Livestock and poultry drug resistance gene risk assessment method based on environmental persistence
By introducing the Environmental Persistence Index (EPI), the risk assessment of antibiotic resistance genes in livestock and poultry farming is dynamically modified, which solves the problem that existing technologies fail to consider the persistence differences of ARGs, and realizes accurate environmental risk assessment and targeted governance, meeting the regulatory requirements for precise pollution control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI JIAOTONG UNIV
- Filing Date
- 2026-03-05
- Publication Date
- 2026-06-05
AI Technical Summary
Existing methods for assessing the risk of antibiotic resistance genes (ARGs) in livestock and poultry farming fail to consider the persistence differences of ARGs after they are released from the anaerobic gut into the aerobic environment, leading to inaccurate risk assessments and potentially over-treatment.
By introducing the Environmental Persistence Index (EPI), the relative abundance ratio of ARGs in emission sources and receiving environments is calculated to dynamically correct their actual persistence capacity in the environment. A dynamic correction model is established to accurately assess the environmental migration and persistence characteristics of ARGs.
It can accurately eliminate "false high-risk" genes that rapidly disappear in the environment, avoid ineffective governance, achieve targeted governance, meet the regulatory needs under the precise pollution control policy, provide a full-phase assessment system, cover risk assessment of water, soil and sediment, and improve the scientificity and comparability of the assessment.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of antibiotic resistance gene risk assessment technology, and particularly relates to a method for assessing antibiotic resistance gene risk in livestock and poultry farming based on environmental persistence index. Background Technology
[0002] Antibiotic resistance (AMR) is a major threat to global public health. Livestock and poultry farm wastewater is a primary point source for the entry of resistance genes (ARGs) into the natural environment. Farm wastewater treated only by simple processes such as oxidation ponds carries large amounts of ARGs into receiving environments such as soil, water bodies, and sediments. These ARGs may spread between environmental bacteria and pathogens through horizontal gene transfer (HGT) mechanisms, using mobile genetic elements such as plasmids, ultimately threatening human health.
[0003] Currently, metagenomic-based risk assessment of ARGs has become mainstream. Existing quantitative assessment frameworks, such as Zhang et al. (Nature Communications, 2022, 13:1553), propose a global health risk assessment system. Its core assessment logic is to assign a comprehensive risk index to each ARG based on four dimensions: human accessibility, mobility, pathogenicity, and clinical availability. When assessing a specific sample, the relative abundance of the ARGs detected in the sample is multiplied by its corresponding risk index and summed to quantify the overall risk load of the sample. While this risk assessment method, based on "abundance and hazard attribute weighting," can clarify the theoretical risk level of ARGs in pollution sources, it fails to consider the migration and retention characteristics of ARGs after they enter the receiving environment from the emission source.
[0004] The gut microbiota of livestock and poultry is mostly an anaerobic environment. When the microorganisms they release enter aerobic soil or water, they often die rapidly due to their inability to adapt to the environment, resulting in a large number of "high-risk" ARGs failing to actually colonize the environment. Conversely, some ARGs may persist or even accumulate for a long time due to environmental adaptability. Therefore, introducing environmental persistence as a key evaluation dimension and establishing a field-based dynamic correction mechanism for traditional static classification is a crucial technical issue that urgently needs to be addressed to overcome the limitations of existing ARG risk assessments and achieve precise control of livestock pollution. Summary of the Invention
[0005] Given that existing risk assessment frameworks for livestock and poultry farming ARGs only perform static classification based on the inherent properties of ARGs, ignoring the differences in persistence of different ARGs after being emitted from the anaerobic gut to the aerobic environment, the purpose of this invention is to propose a dynamic correction model based on the "Environmental Persistence Index (EPI)," which for the first time incorporates the actual persistence of ARGs in the environment into the risk assessment system. By calculating the relative abundance ratio of different ARGs in the receiving environment and the emission source, the actual retention capacity of ARGs during environmental migration is quantified.
[0006] To achieve the above objectives, this invention provides a method for assessing the risk of antibiotic resistance genes in livestock and poultry farming based on an environmental persistence index, comprising the following steps: S1. Obtain livestock and poultry breeding wastewater samples as drug resistance gene emission source samples, and at least one environmental medium sample in the wastewater receiving environment as receiving samples. S2. Perform metagenomic sequencing and data analysis on all samples collected in step S1 to obtain the types and relative abundance of multiple antibiotic resistance genes in each sample. S3. Obtain the basic risk score for each antibiotic resistance gene, wherein the basic risk score is predetermined based on the inherent harmful properties of the resistance gene; S4. For each antibiotic resistance gene detected in both emission source samples and receiving samples, calculate its environmental persistence index in different environmental media. The environmental persistence index is the ratio of the average relative abundance of the resistance gene in the receiving samples to its relative abundance in the emission source samples. S5. Based on the environmental durability index calculated in step S4, the basic risk score of the corresponding drug resistance gene in step S3 is corrected to obtain the corrected risk value of the drug resistance gene in the receiving environment. S6. Based on the modified risk values of each drug resistance gene obtained in step S5, assess the risk of drug resistance gene pollution of the livestock and poultry breeding wastewater to the receiving environment.
[0007] In some embodiments, in step S1, the receiving environmental medium includes at least one of surface water, sediment, and soil. Specifically, the receiving environmental medium includes surface water and bottom sediment collected simultaneously 1 km upstream and downstream of the main river channel connected by the discharge outlet through ditches; for farmland soil irrigated by wastewater return, it is collected in two layers: 0-20 cm topsoil and 20-40 cm middle soil.
[0008] In some implementations, in step S3, the basic risk score This is a quantitative value obtained after a comprehensive assessment of four dimensions: human accessibility, mobility, human pathogenicity, and clinical availability of drug-resistant genes. Referring to the global health risk quantitative data of ARGs in the study by Zhang et al. (Nature Communications, 2022, 13:1553), a baseline risk score was obtained for each ARG after integrating the four dimensions of human accessibility, mobility, human pathogenicity, and clinical availability. ), will base risk score ( ) and the relative abundance of the ARGs ( Multiply by , and obtain the base risk index RI for each ARG.
[0009] In some implementations, in step S4, the Environmental Durability Index (EPI) i, Medium The calculation formula is:
[0010] Among them, RA i, Source RA represents the relative abundance of class i resistance genes in the emission source sample. i, Medium, Avg It represents the average relative abundance of class i drug resistance genes in all or part of the received samples in a specific environmental medium.
[0011] In some implementations, when the receiving environmental media includes multiple types, for each type of resistance gene, the maximum value of its environmental persistence index in different environmental media is taken as the final environmental persistence index (EPI) for that gene. i : , in, This represents the environmental persistence index of class i drug resistance genes in surface water. This represents the environmental persistence index of class i drug resistance genes in the precipitate; This represents the environmental persistence index of class i drug resistance genes in soil.
[0012] In some implementations, in step S5, the corrected risk value RI i, Source The baseline risk index (RI) for class i drug resistance genes in emission sources. i Its final Environmental Durability Index (EPI) i The product of, where the basic risk index RI i The relative abundance of class i drug resistance genes in emission sources Its basic risk score The product of, i.e. .
[0013] In some implementations, step S6 involves calculating a comprehensive environmental risk index. To assess the overall pollution risk, the The sum of the corrected risk values of all drug resistance genes detected in the emission source sample: .
[0014] In some implementations, the relative abundance is standardized in terms of "copies per cell".
[0015] On the other hand, the present invention also provides a system for implementing the above-described evaluation method, comprising: The data acquisition module is used to acquire metagenomic sequencing data from emission source samples and receiving samples; The data processing module is used to analyze sequencing data and obtain the types and relative abundance of antibiotic resistance genes in each sample. The basic risk database module is used to store the basic risk scores of each predefined antibiotic resistance gene; The persistence index calculation module is used to calculate the environmental persistence index of drug-resistant genes in different environmental media based on the relative abundance of drug-resistant genes in emission sources and receiving samples. The risk correction and assessment module is used to correct the basic risk using the environmental durability index, calculate the comprehensive environmental risk index, and output the risk assessment results.
[0016] In another aspect, the present invention also provides a computer-readable storage medium having a computer program stored thereon that, when executed by a processor, implements the above-described evaluation method.
[0017] Technical effect
[0018] To address the potential problem of "over-treatment" caused by traditional ARGs risk assessment in livestock and poultry farming, this invention introduces the Environmental Persistence Index (EPI) to accurately eliminate "pseudo-high-risk" drug-resistant genes that rapidly disappear in the natural environment, thus avoiding companies investing heavily in treating targets that pose no actual ecological threat. Based on the "targeted treatment" strategy guided by this method, only highly persistent genes need to be blocked.
[0019] This technical solution is entirely based on innovative data analysis logic, requiring no development of new detection instruments or changes to existing sampling procedures, and is perfectly compatible with the currently widespread metagenomic high-throughput sequencing (NGS) technology system. Its core algorithm... The logic of (the relative abundance ratio of ARGs in receiving environments and emission sources) is clear.
[0020] To address the shortcomings of existing ARGs environmental monitoring methods that often overlook "hidden pollution" such as sediments, this invention constructs a full-phase assessment system covering "water-soil-sediment" and a maximum risk locking mechanism, providing strong technical support for delineating key ARG risk control areas in aquaculture farms and meeting the rigid regulatory requirements under the precise pollution control policy. Detailed Implementation
[0021] The following describes embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.
[0022] Example 1
[0023] This embodiment selects typical large-scale pig farms and dairy farms in Chongming Island, Shanghai as research objects. Based on the measured data of two batches in spring and summer 2025, it elaborates in detail on the specific operation and application effect of the ARGs risk assessment method based on environmental durability index described in this invention.
[0024] During implementation, a rigorous source-sink sampling system was first established. Wastewater samples were collected from the effluent outlets of oxidation ponds in pig farms and dairy farms as the initial discharge sources for ARGs. Simultaneously, surface water and bottom sediment samples were collected 1 km upstream and downstream of the ditches directly receiving the discharge outlets and the main rivers connected to the ditches. For farmland soils irrigated with wastewater, samples were collected from the 0-20 cm topsoil and the 20-40 cm middle soil layers. All collected samples were transported to the laboratory at low temperatures, where total DNA was extracted and metagenomic high-throughput sequencing was performed using the Illumina PE150 platform. After quality control filtering, the raw sequencing data were used for qualitative annotation and quantitative analysis of drug resistance genes using the ARGs-OAP v3.2.4 workflow. The relative abundance of all ARGs was standardized to "copies / cell" to eliminate background differences in biomass between different environmental media.
[0025] Based on this, and referring to the global health risk quantitative data of ARGs in the study by Zhang et al (Nature Communications, 2022, 13:1553), a basic risk score for each ARG was obtained by integrating four dimensions: human accessibility, mobility, human pathogenicity, and clinical availability. ), will base risk score ( ) and the relative abundance of the ARGs ( Multiply by these to obtain the baseline risk index RI for each ARG. For example, the baseline risk index for class i resistance genes is... , (1) Where i represents different ARG types.
[0026] Subsequently, based on this, the Environmental Persistence Index (EPI) introduced in this embodiment is used for dynamic correction. Unlike traditional assessments that only focus on the basic ARGs risk index RI of the sample, this invention first calculates the "Environmental Persistence Index (EPI)" for each ARG. Specifically, ARGs detected in both wastewater and receiving environments are screened out, and their retention rate in a certain environmental medium migration process is calculated using formula (2). For example, for surface water, the relative abundance average of a certain ARG at 1 km upstream and downstream of the wastewater receiving ditch and the main river channel connected by the ditch is calculated, and then divided by the relative abundance in the wastewater to obtain the EPI. .
[0027]
[0028] For different types of environmental samples, following the "maximum risk lock-in principle", the maximum value among the calculation results of each point is taken as the durability index of the ARG in the receiving environment.
[0029] (3)
[0030] Among them, RA i, Source RA represents the relative abundance of class i resistance genes in the emission source sample. i, Medium, Avg It represents the average relative abundance of class i drug resistance genes in all or part of the received samples in a specific environmental medium.
[0031] Adjusted Risk Value (RI) i, Source The baseline risk index (RI) for class i drug resistance genes in emission sources. i Its final Environmental Durability Index (EPI) i The product of these factors. Finally, a linear weighted model is used to calculate the comprehensive environmental risk index (PARI) of the total ARGs in a given sample: (4) In the study of cattle farm wastewater, the analysis of high-risk ARGs revealed the importance of introducing the Environmental Persistence Index (EPI). The Environmental Persistence Index (EPI) of the top ten basic risk scores (ARGs) is shown in Table 1.
[0032] Table 1. Environmental Persistence Index (EPI) of the Top Ten Basic Risk Scores (ARGs) in Cattle Farm Wastewater
[0033] Table 1 shows that although the genes tet(W / N / W) and tet(B) have high abundance in wastewater and a baseline risk score exceeding 3 × 10⁻⁶, they are associated with certain diseases. 5 However, their corresponding environmental persistence index (EPI) is extremely low. For example, the EPI of tet(W / N / W) is 0 in soil and only around 0.01 in water and sediment. This indicates that such genes disappear rapidly after entering the environment, and relying solely on traditional RI models would significantly overestimate their environmental health risks. Conversely, the gene oqxB, which ranks 10th in baseline risk score, has an absolute abundance of only 9.6 × 10⁻⁶ in wastewater. -6 However, its EPI in soil is as high as 238 and in sediment as high as 104.8, indicating that this gene is extremely easy to colonize and accumulate in the environment. Traditional methods often ignore its risks due to its low initial abundance, while this method can accurately identify such potential risks. In addition, the data also found that the EPI of gene acrF in sediment (2.623) is significantly higher than that in water (0.644), revealing that it mainly follows the sedimentary pathway for environmental fate.
[0034] For pig farm wastewater samples, the distribution of ARGs exhibited distinctly different characteristics compared to cattle farms. This method effectively corrected for the "false positive" risk of high-abundance genes. The aminoglycoside resistance genes APH(6)-Id and APH(3'')-Ib were extremely abundant in pig farm wastewater (0.1583 and 0.1211 copies per cell, respectively), resulting in a traditional risk contribution (RI) as high as 4.19 × 10⁻⁶. 4 and 2.81×10 4 However, assessments showed that the EPIs of these two genes in all three environmental media were far less than 1 (between 0.0002 and 0.002), confirming that although these genes have high emissions, their environmental sustainability is weak, and the actual ecological risk is far lower than theoretical predictions. In contrast, although the abundance of genes mdtE and emrA is low, their EPI values are all greater than 1, with some even exceeding 10, indicating that these multidrug resistance efflux pump genes have extremely strong environmental adaptability and are key targets for subsequent environmental regulation.
[0035] Table 2. Environmental Persistence Index (EPI) of the Top Ten Risk Baseline Scores (ARGs) in Pig Farm Wastewater
[0036] Further based on the formula proposed in this invention The final risk of the four batches of samples was quantitatively calculated. The risk index RI of ARGs in the wastewater of pig farms and cattle farms in the two quarters and the corrected comprehensive environmental risk index PARI are shown in Table 3.
[0037] Table 3. Risk indices (RI) and modified comprehensive environmental risk index (PARI) of ARGs in wastewater from pig farms and cattle farms in two quarters.
[0038] Table 3 shows that the PARI values of pig farm wastewater (157.5 in spring and 71.5 in summer) were significantly higher than those of cattle farm wastewater (1.7 in spring and 6.4 in summer). Compared with the traditional total RI calculation method, the risk value of the pig farm sample obtained by the traditional method is as high as 1.10 × 10⁻⁶. 5 The boundary values were huge and lacked physical meaning, while the PARI value, after EPI correction, calibrated them to the order of 100-200. This result proves that the method proposed in this invention eliminates a large amount of noise interference from "high abundance, low persistence" genes, effectively distinguishes between transient risk and persistent risk from environmental colonization, and makes the risk levels between different farms and seasons more scientifically comparable, correcting the bias caused by neglecting the role of environmental selection in traditional metagenomic risk assessment.
[0039] Based on the aforementioned risk findings, a further analysis of the differences in the persistence coefficient (EPI) of all ARGs in the sample across different environmental media revealed that, within the receiving environment, aquatic environments exhibited the highest persistence coefficient for total ARGs, generally higher than sediments and soils. This phenomenon indicates that in the studied livestock and poultry farming areas, water flow is the primary carrier for the migration and persistence of drug-resistant genes, which may be related to the continuous discharge of wastewater and the high fluidity of the aquatic medium. While sediments and soils have a significant enrichment effect on individual specific genes (such as the aforementioned oqxB), their overall risk contribution is less significant than that of water bodies. This revelation of differences in environmental media is precisely the key to the accurate risk assessment achieved by this invention.
Claims
1. A method for assessing the risk of antibiotic resistance genes in livestock and poultry farming based on an environmental persistence index, characterized in that, Includes the following steps: S1. Obtain livestock and poultry breeding wastewater samples as drug resistance gene emission source samples, and at least one environmental medium sample in the wastewater receiving environment as receiving samples. S2. Perform metagenomic sequencing and data analysis on all samples collected in step S1 to obtain the types and relative abundance of multiple antibiotic resistance genes in each sample. S3. Obtain the basic risk score for each antibiotic resistance gene, wherein the basic risk score is predetermined based on the inherent harmful properties of the resistance gene; S4. For each antibiotic resistance gene detected in both emission source samples and receiving samples, calculate its environmental persistence index in different environmental media. The environmental persistence index is the ratio of the average relative abundance of the resistance gene in the receiving samples to its relative abundance in the emission source samples. S5. Based on the environmental durability index calculated in step S4, the basic risk score of the corresponding drug resistance gene in step S3 is corrected to obtain the corrected risk value of the drug resistance gene in the receiving environment. S6. Based on the modified risk values of each drug resistance gene obtained in step S5, assess the risk of drug resistance gene pollution of the livestock and poultry breeding wastewater to the receiving environment.
2. The evaluation method according to claim 1, characterized in that, In step S1, the receiving environmental medium includes at least one of surface water, sediment, and soil.
3. The evaluation method according to claim 1, characterized in that, In step S3, the basic risk score It is a quantitative value obtained after a comprehensive assessment of four dimensions: human accessibility, mobility, human pathogenicity, and clinical availability of drug-resistant genes.
4. The evaluation method according to claim 1, characterized in that, In step S4, the Environmental Durability Index (EPI) i, Medium The calculation formula is: Among them, RA i, Source RA represents the relative abundance of class i resistance genes in the emission source sample. i, Medium, Avg It represents the average relative abundance of class i drug resistance genes in all or part of the received samples in a specific environmental medium.
5. The evaluation method according to claim 4, characterized in that, When the receiving environmental media include multiple types, for each type of resistance gene, the maximum value of its environmental persistence index in different environmental media is taken as the final environmental persistence index (EPI) for that gene. i : ， in, This represents the environmental persistence index of class i drug resistance genes in surface water. This represents the environmental persistence index of class i drug resistance genes in the precipitate; This represents the environmental persistence index of class i drug resistance genes in soil.
6. The evaluation method according to claim 1, characterized in that, In step S5, the corrected risk value RI i, Source The baseline risk index (RI) for class i drug resistance genes in emission sources. i Its final Environmental Durability Index (EPI) i The product of, where the basic risk index RI i The relative abundance of class i drug resistance genes in emission sources Its basic risk score The product of, i.e. .
7. The evaluation method according to claim 6, characterized in that, In step S6, the comprehensive environmental risk index is calculated. To assess the overall pollution risk, the The sum of the corrected risk values of all drug resistance genes detected in the emission source sample: 。 8. The evaluation method according to any one of claims 1 to 7, characterized in that, The relative abundance is standardized in terms of "copies per cell".
9. A system for implementing the evaluation method according to any one of claims 1 to 8, characterized in that, include: The data acquisition module is used to acquire metagenomic sequencing data from emission source samples and receiving samples; The data processing module is used to analyze sequencing data and obtain the types and relative abundance of antibiotic resistance genes in each sample. The basic risk database module is used to store the basic risk scores of each predefined antibiotic resistance gene; The persistence index calculation module is used to calculate the environmental persistence index of drug-resistant genes in different environmental media based on the relative abundance of drug-resistant genes in emission sources and receiving samples. The risk correction and assessment module is used to correct the basic risk using the environmental durability index, calculate the comprehensive environmental risk index, and output the risk assessment results.
10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the program is executed by the processor, it implements the evaluation method as described in any one of claims 1 to 8.