soil Kit, referring to Kit specifications in specific steps, and storing an extracted DNA sample in a refrigerator at the temperature of-20 ℃ for later use. The concentration of a soil DNA sample is measured by using a Qubit fluorescence quantitative analyzer, the integrity and the purity of the DNA sample are simultaneously detected by adopting a gel electrophoresis method, the agarose concentration is 1 percent, the Marker is Trans 15K plus, the sample loading amount is 1 mu L, the voltage is 100V, and the electrophoresis time is 40 min. The results indicate that the DNA samples can be used for macro-genomic pooling and sequencing analysis.
Metagenomic library construction and sequencing
By using
UltraTMAnd constructing a DNA sequencing library by using the DNA library preparation kit. The total DNA of the soil is randomly broken into fragments with the length of 350bp by an ultrasonic disruptor.
Performing end repairing, adding 55.5 μ L of LDNA fragment (about 1 μ g), 3.0 μ L of end repairing enzyme, and end repairing reaction buffer (10X) into the tube, placing in a thermal cycler, setting the temperature of a thermal cover to be more than or equal to 75 ℃, and performing reaction at 20 ℃ for 30 minutes, 65 ℃ for 30 minutes, and storing at 4 ℃.
Performing linker ligation, adding 15 μ L Blunt/TA ligase, 1 μ L ligation synergist and 2.5 μ L EBNext linker to the end-repair reaction mixture, incubating for 15 min at 20 ℃ in a thermal cycler, adding 3 μ LUSER enzyme to the mixed system, mixing, incubating for 15 min at 37 ℃, and heating to a temperature not lower than 47 ℃.
And (3) screening the sizes of the nucleic acid fragments, and adding 13.5 mu L of distilled water and 40 mu L of LAMPure XP magnetic beads into a connection reaction system. Mix well and incubate at room temperature for 5 minutes. The tube was placed on a magnetic stand and the supernatant was separated from the magnetic beads. After 5 minutes, the supernatant containing the DNA was carefully transferred to a new tube while discarding the magnetic beads containing the larger DNA fragments and adding again 20. mu.L LAMPure XP magnetic beads. Mix well and incubate at room temperature for 5 minutes. The tube was placed on a magnetic stand for 5 minutes and the supernatant was separated from the magnetic beads. After 5 minutes, the supernatant containing the non-target size DNA fragments was carefully removed. On a magnetic stand, 200 μ L of 80% ethanol was added to the tube. And (4) incubating at room temperature for 30 seconds, sucking and removing supernatant, repeating the steps once, and finally uncovering and airing the magnetic beads for 5 minutes. The tube was removed from the magnetic stand and the target sized DNA was eluted from the beads into 17. mu.L of 10 mM Tris-HCl, mixed well with a vortexer and incubated at room temperature for 2 minutes. The tube was placed on a magnetic stand and after 5 minutes 15. mu.L of the liquid was transferred to a new PCR tube for amplification.
PCR amplification was performed by adding 15. mu.L of adaptor-ligated fragment, 25. mu.L of EBNext Q5 Hot Start HiFi PCR Master Mix, 10. mu.L of Landex/universal primer to PCR tubes (total volume 50. mu.l). The PCR reaction condition is pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98 ℃ for 10 seconds; annealing/extension at 65 ℃ for 75 seconds, repeated for 4 cycles; final extension at 65 ℃ for 5 min; storing at 4 ℃.
The PCR amplification product was purified, and 45. mu.L (0.9X) of AMPure XP beads were added to the PCR reaction system. Mix well and incubate at room temperature for at least 5 minutes. The tube was placed on a magnetic stand and the supernatant was separated from the magnetic beads. After 5 minutes, the supernatant was carefully aspirated. On a magnetic stand, 200 μ L of 80% ethanol was added to the tube. Incubate at room temperature for 30 seconds, then carefully aspirate the supernatant and repeat the wash once more. The tube was placed on a magnetic stand, and the beads were air dried for 5 minutes. The tube was removed from the magnetic stand and 33. mu.L of 0.1 XTE was added to elute the DNA from the beads. Mix well with a vortex apparatus and incubate for 2 minutes at room temperature. The tube was placed on a magnetic stand and after 5 minutes 28. mu.L of the liquid was transferred to a new PCR tube and stored at-20 ℃. Finally, the size range of the DNA fragments in the library was confirmed using an agilent 2100 chip bioanalyzer.
Finally, metagenomic sequencing is carried out on the constructed library by adopting a PE 150 sequencing strategy in an IlluminaNovaSeq sequencer.
The sequence of the adaptor involved in library construction consists of an "anchor sequence + index + primer", and comprises two sequences (the sequences are shown below), wherein the underline is the anchor sequence, and the non-underline is the primer sequence:
5’-AATGATACGGCGACCACCGAGATCTACAC(index)TCTTTCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO：1)
5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(index)ATCTCGTATGCCGTCTTCTGCTTG-3’(SEQ ID NO：2)
sequencing data analysis and ARGs control effect evaluation
For metagenome original sequence data of the off-line sequencing, the quality control is carried out by using fastp software. The sequences were then aligned to The Antibiotic Resistance gene Database (CARD) using BLASTX with The alignment parameters E-value set to 1E-5 and max _ target _ seqs set to 1. And removing sequences with the similarity lower than 80% and the matching length less than 25 amino acids in the comparison result by using a self-defined python script, and filtering to obtain an ARGs similar sequence set. And finally, calculating the relative abundance of the ARGs by adopting a self-defined python script, classifying the drug resistance types of the ARGs, and counting the control effect of chitosan treatment on various types of ARGs pollution in the soil.
The relative abundance (ppm) of ARGs is calculated as follows:
wherein: n is a radical ofARGs like sequencesThe number of ARGs similar sequences which meet the alignment screening parameters; n is a radical ofTotal number of sequencesThe total number of sequencing data; n is the total number of ARGs classified into a particular antibiotic type.
In the preliminary experiment stage, the control effect of ARGs pollution under the treatment of chitosan and derivatives thereof with different addition ratios (20-50 g/kg of dry soil weight) is preliminarily researched, several typical ARGs in the soil are quantitatively analyzed, and the chitosan and derivatives thereof with the addition ratio of 20-50 g/kg of dry soil weight have certain reduction effect on the ARGs pollution, the reduction efficiency and the material cost are comprehensively considered, particularly the chitosan and derivatives thereof with the ratio of 30g/kg of dry soil weight have the most advantage on the control effect of the ARGs pollution, and the reduction efficiency of the resistant gene pollution is highest.
As a result, as shown in FIG. 1, the total abundance and the relative abundance of each type of ARGs in the soil treated with chitosan were significantly decreased (p <0.01) compared to the control group
The decrease rate of the relative abundance of each type of antibiotic resistance gene is shown in fig. 2, wherein the abundance of the diaminopyrimidine antibiotic resistance gene is decreased by 100%, the abundance of the fosfomycin antibiotic resistance gene is decreased by 95.9%, the abundance of the nucleoside antibiotic resistance gene is decreased by 90.2%, the abundance of the MLSB antibiotic resistance gene is decreased by 86.4%, the abundance of the glycopeptide antibiotic resistance gene is decreased by 83.2%, the abundance of the tetracycline antibiotic resistance gene is decreased by 81.6%, the abundance of the chloramphenicol antibiotic resistance gene is decreased by 79.3%, the abundance of the aminoglycoside antibiotic resistance gene is decreased by 70.0%, the abundance of the fluoroquinolone antibiotic resistance gene is decreased by 68.4%, the abundance of the rifamycin antibiotic resistance gene is 56.0%, the abundance of the aminocoumarin antibiotic resistance gene is decreased by 43.8%, the abundance of the sulfonamide antibiotic resistance gene is decreased by 28.3%, and the abundance of the peptide antibiotic resistance gene is decreased by 17.9%.
Test results show that the application of the chitosan with the addition proportion of 30g/kg of soil dry weight has a strong control effect on ARGs pollution, and the abundance of various types of antibiotic resistance genes in the soil can be effectively reduced.
Example 2
The invention simultaneously verifies and evaluates the application effect of the chitosan derivative carboxymethyl chitosan in controlling the antibiotic gene pollution in soil through an indoor simulation test, and the specific operation steps are as follows:
harvesting of soil
The procedure is as in example 1.
Microcosm test
The procedure of example 1 was followed except that the experimental additive was carboxymethyl chitosan, a chitosan derivative.
Extraction and quality detection of total DNA of soil
The procedure is as in example 1.
Metagenomic sequencing
The procedure is as in example 1.
Sequencing data analysis and ARGs control effect evaluation
The procedure is as in example 1.
As a result, as shown in FIG. 1, the total abundance of ARGs and the relative abundance of each type of ARGs in the soil treated with carboxymethyl chitosan were significantly decreased (p <0.05) compared to the control group
The decrease rate of the relative abundance of each type of antibiotic resistance gene is shown in fig. 3, wherein the abundance of the nucleoside antibiotic resistance genes is decreased by 80.6%, the abundance of the fluoroquinolone antibiotic resistance genes is decreased by 78.9%, the abundance of the diaminopyrimidine antibiotic resistance genes is decreased by 70.2%, the abundance of the fosfomycin antibiotic resistance genes is decreased by 58.3%, the abundance of the aminocoumarin antibiotic resistance genes is decreased by 51.3%, the abundance of the glycopeptide antibiotic resistance genes is decreased by 34.9%, the abundance of the rifamycin antibiotic resistance genes is decreased by 33.6%, and the abundance of the MLSB antibiotic resistance genes is decreased by 24.8%.
Test results show that the application of the carboxymethyl chitosan with the addition proportion of 30g/kg of dry soil has a strong control effect on ARGs pollution, and the abundance of various types of antibiotic resistance genes in the soil can be effectively reduced.
Appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and teachings of the foregoing description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
SEQUENCE LISTING
<110> Zhejiang university
Application of chitosan and derivatives thereof in reducing antibiotic resistance gene pollution in soil
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 58
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 2
<211> 57
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gatcggaaga gcacacgtct gaactccagt cacatctcgt atgccgtctt ctgcttg 57
Claims (9)
1. Application of chitosan and derivatives thereof in reducing antibiotic resistance gene pollution in soil.
2. Use according to claim 1, characterized in that chitosan and its derivatives are used to reduce the abundance of antibiotic resistance genes in soil.
3. Use according to claim 1, characterized in that the antibiotic resistance genes are diaminopyrimidines, fosfomycins, nucleosides, macrolides-lincomycins-streptogramins B, glycopeptides, tetracyclines, chloramphenics, aminoglycosides, fluoroquinolones, rifamycins, aminocoumarins, sulfonamides and peptides.
4. The use according to claim 1, comprising: adding chitosan and its derivatives into the soil to be treated.
5. The use of claim 4, wherein the chitosan and its derivatives are added in an amount of 20-50 g/kg dry weight of soil.
6. Use according to claim 4, characterized in that the chitosan and its derivatives are added to the soil in powder form.
7. The use according to claim 1, wherein the chitosan and its derivatives have a degree of deacetylation of 95% or more, a viscosity of less than 200 mPa-s and a molecular weight of 10000 to 50000 Da.
8. Use according to claim 1, characterized in that the chitosan derivatives comprise carboxylated chitosan, hydroxylated chitosan, chitosan salts, chitosan oligosaccharides.
9. Use according to claim 8, wherein the carboxylated chitosan is carboxymethyl chitosan, carboxyethyl chitosan; the hydroxylated chitosan is hydroxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, and methyl glycol chitosan; the chitosan salt is chitosan lactate, chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan nitrate, chitosan sulfate, chitosan acetate, chitosan citrate, chitosan succinate, chitosan glutamate, chitosan tartrate or chitosan acetylsalicylate.
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| CN115181572A (en) * | 2022-07-22 | 2022-10-14 | 浙江大学 | Application of theaflavin and composition thereof in reduction of antibiotic resistance genes in soil |
| CN116426445A (en) * | 2023-06-08 | 2023-07-14 | 南京农业大学三亚研究院 | Pseudomonas bacteria NJAU-T102 and application thereof |
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| CN116426445B (en) * | 2023-06-08 | 2023-08-04 | 南京农业大学三亚研究院 | Pseudomonas bacteria NJAU-T102 and application thereof |
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