Method for water body microbial safety risk assessment based on metagenomics and viromics

Abstract

This invention discloses a method for assessing the microbial safety risk of water bodies based on metagenomics and viromics, comprising: metagenomic detection of the relative abundance F of each pathogenic microorganism in the water sample to be assessed. i Where i represents the pathogenic microorganism label; for pathogenic microorganism i, determine its hazard level PM. i Antibiotic resistance gene risk value (ARG) i and virulence factor risk value (VF) i ; Determine the risk coefficient α of increased transmission due to water sample treatment methods, including antibiotic resistance gene risk values ​​and virulence factor risk values; For pathogenic microorganism i, determine the overall hazard level PM i Antibiotic resistance gene risk value (ARG) i Virulence factor risk value (VF) i The risk coefficient α and relative abundance F of increased transmission due to water sample treatment methods i Determine the overall risk value R of pathogenic microorganism i i The comprehensive risk value of all pathogenic microorganisms detected by the metagenomics is used to obtain the microbial safety risk assessment result R of the water sample to be evaluated.

Description

Technical Field

[0001] This invention relates to the field of aquatic microbial monitoring technology, specifically to a method for assessing the safety risks of aquatic microorganisms based on metagenomics and virology. Background Technology

[0002] Current drinking water standards stipulate that drinking water should be free of pathogenic microorganisms. However, the existing standard (GB 5749-2022) only tests for total bacterial count, total coliform count, and Escherichia coli count, without mentioning other pathogenic microorganisms and their hazards. Modern metagenomics and metaviromyctics studies show that pathogenic microorganisms and related risk genes pose potential hazards in various water bodies that are directly related to human health and may cause foodborne illnesses (including but not limited to drinking water, water used in food processing, and water that comes into contact with humans during production).

[0003] Food processing involves the use of large quantities of production water. A typical example is the production of sprouts, where tap water or well water is used in the seed selection, soaking, and germination stages, and production wastewater is treated biochemically and disinfected before being reused. If this production water is only supplied according to the standards mentioned above, it can easily create a risk of foodborne illnesses, threatening human health, and urgently needs to be addressed.

[0004] The following existing technologies were found through a search: The patent specification with publication number CN113897411A discloses a rapid and simple method for evaluating the microbial safety of water sources and drinking water. It utilizes the relationship between the growth rate of microorganisms in water and biodegradable organic matter to evaluate the microbial safety of water sources and drinking water by detecting the bacterial growth potential of water samples.

[0005] The patent specification with publication number CN109777854A discloses a rapid detection method for microorganisms in drinking water. The method involves placing a microbial filter membrane in the water to be tested for 30-40 minutes, removing it, and then letting it stand in corn syrup stained with shellac for 30-40 minutes. The color change or absorbance change of the corn syrup is then detected at 530-535 nm.

[0006] Metagenomic and metaviromic analysis methods are commonly used in scientific research, but a standardized monitoring method that can be used for routine surveillance has not yet been developed. Summary of the Invention

[0007] The inventors conducted metagenomic and metaviromic analyses on microorganisms in raw water (tap water or well water), rinsing water, primary treatment effluent (rinsing water after biochemical treatment), and disinfection effluent (primary treatment effluent after ultraviolet disinfection) used in sprout production. The analysis revealed changes in microbial composition, virulence factors, and resistance genes. The results showed that the disinfection effluent contained high-risk pathogenic microorganisms such as Bacillus and Salmonella. Furthermore, the abundance of certain microbial genes increased after disinfection, indicating that horizontal gene transfer may have occurred, and that energy metabolism and the two-component regulatory system were significantly activated, while the oxidative stress repair capacity decreased. This further enhances the adaptability of pathogens and increases public health risks, which are not included in the risk assessment of existing standards.

[0008] This invention provides a method for assessing the safety risks of aquatic microorganisms based on metagenomics and viromics. It assesses the potential pathogenicity of pathogenic microorganisms and detects the transfer and spread risks of antibiotic resistance genes and virulence factors through metagenomics, thereby assessing the risks in the environment.

[0009] The specific technical solution is as follows: A method for assessing the microbial safety risks of aquatic bodies based on metagenomics and virology includes: Metagenomic analysis of the relative abundance F of various pathogenic microorganisms in the water sample to be evaluated i , where i represents the pathogenic microorganism label; For pathogenic microorganism i, determine its degree of harm PM i (Pathogenic Microorganism), Antibiotic Resistance Gene Risk Value (ARG) i (Antibiotic Resistance Gene) and virulence factor risk value (VF) i (VirulenceFactor); Determine the risk coefficient α of increased transmission due to water sample treatment methods for antibiotic resistance gene risk values ​​and virulence factor risk values; Regarding pathogenic microorganisms i, the overall hazard level PM i Antibiotic resistance gene risk value (ARG) i Virulence factor risk value (VF) i The risk coefficient α and relative abundance F of increased transmission due to water sample treatment methods i Determine the overall risk value R of pathogenic microorganism i i ; The comprehensive risk value of all pathogenic microorganisms detected by metagenomics is used to obtain the microbial safety risk assessment result R of the water sample to be evaluated.

[0010] The method of this invention is applicable to assessing the potential hazards of pathogenic microorganisms and related risk genes in various water bodies that may cause foodborne diseases and are directly related to human health (including but not limited to drinking water, water used for food processing, and water bodies that come into contact with humans during production).

[0011] This invention involves collecting, enriching, extracting nucleic acids, and sequencing water samples to be evaluated, establishing a metagenomic and metavirographic library, and further evaluating and quantifying it. Metagenomic data is complex and requires classification and scoring of species, virulence factors, and resistance genes to assess its overall environmental risk.

[0012] In some embodiments, the higher the degree of hazard, the higher the PM2.5 risk assessment method for aquatic microbial safety. i The larger the value.

[0013] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology classifies the degree of hazard into four categories, wherein: Pathogens that can cause serious disease in humans or animals and for which there are usually no effective prevention and treatment measures are identified as high-risk. Microorganisms that can cause serious human or animal diseases and may be transmitted via aerosols, but which usually have preventive or therapeutic treatments, are identified as high-risk. Microorganisms that can cause diseases in humans or animals but are not usually highly contagious and for which there are effective preventive or therapeutic measures are identified as low-risk. Microorganisms that do not cause disease in humans or animals are classified as low-hazard.

[0014] Antibiotic-resistant genes (ARGs) can exist in various environments, including medical settings, drinking water, natural environments, and permafrost. The Comprehensive Antibiotic Resistance Database (CARD) and gene sequence alignment can accurately identify antibiotic resistance gene categories, drug targets, and antibiotic resistance mechanisms, providing a basis for antibiotic risk assessment.

[0015] This invention mainly relies on antibiotic resistance gene databases for gene identification and functional annotation, and constructs an evaluation system.

[0016] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology includes the antibiotic resistance gene risk value (ARG). i This includes transmission risk S and genetic risk G, where: Transmission risk S includes mobility and pathogenicity potential; mobility assessment criteria include whether it coexists with mobile genetic elements (such as integrons, insertion sequences, etc.) and whether it exists on mobile plasmids; pathogenicity potential assessment criteria include whether it coexists with genes encoding virulence factors. Genetic risk G is graded and assessed based on the antibiotic resistance mechanism mediated by antibiotic resistance genes and the clinical importance of the antibiotics corresponding to the antibiotic resistance genes.

[0017] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology includes three levels of genetic risk, wherein: Antibiotic resistance genes encoding sites related to resistance mechanisms of broad-spectrum antibiotics (such as carbapenems, vancomycin, and third-generation cephalosporins) are assessed as high-risk. Antibiotic resistance genes encoding sites related to resistance mechanisms against commonly used antibiotics (such as fluoroquinolones, macrolides, aminoglycosides, etc.) are assessed as medium risk. Genes encoding resistance mechanisms against less frequently used antibiotics or inherent, low-level antibiotic resistance are assessed as low risk.

[0018] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology indicates that the higher the risk, the greater the genetic risk G value.

[0019] The strength of a pathogen's ability to cause disease is called virulence, usually categorized as highly virulent, weakly virulent, or non-virulent, and is determined by the virulence factors encoded by the pathogen. Virulence factors can be classified into various types, including adhesion factors, invasion factors, secretion systems, toxins, and iron transporters. Different virulence factors work synergistically to enable pathogens to successfully adhere to and multiply in host cells, and to cause damage to host cells. With the rapid development of science and technology, more and more virulence factors have been discovered, and the pathogenic mechanisms of many pathogens have been elucidated. The Virulence Factors Database (VFDB) contains virulence factors that have been validated through biological experiments.

[0020] This invention takes into account the mechanism of action of virulence factors, the clinical severity of the diseases they cause (referencing the World Health Organization and relevant infectious disease diagnosis and treatment guidelines), and especially considers their transmission potential and adaptability in aquatic environments.

[0021] In some embodiments, the water microbial safety risk assessment method based on metagenomics and viromics, virulence factor risk value (VF) iThis includes the pathogenicity C of virulence factors and the environmental transmission risk E of virulence factors. The pathogenicity C of virulence factors is graded and assessed according to the characteristics of the toxin and its clinical harm, while the environmental transmission risk E of virulence factors is judged based on gene mobility and environmental adaptability.

[0022] In some embodiments, the water microbial safety risk assessment method based on metagenomics and viromics classifies the pathogenicity of virulence factor genes into three levels: low-risk toxins, intermediate-risk toxins, and high-risk toxins, wherein: Low-risk toxins are characterized by local effects or self-limiting diseases, with clinical hazards including mild diarrhea or skin infections, such as Staphylococcus aureus enterotoxin. Medium-risk toxins are characterized by systemic effects or require medical intervention, and their clinical hazards include fever, severe diarrhea, or tissue necrosis, such as anthrax toxins. High-risk toxins are characterized by their lethality or disabling effects, with clinical harms including organ failure, nervous system damage, or death, such as botulinum toxin.

[0023] In some embodiments, the water microbial safety risk assessment method based on metagenomics and viromics indicates that the higher the risk, the greater the pathogenicity C value of the virulence factor gene.

[0024] In some embodiments, the water microbial safety risk assessment method based on metagenomics and viromics includes criteria for judging gene mobility, such as whether the gene encoding virulence factors is located on a known mobile genetic element.

[0025] In some embodiments, the environmental adaptability assessment criteria of the metagenomics and viromics-based aquatic microbial safety risk assessment method include whether virulence factors have been reported in the literature to enhance the survival of microorganisms in water or biofilms.

[0026] In some embodiments, the water microbial safety risk assessment method based on metagenomics and viromics assesses the risk coefficient α of increased transmission due to water sample treatment methods by classifying and evaluating it according to the stress response characteristics of microorganisms caused by the water sample treatment process.

[0027] In some embodiments, the water microbial safety risk assessment method based on metagenomics and viromics includes microbial stress response characteristics such as gene transfer potential and upregulated expression of virulence factors.

[0028] In some embodiments, the more and stronger the stress response characteristics of the microorganisms in the water body based on metagenomics and viromics, the greater the risk coefficient α value of increased transmission due to water sample treatment methods.

[0029] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology is applied in scenarios including, but not limited to, microbial risk assessment of water used in food processing (such as water used in sprout production). Sprout production involves contact with or use of various types of water, such as raw water (e.g., tap water or well water), rinsing water from the production process, primary effluent after simple treatment, and disinfected reclaimed water. Based on the selection pressure exerted on the microbial community by different treatment processes, the stress response characteristics of microorganisms include the following classifications: Level A: Microorganisms are in their natural state with a low risk of gene transfer, such as untreated raw water like tap water or well water. Level B involves the destruction of some microorganisms, releasing DNA and promoting horizontal gene transfer. Examples include rinse water and primary treatment effluent. The corresponding water treatment processes are physical filtration, sedimentation, and biological treatment. Alternatively, long-term low-dose disinfection can lead to enhanced microbial adaptability and a persistent risk of gene transfer. Examples include reclaimed water and greywater reuse. The corresponding water treatment processes are multi-stage treatments such as membrane and disinfection.

[0030] Level C indicates enhanced microbial stress response, upregulated expression of virulence factors, and a high risk of resistance gene transfer, such as in effluent after disinfection. The corresponding water treatment processes are ultraviolet, chlorine, ozone, etc.; or high selective pressure, which easily leads to the coexistence of multidrug-resistant bacteria and virulence genes, such as in medical wastewater effluent. The corresponding water treatment processes are biochemical and disinfection, etc.

[0031] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology, R i =[PM i +α×(ARG i +VF i )]×F i .

[0032] In some embodiments, the water microbial safety risk assessment method based on metagenomics and virology is described. Where n represents the type of pathogenic microorganism.

[0033] Compared with the prior art, the beneficial effects of this invention are as follows: This invention not only detects pathogens and gene changes, but also dynamically monitors the dynamic response of the microbial community caused by disinfection and other treatment processes, using these dynamic indicators as a basis for assessing safety risks. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Example 1: A method for assessing the microbial safety risks of aquatic bodies based on metagenomics and virology includes: Metagenomic analysis of the relative abundance F of various pathogenic microorganisms in the water sample to be evaluated i (unit: %), where i represents the pathogenic microorganism label; For pathogenic microorganism i, determine its degree of harm PM i Antibiotic resistance gene risk value (ARG) i and virulence factor risk value (VF) i ; Determine the risk coefficient α of increased transmission due to water sample treatment methods for antibiotic resistance gene risk values ​​and virulence factor risk values; Regarding pathogenic microorganisms i, the overall hazard level PM i Antibiotic resistance gene risk value (ARG) i Virulence factor risk value (VF) i The risk coefficient α and relative abundance F of increased transmission due to water sample treatment methods i Determine the overall risk value R of pathogenic microorganism i i ; The comprehensive risk value of all pathogenic microorganisms detected by metagenomics is used to obtain the microbial safety risk assessment result R of the water sample to be evaluated.

[0036] The degree of harm is divided into four categories, among which: Pathogens that can cause serious disease in humans or animals and for which there are usually no effective prevention or treatment measures are identified as high-hazard pathogens, corresponding to PM2.5. i Assign 3 points; Microorganisms that can cause serious human or animal diseases and may be transmitted via aerosols, but for which there are usually preventative or therapeutic methods, are identified as having a high level of hazard, corresponding to PM2.5. i Assign 2 points; Microorganisms that can cause diseases in humans or animals but are not usually highly contagious, and for which effective prevention or treatment measures are available, are classified as low-hazard microorganisms, corresponding to PM2.5. i Assign 1 point; Microorganisms that do not cause disease in humans or animals are classified as low-hazard, corresponding to PM2.5. i Assign a score of 0.

[0037] For example, referring to the classification of the "List of Pathogenic Microorganisms Transmissible to Humans" formulated by the National Health Commission: Secondary pathogenic microorganisms (PM) such as Mycobacterium tuberculosis and Vibrio cholerae were found in water samples during metagenomic testing. i (Assigned 2 points) The degree of harm is relatively high. Other pathogens, such as *Escherichia coli* and *Pseudomonas aeruginosa*, are mostly classified as Class III pathogens with a lower degree of harm (PM). i (Assign 1 point).

[0038] Antibiotic resistance gene risk value (ARG) i Including transmission risk S and genetic risk G, expressed as: ARG i =S+G, where: Transmission risk S includes mobility and pathogenicity potential, expressed as S = mobility assignment + pathogenicity potential; the criteria for mobility assessment include whether it coexists with mobile genetic elements and whether it exists on mobile plasmids. If yes, mobility is assigned 1 point; if no, mobility is assigned 0 points. The criteria for pathogenicity assessment include whether it coexists with genes encoding virulence factors. If yes, pathogenicity potential is assigned 1 point; if no, pathogenicity potential is assigned 0 points. Genetic risk G is graded and assessed based on the antibiotic resistance mechanism mediated by antibiotic resistance genes and the clinical importance of the antibiotics corresponding to the antibiotic resistance genes.

[0039] Genetic risks are classified into three levels, including: Antibiotic resistance genes encoding sites related to resistance mechanisms of broad-spectrum antibiotics were assessed as high-risk, corresponding to a G score of 3. Antibiotic resistance genes encoding sites related to resistance mechanisms of commonly used antibiotics are assessed as medium risk, corresponding to a G score of 2. Encoding antibiotic resistance mechanisms that are less frequently used or that are inherent, low-level antibiotic resistance genes is assessed as low risk and assigned a G value of 1.

[0040] When pathogenic microorganism i lacks antibiotic resistance genes, the antibiotic resistance gene risk value ARG is calculated. i Assign a score of 0.

[0041] Virulence Factor Risk Value (VF) i This includes the pathogenicity C of the virulence factor gene and the environmental transmission risk E of the virulence factor, denoted as: VF i =C+E, where the pathogenicity C of the virulence factor gene is assessed according to the characteristics of the toxin and its clinical harm, and the environmental transmission risk E of the virulence factor is assessed based on gene mobility and environmental adaptability. The environmental transmission risk E of the virulence factor = gene mobility assignment + environmental adaptability assignment.

[0042] The pathogenicity of virulence factor genes is classified into three levels: low-risk toxins, intermediate-risk toxins, and high-risk toxins. Low-risk toxins are characterized by local effects or self-limiting diseases, with clinical hazards including mild diarrhea or skin infection, and are assigned a score of 1 for C. Medium-risk toxins are characterized by systemic effects or the need for medical intervention, and their clinical hazards include fever, severe diarrhea, or tissue necrosis. They are assigned a score of 2 for the C category. High-risk toxins are characterized by lethality or disability, and their clinical harms include organ failure, nervous system damage, or death. They are assigned a score of 3 for the C category.

[0043] The criteria for assessing gene mobility include whether the gene encoding a virulence factor is located on a known mobile genetic element. If so, gene mobility is assigned 1 point; otherwise, gene mobility is assigned 0 points.

[0044] The criteria for evaluating environmental adaptability include whether the virulence factor has been reported in the literature to enhance the survival of microorganisms in water or biofilms. If so, environmental adaptability is assigned 1 point; if not, it is assigned 0 points.

[0045] The risk coefficient α of increased transmission due to water sample treatment methods is graded and assessed based on the stress response characteristics of microorganisms caused by the water sample treatment process.

[0046] Microbial stress response characteristics include gene transfer potential and upregulation of virulence factor expression.

[0047] Microbial stress response characteristics include the following gradations: Level A: Microorganisms are in a natural state with a low risk of gene transfer, corresponding to an α score of 1 point; Level B: Some microorganisms are destroyed, releasing DNA and promoting horizontal gene transfer, or long-term low-dose disinfection leads to enhanced microbial adaptability and persistent risk of gene transfer, corresponding to an α score of 2. Level C indicates enhanced microbial stress response, upregulated expression of virulence factors, high risk of resistance gene transfer, or high selective pressure, and a tendency for multidrug-resistant bacteria to coexist with virulence genes, corresponding to an α score of 3. R i =[PM i +α×(ARG i +VF i )]×F i =[PM i +α×((G+S)+(C+E))]×F i .

[0048] Where n represents the type of pathogenic microorganism.

[0049] Application Example 1: Using the metagenomics and virology-based aquatic microbial safety risk assessment method described in Example 1, Escherichia coli (E. coli) was found in the rinsing water from sprout production after biochemical treatment and ultraviolet sterilization. Escherichia coli The relative abundance was 0.052% (F i =0.052), belonging to the Class III hazardous pathogenic microorganisms (PM i=1), the main virulence factor includes Shiga toxin, which can bind to receptors on the surface of host cells to enter the cell, inhibit protein synthesis, induce inflammatory responses and apoptosis; it can encode a series of adhesion and invasion-related proteins, causing host cell lesions, and has flagella and other structures to promote lesion metastasis and colonization; it secretes a variety of effector proteins to promote participation in toxin transport and regulate host immune responses, etc. Therefore, Shiga toxin is rated as a medium-risk toxin (pathogenicity C=2), and the toxin gene is located on a mobile genetic element (gene mobility assigned 1 point, environmental adaptability assigned 0 points, virulence factor environmental transmission risk E=1), hence the virulence factor risk value (VF) is... i The value is 3; no antibiotic resistance gene was found after comparison with the CARD database, therefore ARG... i The risk level is 0; combined with the gene transfer risk coefficient (α=3) brought about by the disinfection process, the comprehensive risk value (R) is calculated. i The value is 0.52.

[0050] R i =[PM i +α×(ARG i +VF i )]×F i =[1+3×(0+3)]×0.052=0.52.

[0051] Therefore, the biohazard risk of Escherichia coli in the water sample is 0.52.

[0052] Application Example 2: Using the metagenomics and viromics-based aquatic microbial safety risk assessment method of Example 1, Pseudomonas aeruginosa was found in the rinsing water from sprout production after biochemical treatment and ultraviolet sterilization (the same water sample as in Application Example 1). Pseudomonas aeruginosa The relative abundance was 1.225% (F i =1.225), the abundance increased by 3.45 times compared with before disinfection (untreated, α=1) (abundance before disinfection was 0.355%), belonging to the Class III hazardous pathogenic microorganism (PM). i =1), its elastase and other virulence factors are rated as medium risk (C=2) and are located on a mobile plasmid (gene mobility assigned 1 point, environmental adaptability assigned 0 points, virulence factor environmental transmission risk E=1), therefore VF i The risk score is 3; the blaVIM gene it carries mediates carbapenem resistance, which is high-risk (inherent gene risk G=3), and it is located on a mobile plasmid (mobility +1) and coexists with a gene encoding a virulence factor (pathogenic potential +1). Therefore, the additional risk score for transmission is 2, hence ARG. i The α value was 5. Metagenomic analysis showed upregulated expression of virulence genes and horizontal transfer of resistance genes in water samples after UV disinfection (α=3).

[0053] After disinfection: R i =[PM i +α×(ARG i +VF i )]×F i =[1+3×(5+3)]×1.225=30.625.

[0054] Before disinfection: Ri=[PM i +α×(ARG i +VF i )]×F i =[1+1×(5+3)]×0.355=3.195.

[0055] In the disinfected water sample, the composite risk value of Pseudomonas aeruginosa was 30.625, which was significantly higher than the risk value before disinfection (3.195), showing a significant increase, and was much higher than the risk value of Escherichia coli in the same sample (0.52).

[0056] The above results indicate that low-pressure ultraviolet disinfection failed to effectively control the risk of Pseudomonas aeruginosa. On the contrary, it may have enhanced its virulence and resistance through stress response and horizontal gene transfer. Furthermore, Pseudomonas aeruginosa exhibited stronger adaptability and public health risks in the post-disinfection environment and should be considered a key pathogen for monitoring.

[0057] As can be seen from the above cases, the traditional assessment system using Escherichia coli as an indicator bacterium may underestimate the actual microbial risk. It is necessary to adopt the method of this invention and combine metagenomics / viromics to conduct a comprehensive risk assessment at the multi-pathogen and multi-gene levels.

[0058] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology, characterized in that, include: Metagenomic analysis of the relative abundance F of various pathogenic microorganisms in the water sample to be evaluated i , where i represents the pathogenic microorganism label; For pathogenic microorganism i, determine its degree of harm PM i Antibiotic resistance gene risk value (ARG) i and virulence factor risk value (VF) i ; Determine the risk coefficient α of increased transmission due to water sample treatment methods for antibiotic resistance gene risk values ​​and virulence factor risk values; Regarding pathogenic microorganisms i, the overall hazard level PM i Antibiotic resistance gene risk value (ARG) i Virulence factor risk value (VF) i The risk coefficient α and relative abundance F of increased transmission due to water sample treatment methods i Determine the overall risk value R of pathogenic microorganism i i ; The comprehensive risk value of all pathogenic microorganisms detected by metagenomics is used to obtain the microbial safety risk assessment result R of the water sample to be evaluated.

2. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 1, characterized in that, The higher the level of harm, the more PM... i The larger the value; The degree of harm is divided into four categories, among which: Pathogens that can cause serious disease in humans or animals and for which there are usually no effective prevention and treatment measures are identified as high-risk. Microorganisms that can cause serious human or animal diseases and may be transmitted via aerosols, but which usually have preventive or therapeutic methods, are identified as high-risk. Microorganisms that can cause diseases in humans or animals but are not usually highly contagious and for which there are effective preventive or therapeutic measures are identified as low-risk. Microorganisms that do not cause disease in humans or animals are classified as low-hazard.

3. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 1, characterized in that, Antibiotic resistance gene risk value (ARG) i This includes transmission risk S and genetic risk G, where: Transmission risk S includes mobility and pathogenicity potential; mobility assessment criteria include whether it coexists with mobile genetic elements and whether it exists on mobile plasmids; pathogenicity potential assessment criteria include whether it coexists with genes encoding virulence factors; Genetic risk G is graded and assessed based on the antibiotic resistance mechanism mediated by antibiotic resistance genes and the clinical importance of the antibiotics corresponding to the antibiotic resistance genes.

4. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 3, characterized in that, Genetic risks are classified into three levels, including: Antibiotic resistance genes encoding sites related to resistance mechanisms to broad-spectrum antibiotics were assessed as high-risk. Antibiotic resistance genes encoding sites related to resistance mechanisms to commonly used antibiotics were assessed as medium risk. Encoding antibiotic resistance mechanisms that target less frequently used antibiotics or inherent, low-level antibiotic resistance genes is assessed as low risk. The higher the risk, the greater the genetic risk G value.

5. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 1, characterized in that, Virulence Factor Risk Value (VF) i This includes the pathogenicity C of virulence factors and the environmental transmission risk E of virulence factors. The pathogenicity C of virulence factors is graded and assessed according to the characteristics of the toxin and its clinical harm, while the environmental transmission risk E of virulence factors is judged based on gene mobility and environmental adaptability.

6. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 5, characterized in that, The pathogenicity of virulence factor genes is classified into three levels: low-risk toxins, intermediate-risk toxins, and high-risk toxins. Low-risk toxins are characterized by local effects or self-limiting diseases, with clinical hazards including mild diarrhea or skin infection. Medium-risk toxins are characterized by systemic effects or require medical intervention, and their clinical hazards include fever, severe diarrhea, or tissue necrosis. High-risk toxins are characterized by lethality or disability, and their clinical harms include organ failure, nervous system damage, or death. The higher the risk, the greater the pathogenicity C value of the virulence factor gene.

7. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 5, characterized in that, Criteria for assessing gene mobility include whether the gene encoding virulence factors is located on a known mobile genetic element; The criteria for assessing environmental adaptability include whether virulence factors have been reported in the literature to enhance the survival of microorganisms in water or biofilms.

8. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 1, characterized in that, The risk coefficient α of increased transmission due to water sample treatment methods is graded and assessed based on the stress response characteristics of microorganisms induced by the water sample treatment process; Microbial stress response characteristics include gene transfer potential and upregulation of virulence factor expression; The more numerous and stronger the stress response characteristics of microorganisms, the greater the risk coefficient α value of increased transmission due to water sample treatment methods.

9. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 8, characterized in that, Microbial stress response characteristics include the following gradations: Level A: Microorganisms are in a natural state with a low risk of gene transfer. Level B: Some microorganisms are destroyed, releasing DNA and promoting horizontal gene transfer, or long-term low-dose disinfection leads to enhanced microbial adaptability and a persistent risk of gene transfer. Level C: Enhanced microbial stress response, upregulated expression of virulence factors, high risk of resistance gene transfer, or high selective pressure, making it easy for multidrug-resistant bacteria to coexist with virulence genes.

10. The method for assessing the microbial safety risk of aquatic bodies based on metagenomics and virology according to claim 1, characterized in that, R i =[PM i +α×(ARG i +VF i )]×F i ; Where n represents the type of pathogenic microorganism.

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