Method for evaluating potential health risk of ARGs in air medium and application thereof

Abstract

The invention belongs to the technical field of air pollution evaluation, and particularly relates to a potential health risk assessment method for ARGs in an air medium and application of the potential health risk assessment method. The method comprises the following steps: firstly evaluating human accessibility, inhalability, mobility, host pathogenicity and clinical availability of each ARG in an air medium, then calculating a potential health risk value of each ARG by integrating the parameters, and then calculating a total health risk value of ARGs in the air medium of a target site. Therefore, the method can comprehensively and quantitatively evaluate the potential health risk when the exposed crowd in the target place is exposed to the ARGs in the air medium, and has more reference compared with a conventional toxicology method, a QMRA method and a risk grading evaluation method applied to ARGs risk evaluation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of air pollution evaluation, and particularly relates to a method for evaluating the potential health risk of ARGs in air medium and application thereof. BACKGROUND

[0002] Antibiotic Resistance Genes (ARGs) are gene fragments produced by gene mutation of bacteria under the selective pressure of antibiotics, which can confer drug resistance to bacteria. The overuse of antibiotics leads to the widespread distribution of ARGs in the environment and the induction of superbugs, which increases the difficulty of curing bacterial infectious diseases and causes serious drug resistance threats. Compared with relatively fixed media such as water and soil, ARGs in air medium can be directly inhaled by human body, and the potential health risks brought by inhalation include the following aspects: (1) ARGs are carried by biological aerosol particles, which can stimulate the respiratory mucosa after being inhaled into the human body, causing stress reactions such as cough, sore throat, shortness of breath, and some small particle size particles can penetrate into the alveolar region of the human body and diffuse to the deep lung tissue; (2) pathogenic host bacteria carrying ARGs will cause diseases such as allergy, respiratory tract, digestive tract and blood infection after entering the human body; (3) ARGs themselves have mobility, in addition to the generation-by-generation proliferation of ARGs caused by host microorganism reproduction, ARGs will also be carried by Mobile Genetic Elements (MGEs) to induce interspecies transmission of ARGs, and put the drug resistance in the human body.

[0003] Existing risk assessment methods for ARGs include toxicological methods primarily based on toxicity testing and model-based risk assessment methods, including Quantitative Microbial Risk Assessment (QMRA) and graded risk assessment. Toxicity testing involves exposing experimental animals to antibiotics via different routes and for different durations, and detecting various toxicity endpoints. While mouse toxicity testing can detect changes in ARG levels in mice after antibiotic exposure, quantifying ingested ARGs and conducting risk assessment remains a challenging problem. Furthermore, the significant size difference between mice and humans, and their different tolerance thresholds to antibiotics, make it difficult to characterize the degree of harm to humans from antibiotic or ARG intake using mice as test subjects. QMRA is a quantitative method for assessing the likelihood of adverse health risks in humans after exposure to pathogenic microorganisms. It can estimate the human health risk posed by a specific pathogen based on data on occurrence, exposure, and dose-response relationships. The QMRA risk assessment process includes four steps: hazard identification, exposure assessment, dose-response evaluation, and risk characterization. The main routes of pathogen exposure are oral ingestion, nasal inhalation, and skin contact. The final risk assessment is measured in annual Disability Adjusted Life Years (DALYs), which represent the total healthy life years lost from the onset of illness to death due to a toxic substance. In the QMRA method for ARGs, during hazard identification, antibiotic-resistant bacteria (ARBs) carrying ARGs or pathogenic ARGs are identified as risk targets, and the risk is then calculated using relevant exposure parameters. Using the host bacteria or pathogenic host bacteria of ARGs as risk assessment targets only indirectly calculates the potential risk of ARGs. Furthermore, due to the lack of exposure assessment and dose-response data, the accuracy of the QMRA method in assessing the potential health risks posed by antibiotic-resistant bacteria and ARGs needs significant improvement.

[0004] Risk grading methods use ARGs as direct risk assessment targets and simultaneously consider multiple risk factors, including ARG detection levels, host pathogenicity, and the ability to be carried by mobile genetic elements (MGEs) to spread among different microorganisms, especially pathogenic ones. The risk posed by ARGs is assessed through a tiered evaluation. ARGs with pathogenic host bacteria and the ability to be carried by MGEs to spread among different microorganisms, especially pathogenic ones, are assessed as high-risk ARGs. While grading and screening methods based on ARG abundance and characteristics can identify high-risk ARGs, the risk value of ARGs remains unquantified. Furthermore, current risk grading methods typically assess the risk of ARGs in water and soil and are not applicable to the risk assessment of ARGs in airborne media. Summary of the Invention

[0005] To address the above-mentioned technical problems, this invention provides a method for assessing the potential health risks of ARGs in air and its application. This assessment method comprehensively considers the human accessibility, inhalability, mobility, host pathogenicity, and clinical usability of ARGs in air. By screening and calculating parameters, it can achieve the assessment of the potential health risks of ARGs in air. This assessment method determines the levels of relevant parameters based on absolute quantification using metagenomic sequencing and quantitative real-time PCR, providing more accurate ARG risk values ​​and overcoming the limitations of existing ARG assessment methods.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a method for assessing the potential health risks of ARGs in air, specifically including the following steps: S1. Use metagenomic sequencing to obtain qualitative data on the types of ARGs in the air medium of the target location. Through ARG type analysis, screen out the types of ARGs that co-occur in the air medium of the target location and the human environment, i.e., human-accessible ARGs in the air medium of the target location. Calculate the possibility of each human-accessible ARG in the air medium of the target location being transferred to the human environment and causing harm to the human body according to formula (1), i.e., the human accessibility HA of the human-accessible ARG. HA = Average Abundance 人体环境 × Detection frequency 人体环境 ...(1); In the formula, the average abundance 人体环境 The mean abundance of this human-accessible ARG in the human environment is expressed in RPKM; detection frequency. 人体环境 The detection rate of this human-accessible ARG in the human environment is expressed in %; HA is dimensionless. S2. Use the real-time PCR method to obtain the absolute quantitative copy number of each human-accessible ARG in the air medium of the target location per unit volume of air, that is, the absolute abundance of the human-accessible ARG, and calculate the inhalability IH of the human-accessible ARG according to formula (2). IH = Absolute Abundance ARG × inhalable proportion × retention rate × respiratory rate of exposed population × exposure time of exposed population……(2); In the formula, absolute abundance ARG The absolute abundance of this human-accessible ARG is expressed in copies / m³. 3 空气 The inhalable proportion is the inhalable proportion of the human-accessible ARG, expressed as a percentage; the retention rate is the retention rate of the aerosol in the human environment after inhalation, taken as 0.5; the respiratory rate of the exposed population is the respiratory rate of the exposed population, expressed as m³ / s. 3 / d; Exposure time for the exposed population is the annual exposure time of the exposed population, in hours; IH is dimensionless; S3, using quantity MGE The MB represents the mobility of ARGs in the air of the target location to carry horizontal gene transfer between different host microorganisms via MGEs, i.e., MB = quantity. MGE The quantity MGE The number of MGEs in the air medium of the target location that are significantly correlated with each human-accessible ARG (p<0.05, r>0.7); MB is dimensionless; S4. Screen all host bacteria of each human-accessible ARG through the correlation network between ARGs and bacteria in the air medium of the target location, screen pathogenic hosts, calculate the proportion of pathogenic hosts in all host bacteria, and calculate the host pathogenicity HP of the human-accessible ARG according to formula (3). ... (3); In the formula, The proportion of pathogenic host bacteria of this human-accessible ARG among all host bacteria; The pathogenicity of this pathogenic host bacterium; HP is dimensionless; S5. The clinical usage of antibiotics resistant to each human-accessible ARG in the air medium of the target location is statistically analyzed, and the clinical availability CA of the human-accessible ARG is calculated according to formula (4). ... (4); In the formula, the amount of antibiotics used is... i The total global medical consumption of all antibiotic types resistant to the human-accessible ARG; CA is dimensionless; S6. Taking into account human accessibility, inhalability, mobility, host pathogenicity and clinical availability, calculate the potential health risk value (HR) of each human-accessible ARG in the air medium of the target location according to formula (5); ... (5); S7. Calculate the total health risk value (HR) of ARGs in the air medium of the target location according to formula (6). S ; ... (6); where HR S The total health risk of ARGs in the air, HR i The potential health risk value for each human-accessible ARG in the air.

[0007] The method's S1 analyzes the types of ARGs in the air medium of the target location to assess the human accessibility of ARGs in the air medium of the target location, that is, to assess whether ARGs can be transferred from the environment to the human body through host bacteria and subsequently have a negative impact on human health. For ARGs in the air, inhalability is an important quantitative indicator. S2 analyzes the concentration and inhalability of ARGs in the air of the target location. Through multi-parameter coupling, it assesses the possibility of inhaling ARGs in the air, the retention after inhalation, and the exposure dose after inhalation. S3 analyzes the correlation between ARGs and MGEs in the air medium of the target site, screens out MGEs that may be involved in the horizontal gene transfer of human accessible ARGs, and assesses the mobility of ARGs through horizontal gene transfer between different host microorganisms by the number of MGEs, that is, whether ARGs can be transferred from non-pathogens to pathogens by MGEs. Different pathogens have varying degrees of pathogenicity. S4 analyzes the correlation between ARGs in the air medium of the target location and all airborne bacteria and pathogenic airborne bacteria to assess the pathogenicity of ARGs after they are transferred from non-pathogenic hosts to pathogenic hosts, i.e. whether the host bacteria of ARGs are human pathogens. S5 statistically analyzes the clinical usage of antibiotics resistant to ARGs in the air of the target location and assesses whether ARGs in the air of the target location are resistant to commonly used antibiotics.

[0008] S6 can filter out ARGs that simultaneously meet the risk conditions by multiplying the various values. This allows HR to classify the potential health risks of a certain ARG in the air medium. For example, the first 25% is classified as high risk, the first 50% to 25% as relatively high risk, the first 75% to 50% as medium risk, the last 25% as low risk, and those with a risk value of 0 are considered no risk.

[0009] S7 calculates the total health risk value of ARGs in the air medium of the target location based on the potential health risk value of each ARG. This allows for the identification of high-risk points and times of exposure to ARGs in the air medium of the target location, enabling the development of effective preventive measures and response strategies to improve public health and safety.

[0010] Preferably, in S1, ARGs in the air medium of the target location are collected by a sampler for analysis and detection of their species and abundance.

[0011] For example, the sampler may be a medium-sized particulate sampler. After sampling, the actual sampling volume of the sampler is recorded for quantitative calculation of ARGs in the air medium.

[0012] More preferably, the sampling height of the sampler is the average breathing height of the exposed population in the air medium.

[0013] More preferably, the sampling time of the sampler is the exposure time of the exposed population in the air medium.

[0014] Preferably, after preprocessing the collected samples in S1, the types of ARGs co-occurring in the air medium and human environment of the target location are screened by comparing them with the data on the types and abundance of ARGs in the human environment recorded in the literature (Zhang Z, Zhang Q, Wang T, et al., 2022. Assessment of global health risk of antibiotic resistance genes[J]. Nature Communications, 13(1), 1553.). The human environment includes the digestive tract and skin. The average abundance and detection frequency of each ARG in the human environment are obtained by querying the data on the types and abundance of ARGs in the human environment compiled by Zhang et al. (2022). The average abundance of each ARG in the air medium of different locations is... 人体环境 × Detection frequency 人体环境 The ARGs are the same in all locations, but the types of ARGs in the air medium are different in different locations. Therefore, the HA of ARGs in the air medium in different locations consists of different values.

[0015] Preferably, in S2, the parameters used to calculate the IH value are all assumed, fitted and tested using Monte Carlo simulation. The Monte Carlo simulation of the data is completed using Oracle Crystal Ball risk management software. All input variables are randomly selected from their probability distributions, and the output variables are iterated 10,000 times to make the parameter distribution reach a stable state.

[0016] Preferably, the inhalable ratio in formula (2) of S2 is the ratio of the concentration of inhalable bacterial aerosol (<3.3 μm) to the concentration of total bacterial aerosol.

[0017] More preferably, the method for obtaining the concentration of inhalable bacterial aerosol and the total bacterial aerosol is as follows: bacterial aerosols are collected at each sampling point using an impact sampler, wherein the cutting diameters corresponding to the impact sampler levels I to VI are >7, 4.7~7.0, 3.3~4.7, 2.1~3.3, 1.1~2.1 and 0.65~1.1 μm, respectively; bacteria on each level of the sampler are isolated and cultured, and the colony count results on each level of the culture dish of each sampler are revised using the orifice correction method, and the bacterial aerosol concentration at each sampling point is calculated using formula (7); ... (7); In the formula, C represents the bacterial aerosol concentration, with units of CFU / m³. 3 空气 ,Pr i The effective colony count on each sampler is expressed in CFU, Q is the sampling gas flow rate in L / min, and T is the sampling time in min. When calculating the concentration of the inhalable bacterial aerosol, Pr i The effective colony count on level IV to VI samplers; when calculating the total bacterial aerosol concentration, Pr i The effective colony count on samplers of levels I to VI.

[0018] Alternatively, a correction formula can be used. The formula is revised; where Pr is the effective colony count, i.e., the corrected colony count (CFU), r is the actual colony count (CFU), and N is the number of sampling wells at each level of the sampler. Preferably, the respiratory rate of the exposed population in S2 is calculated separately based on the respiratory volume and respiratory rate of different exposed populations.

[0019] Preferably, in S3, the cor() function in the igraph package of R language is used to calculate the Spearman correlation between ARGs and MGEs, and to screen the types of MGEs that are significantly associated with each human-accessible ARG, with the screening criteria being p<0.05 and r>0.7.

[0020] Preferably, the types of MGEs included in the correlation calculation include inserted sequences and common regions of inserted sequences, integrases, transposons, transposases, and plasmids.

[0021] Preferably, Monte Carlo simulation is used in S3 to simulate the quantity under different spatiotemporal distributions. MGE Make assumptions, fit and test the data.

[0022] Preferably, the method for screening pathogenic hosts in S4 is as follows: The Spearman correlation between each human-accessible ARG and bacteria is calculated using the `cor()` function in the `igraph` package of the R language. Bacterial species significantly associated with the human-accessible ARG are screened as potential host bacteria for that human-accessible ARG, with screening conditions of p < 0.05 and r > 0.7. The obtained potential host bacteria are compared with the Global Catalogue of Microorganisms (gc Pathogen) database to screen for pathogenic hosts. The proportion of pathogenic host bacteria of the human-accessible ARG among all host bacteria is the pathogenic host bacteria of that human-accessible ARG. .

[0023] Preferably, This refers to the total number of VFs that this pathogenic host bacterium may carry. VF The quantity VF The method for obtaining the data is as follows: Diamond compares the amino acid sequences of the non-redundant gene set obtained from metagenomics with the Virulence Factors of Pathogenic Bacteria Database (VFDB), and obtains pathogenic VFs in the air medium of the target location by screening annotation information. The comparison parameter is set with an expected value e-value of 1e-5. The pathogenic host bacteria and pathogenic VFs of the human accessible ARG are screened through the correlation network of pathogenic host bacteria and pathogenic VFs. The correlation coefficient is calculated using the igraph package of R language. The Spearman correlation between pathogenic host bacteria and VFs is calculated using the cor() function. After calculation, VFs that are significantly related to the pathogenic host bacteria are screened as pathogenic VFs that may be carried by the pathogenic host bacteria and counted. The screening conditions are p<0.05 and r>0.7.

[0024] Preferably, Monte Carlo simulation is used for... and Make assumptions, fit and test the data.

[0025] Preferably, the data on global medical consumption mentioned in S5 is sourced from the ResistanceMap database (https: / / resistancemap.onehealthtrust.org / AntibioticUse.php).

[0026] Preferably, Monte Carlo simulations are used to measure antibiotic usage. i Make assumptions, fit and test the data.

[0027] A second aspect of the present invention provides the application of the above-described method for assessing the potential health risks of ARGs in airborne media in assessing the potential health risks of ARGs in airborne media in areas where wastewater is located.

[0028] Preferably, the area where the wastewater is located is a wastewater treatment plant.

[0029] The beneficial effects of this invention are as follows: 1. The assessment method provided by this invention comprehensively considers the various characteristics of ARGs in the air and can comprehensively assess the potential health risks of people exposed to ARGs in the air in a target location.

[0030] 2. This invention determines the levels of relevant parameters based on absolute quantification using metagenomic sequencing and quantitative real-time PCR, which can provide more accurate ARGs risk values.

[0031] 3. Compared with conventional toxicological methods, QMRA methods, and risk grading assessment methods used for ARGs risk assessment, the assessment method provided by this invention can quantitatively assess the potential health risks of ARGs in air, and the assessment results are more reliable. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0033] ARGs in the air can be directly inhaled by humans. After inhalation, they will be transferred and amplified within the body, increasing drug resistance. Their potential harm cannot be ignored. Currently, there is a lack of assessment methods and systems for the potential risks of ARGs in the air, making it impossible to quantify the potential health risks posed by ARGs in the air.

[0034] This invention provides a method for assessing the potential health risks of ARGs in air, specifically including the following steps: S1. Use metagenomic sequencing to obtain qualitative data on the types of ARGs in the air medium of the target location. Through ARG type analysis, screen out the types of ARGs that co-occur in the air medium of the target location and the human environment, i.e., human-accessible ARGs in the air medium of the target location. Calculate the possibility of each human-accessible ARG in the air medium of the target location being transferred to the human environment and causing harm to the human body according to formula (1), i.e., the human accessibility HA of the human-accessible ARG. HA = Average Abundance 人体环境 × Detection frequency 人体环境 ...(1); In the formula, the average abundance 人体环境 The mean abundance of this human-accessible ARG in the human environment is expressed in RPKM; detection frequency. 人体环境 The detection rate of this human-accessible ARG in the human environment is expressed in %; HA is dimensionless. S2. Use the real-time PCR method to obtain the absolute quantitative copy number of each human-accessible ARG in the air medium of the target location per unit volume of air, that is, the absolute abundance of the human-accessible ARG, and calculate the inhalability IH of the human-accessible ARG according to formula (2). IH = Absolute Abundance ARG × inhalable proportion × retention rate × respiratory rate of exposed population × exposure time of exposed population……(2); In the formula, absolute abundance ARG The absolute abundance of this human-accessible ARG is expressed in copies / m³. 3 空气 The inhalable proportion is the inhalable proportion of the human-accessible ARG, expressed as a percentage; the retention rate is the retention rate of the aerosol in the human environment after inhalation, taken as 0.5; the respiratory rate of the exposed population is the respiratory rate of the exposed population, expressed as m³ / s. 3 / d; Exposure time for the exposed population is the annual exposure time of the exposed population, in hours; IH is dimensionless; S3, using quantity MGE The MB represents the mobility of ARGs in the air of the target location to carry horizontal gene transfer between different host microorganisms via MGEs, i.e., MB = quantity. MGE The quantity MGE The number of MGEs in the air medium of the target location that are significantly correlated with each human-accessible ARG (p<0.05, r>0.7); MB is dimensionless; S4. Screen all host bacteria of each human-accessible ARG through the correlation network between ARGs and bacteria in the air medium of the target location, screen pathogenic hosts, calculate the proportion of pathogenic hosts in all host bacteria, and calculate the host pathogenicity HP of the human-accessible ARG according to formula (3). ... (3); In the formula, The proportion of pathogenic host bacteria of this human-accessible ARG among all host bacteria; The pathogenicity of this pathogenic host bacterium; HP is dimensionless; S5. The clinical usage of antibiotics resistant to each human-accessible ARG in the air medium of the target location is statistically analyzed, and the clinical availability CA of the human-accessible ARG is calculated according to formula (4). ... (4); In the formula, the amount of antibiotics used is... i The total global medical consumption of all antibiotic types resistant to the human-accessible ARG; CA is dimensionless; S6. Taking into account human accessibility, inhalability, mobility, host pathogenicity and clinical availability, calculate the potential health risk value (HR) of each human-accessible ARG in the air medium of the target location according to formula (5); ... (5); S7. Calculate the total health risk value (HR) of ARGs in the air medium of the target location according to formula (6). S ; ... (6); where HR S The total health risk of ARGs in the air, HR i The potential health risk value for each human-accessible ARG in the air.

[0035] This method comprehensively considers the human accessibility, inhalability, mobility, host pathogenicity, and clinical usability of ARGs in the air. By screening and calculating parameters, it can achieve the assessment of the potential health risks of ARGs in the air.

[0036] This invention also provides the application of the above-described method for assessing the potential health risks of ARGs in airborne media in assessing the potential health risks of ARGs in airborne media in areas where wastewater is located.

[0037] The following detailed description, using specific embodiments, will further illustrate this point.

[0038] Unless otherwise specified in the following embodiments, the experimental steps or conditions can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials and instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0039] Example 1 Wastewater treatment plants are important reservoirs of aerosols (ARGs). During wastewater and sludge treatment, ARGs are released into the air as bioaerosols, polluting the environment and posing potential health threats to wastewater treatment plant workers and nearby residents. This embodiment uses an application A as an example. 2 Taking the air medium of a typical wastewater treatment plant using the / O process as an example, this paper provides a method for assessing the potential health risks of ARGs in the air medium. The specific steps of this method are as follows: S1: Human accessibility (HA) analyzes the types and abundance of ARGs in the air of wastewater treatment plants to assess the possibility of ARGs transferring from the gas phase to the human environment. The specific methods are as follows: Sample collection followed these steps: ARGs in the air from the wastewater treatment plant were enriched onto the surface of a sterilized quartz fiber filter membrane (90 mm, Whatman™, UK) using a medium-sized particulate matter sampler (TH-150, Tianhong, Wuhan, China). The sampler flow rate was 100 L / min (the actual sampling flow rate fluctuated within the range of 100 ± 0.3 L / min due to changes in environmental conditions during sampling). After sampling, the actual sampling volume was recorded for quantitative calculation of ARGs in the air. Sampling points were set up in the main wastewater and sludge treatment units, including the screen room, aeration tank, and sludge dewatering room. The sampling duration was 8 hours, conducted during the working hours of wastewater treatment plant workers (9:00-17:00). The sampling height was set at the average breathing height of wastewater treatment plant workers, i.e., 1.5 m.

[0040] After sampling, the collected samples were pretreated: Under aseptic conditions, the filter membrane was cut and placed into a 50 mL centrifuge tube. Sterile water was added to the centrifuge tube to submerge the filter membrane, the cap was tightened, and the tube was shaken for 30 min. Then, it was centrifuged at 4 ℃ and 200 g for 3 h. After centrifugation, the suspension in the tube was filtered and enriched onto a 50 mm diameter, 0.22 μm pore size polyethersulfone (PES) membrane for the analysis and detection of ARGs.

[0041] Qualitative data on the species of ARGs in the air were obtained using metagenomic sequencing. The sequencing results were compared with the SARG database (version 2.0) (BLASTP alignment parameter was set with an expected e-value of 1e-5) to obtain antibiotic resistance functional annotation information for the corresponding genes.

[0042] By comparing the data on the types and abundance of ARGs in the human environment (including the digestive tract and skin) as recorded in the literature (Zhang Z, Zhang Q, Wang T, et al., 2022. Assessment of global health risk of antibiotic resistance genes[J]. Nature Communications,13(1), 1553.), the types of ARGs co-occurring in the air medium of the wastewater treatment plant and the human environment were screened out, that is, the human accessible ARGs in the air medium of the wastewater treatment plant. By using the detection abundance and detection frequency of each human accessible ARG in the human environment, the possibility of each human accessible ARG in the air medium of the wastewater treatment plant transferring to the human environment and causing harm to the human body was calculated by formula (1), that is, the human accessibility HA of each human accessible ARG in the air medium of the wastewater treatment plant.

[0043] HA = Average Abundance 人体环境 × Detection frequency 人体环境 ...(1); In the formula, the average abundance 人体环境 The average abundance of this human-accessible ARG in the air medium of the wastewater treatment plant in the human environment is expressed in RPKM, and the detection frequency is [missing information]. 人体环境 The detection rate of the human-accessible ARG in the air medium of the wastewater treatment plant in the human environment is expressed as %; HA is dimensionless. The average abundance and detection frequency of each human-accessible ARG in the human environment were obtained by querying the data on the types and abundance of ARGs in the human environment as recorded in the literature (Zhang Z, Zhang Q, Wang T, et al., 2022. Assessment of global health risk of antibiotic resistance genes[J]. Nature Communications, 13(1), 1553.).

[0044] A total of 331 human-accessible ARGs were detected in the air at the wastewater treatment plant, accounting for 65.8% of the total number of ARGs detected in the air. The abundance of these human-accessible ARGs in the human environment ranged from 0.035 to 205.010 RPKM, and the detection frequency ranged from (9.8E-02)% to 100%. The final calculated human accessibility range of the human-accessible ARGs was 3.46E-05 to 183.30.

[0045] S2: Inhalability (IH) – This analyzes the concentration and inhalability of ARGs in the air of wastewater treatment plants to assess the likelihood of inhaling ARGs from the air, their retention after inhalation, and the exposure dose after inhalation. The specific methods are as follows: The absolute quantitative copy number of each human-accessible ARG in each unit volume of air was obtained using real-time PCR, i.e., the absolute abundance of that human-accessible ARG, expressed as copies / m³. 3 空气 count.

[0046] The ratio of inhalable bacterial aerosol (<3.3 μm) concentration to total bacterial aerosol concentration was used instead of the ratio used to calculate the inhalable proportion (%) of ARGs in the air medium. The inhalable bacterial aerosol concentration and the total bacterial aerosol concentration were obtained by the following steps: (1) A six-stage impactor sampler was used to collect bacterial aerosols at each sampling point 1.5 m from the ground. The cutting diameters corresponding to sampler stages I to VI were >7, 4.7~7.0, 3.3~4.7, 2.1~3.3, 1.1~2.1, and 0.65~1.1 μm, respectively. The sampler flow rate was set to 28.3 L / min, the sampling time was 3 min, and three parallel samples were collected from each sampling point. Bacteria on each stage of the sampler were isolated and cultured using nutrient agar medium (Haibo Biotechnology, Qingdao, China). After sampling, the culture dishes were placed in an incubator at 37 ℃ for 24 h and then counted. (2) The colony count results on each stage of the sampler's culture dish were revised using the positive well correction method. The correction formula is as follows: In the formula, Pr is the effective colony count, i.e. the corrected colony count (CFU), r is the actual colony count (CFU), and N is the number of sampling wells at each level of the sampler. The formula for calculating the bacterial aerosol concentration at the sampling point is: In the formula, C is the bacterial aerosol concentration (CFU / m³). 3 空气 ), Pr i Q represents the effective colony count (CFU) on each sampler, Q represents the sampling gas flow rate (L / min), and T represents the sampling time (min).

[0047] When calculating the concentration of inhalable bacterial aerosols, Pr i The effective colony count on level IV to VI samplers; when calculating the total bacterial aerosol concentration, Pr i This refers to the effective colony count on samplers of levels I to VI. In other cases, if the inhalable ARGs portion of the airborne medium is directly collected by controlling the sampler orifice size, it is not necessary to calculate the inhalable proportion; IH = absolute abundance of inhalable ARGs. ARG × Retention rate × Breathing rate of exposed population × Exposure time of exposed population; The retention rate of aerosols in the human environment after inhalation is calculated as 0.5, based on the reference (Ma JX, Wang X, Pan YR, et al., 2024. Data-driven systematic analysis of waterborne viruses and health risks during the wastewater reclamation process[J]. Environmental Science and Ecotechnology, 19, 100328.).

[0048] The respiratory rates of the exposed population were obtained from the "Handbook of Exposure Parameters for Chinese Populations (Adult Volume)" published by the Ministry of Environmental Protection of China in 2013, and were calculated separately for adult male and adult female workers according to their different respiratory volumes and respiratory rates.

[0049] The exposure time of the exposed population is calculated based on the actual daily working hours of the wastewater treatment plant workers. The exposure time of the exposed population = annual exposure frequency (days) × average daily exposure duration (hours / day). The annual exposure frequency is calculated based on the actual number of working days of the wastewater treatment plant workers, and is taken as 250 days.

[0050] The formula for calculating the inhalability of each human-accessible ARG in the air medium of a wastewater treatment plant is: IH = Absolute Abundance ARG × Inhalable abundance × Retention rate × Respiratory rate of exposed population × Exposure time of exposed population; Where, absolute abundance ARG The absolute abundance of this human-accessible ARG (copies / m²) 3 空气 The inhalable proportion is the inhalable proportion (%) of the human-accessible ARG; the retention rate is the retention rate of the aerosol in the human environment after inhalation, taken as 0.5; and the respiratory rate of the exposed population is the respiratory rate (m³) of the exposed population. 3 / d), the exposure time for the exposed population is the annual exposure time (h) for the exposed population; IH is dimensionless. The parameters used to calculate the inhalability of ARGs in the air were hypothesized, fitted, and tested using Monte Carlo simulation. The Monte Carlo simulation was performed using Oracle Crystal Ball risk management software. All input variables were randomly selected from their probability distributions, and the output variables underwent 10,000 iterations to achieve a stable parameter distribution. In this wastewater treatment plant, the mean inhalability index of different human-accessible ARGs in the air ranged from 1.13E+06 to 4.90E+08.

[0051] S3: Mobility (MB): The correlation between ARGs and horizontal gene transfer elements (MGEs) in the air media of wastewater treatment plants was analyzed to assess the transferability of ARGs via MGEs for horizontal gene transfer between different host microorganisms. The specific methods are as follows: The MGE information corresponding to genes in the air medium of the wastewater treatment plant was obtained by comparing the amino acid sequences of the non-redundant gene set obtained from metagenomic sequencing in S1 with the Mobile Genetic Elements database using Diamond (http: / / www.diamondsearch.org / index.php, version 0.8.35). The comparison parameter was set with an expected e-value of 1e-3.

[0052] The correlation coefficient between ARGs and MGEs was calculated using the igraph package in R. The cor() function was used to calculate the Spearman correlation between ARGs and MGEs. After calculation, the types of MGEs that were significantly correlated with ARGs were screened and counted. The screening criteria were p<0.05 and r>0.7.

[0053] The types of airborne MGEs included in risk management, i.e., those included in correlation calculations, include inserted sequences and common regions of inserted sequences, integrases, transposons, transposases, and plasmids.

[0054] The number of mobile MBs used for each human-accessible ARG in the air medium of a wastewater treatment plant. MGE This means: MB = quantity MGE In the formula, the quantity MGE The number of MGE species that were significantly correlated with each human-accessible ARG in this air medium (p<0.05, r>0.7) was determined. Monte Carlo simulations were used to analyze the number of species under different spatiotemporal distributions. MGE Make assumptions, fit and test the data.

[0055] A total of 314 mobile ARGs were screened in the air medium of the wastewater treatment plant, accounting for 94.9% of the total number of ARGs accessible to humans.

[0056] S4: Host pathogenicity (HP): The correlation between ARGs in the air of wastewater treatment plants and all airborne bacteria and pathogenic airborne bacteria was analyzed to assess the pathogenicity of ARGs after transfer from non-pathogenic hosts to pathogenic hosts. The specific methods are as follows: The population structure of microorganisms in the air was obtained using metagenomic sequencing. Using Diamond (http: / / www.diamondsearch.org / index.php, version 0.8.35), the amino acid sequences of the non-redundant gene set obtained from metagenomic sequencing in S1 were aligned with a non-redundant database (NR) (BLASTP alignment parameter set with an expected e-value of 1e-5). Species annotations were obtained from the taxonomic information database corresponding to the NR database, and the abundance of each species was calculated using the sum of gene abundances corresponding to that species.

[0057] All host bacteria for each human-accessible ARG were screened using a correlation network between ARGs and bacteria. The correlation coefficients were calculated using the igraph package in R. The Spearman correlation between each human-accessible ARG and bacteria was calculated using the cor() function. After calculation, bacterial species that were significantly correlated with each human-accessible ARG were selected as potential host bacteria for ARGs. The selection criteria were p<0.05 and r>0.7.

[0058] The obtained potential host bacteria were compared with the Global Catalogue of Microorganisms (gc Pathogen) database to screen for pathogenic hosts. Finally, the proportion of pathogenic hosts among all hosts was calculated. .

[0059] The pathogenicity of the host itself is quantified by the number of VFs it may carry.

[0060] The VFs corresponding to genes in the air medium of wastewater treatment plants were obtained by Diamond by comparing the amino acid sequences of the non-redundant gene set obtained from metagenomics with the Virulence Factors of Pathogenic Bacteria Database (VFDB). The comparison parameter was set with an expected value of 1e-5. Pathogenic VFs with bacterial virulence were obtained by screening through annotation information.

[0061] The correlation network between pathogenic host bacteria and pathogenic virus fairings (VFs) in ARGs was used to screen for pathogenic VFs that may be carried by pathogenic host bacteria. The correlation coefficient was calculated using the igraph package in R language. The Spearman correlation between pathogenic host bacteria and VFs was calculated using the cor() function. After calculation, VFs that were significantly correlated with pathogenic host bacteria were selected as potential pathogenic VFs carried by pathogenic host bacteria and counted. The screening criteria were p < 0.05 and r > 0.7.

[0062] The formula for calculating the pathogenicity of a pathogenic host is: In the formula, The pathogenicity of this pathogenic host bacterium is determined by the total number of VFs it carries. VFs Obtained through calculation.

[0063] Using Monte Carlo simulation and Make assumptions, fit and test the data.

[0064] The formula for calculating the host pathogenicity of each human-accessible ARG in the air medium of a wastewater treatment plant is as follows: .

[0065] In the air medium of this wastewater treatment plant, a total of 329 human-accessible ARGs with pathogenic hosts were screened out, and their host pathogenicity ranged from 0.03 to 436.

[0066] S5: Clinical Availability (CA): This involves statistically analyzing the clinical usage of antibiotics resistant to ARGs in the air of the wastewater treatment plant to assess whether ARGs in the air are resistant to commonly used antibiotics. The specific methods are as follows: This section calculates the total global medical consumption of all antibiotic types resistant to each human-accessible ARG. Global antibiotic consumption data is sourced from the ResistanceMap database (https: / / resistancemap.onehealthtrust.org / AntibioticUse.php).

[0067] The formula for calculating the clinical availability of each human-accessible ARG in the air medium of a wastewater treatment plant is as follows: In the formula, the amount of antibiotics used is... i This represents the total global medical consumption of all antibiotic types resistant to the human-accessible ARG. The clinical availability of each human-accessible ARG is calculated separately, with the clinical availability of all ARGs being the sum of the clinical availability of each individual ARG. Antibiotic usage is simulated using Monte Carlo simulations. i Make assumptions, fit and test the data.

[0068] In the air medium of this wastewater treatment plant, the clinical availability of all human-accessible ARGs ranges from 0.03 to 1.48E+06.

[0069] S6: Calculate the potential health risk (HR) value for each human-accessible ARG in the air of this wastewater treatment plant, taking into account human accessibility, inhalability, mobility, host pathogenicity, and clinical availability. The specific method is as follows: In the formula, HA represents the human accessibility of the human-accessible ARG calculated by S1, IH represents the inhalability of the human-accessible ARG calculated by S2, MB represents the mobility of the human-accessible ARG calculated by S3, HP represents the host pathogenicity of the human-accessible ARG calculated by S4, and CA represents the clinical availability of the human-accessible ARG calculated by S5. All five parameters are dimensionless.

[0070] The potential health risks of ARGs in the air of the wastewater treatment plant were classified into five levels by calculating risk values: R4 (high risk; top 25%), R3 (relatively high risk; top 50% to 25%), R2 (medium risk; top 75% to 50%), R1 (low risk; bottom 25%), and R0 (no risk; risk value is 0).

[0071] In the air medium of this wastewater treatment plant, based on a comprehensive assessment of human accessibility, inhalability, mobility, host pathogenicity, and clinical usability, a total of 313 ARGs with potential health risks were screened, accounting for 94.6% of the total number of human-accessible ARGs. The total calculated risk values ​​and their risk categories are shown in Table 1, with risk values ​​ranging from 1.99E-03 to 2.30E+12. Based on the calculated risk values, all risk ARGs were divided into five levels: high risk R4, relatively high risk R3, moderate risk R2, relatively low risk R1, and no risk R0. No-risk ARGs were those that did not carry MGEs or were not significantly correlated with pathogenic host bacteria.

[0072] S7: The total health risk of ARGs in the air is the sum of the potential risk values ​​of all detected ARGs in the air.

[0073] The formula for calculating the total health risk value of ARGs in the air at a certain sampling point or sampling time in this wastewater treatment plant is as follows: In the formula, HR i The potential health risk value (HR) for each human-accessible ARG in the air medium of a wastewater treatment plant at a certain sampling point or sampling time.

[0074] Based on the total health risk value of ARGs in the air at different sampling points and sampling times, risk points and times with high ARG exposure risk in the air of wastewater treatment plants can be screened out, so as to formulate effective prevention measures and response strategies and improve public health and safety.

[0075] Based on the detection of accessible ARGs and their potential health risks at different sampling points in the air of this wastewater treatment plant, the total potential health risk of ARGs at different sampling points was calculated, as shown in Table 2. The aeration tank and sludge dewatering room were the units with the highest potential health risk indices for workers exposed to airborne ARGs. The median health risk indices for male workers were 1.73E+12 and 3.90E+11, respectively, while those for female workers were 1.38E+12 and 3.17E+11, respectively. Therefore, the aeration tank and sludge dewatering room are the main potential risk points for airborne ARGs in this wastewater treatment plant, and workers working in these units should pay more attention to their personal protection.

[0076] Based on the detection of accessible ARGs and their potential health risks in the air at different sampling times at this wastewater treatment plant, the total potential health risk of ARGs in the air at different sampling times was calculated, as shown in Table 3. The highest potential health risk indices of accessible ARGs in the air were observed in July and October, with median values ​​of 1.47E+15 and 4.77E+11, respectively. Therefore, wastewater treatment plant workers should take extra precautions during these two seasons to reduce the risk of exposure to airborne ARGs.

[0077] In addition to workers' personal protection, wastewater treatment plants should also take corresponding control and response measures. For example, in indoor wastewater and sludge treatment units, ventilation can be enhanced to promote the diffusion of airborne microorganisms and reduce the concentration of ARGs in the air, or ARG reduction technologies can be developed to control the escape and spread of ARGs during wastewater and sludge treatment.

[0078] Table 1. Health risk values ​​and risk categories of accessible ARGs in the air of wastewater treatment plants.

[0079] Table 2. Total Health Risk Values ​​of Human-Accessible ARGs in Air at Different Sampling Points in Wastewater Treatment Plant

[0080] Table 3. Total health risk values ​​of accessible ARGs in the air at different sampling times at wastewater treatment plants.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for assessing potential health risks of ARGs in air medium, characterized in that, Specifically comprising the following steps: S1, obtaining the qualitative data of ARGs in the air medium of the target place by using metagenomic sequencing method, screening the ARGs type co-occurring in the air medium of the target place and the human environment, i.e. the human accessible ARGs in the air medium of the target place, through ARGs type analysis, calculating the possibility of each human accessible ARG in the air medium of the target place transferring to the human environment and bringing harm to the human body, i.e. the human accessibility HA of the human accessible ARG according to formula (1); HA = average abundance 人体环境 x frequency of detection 人体环境 … (1); wherein the average abundance 人体环境 is the average abundance of the human accessible ARG in the human environment in RPKM; the detection frequency 人体环境 is the detection rate of the human accessible ARG in the human environment in %; HA is dimensionless; S2, obtaining the absolute quantitative copy number of each human accessible ARG in per unit volume of air in the air medium of the target place, i.e. the absolute abundance of the human accessible ARG by using fluorescent quantitative PCR method, calculating the inhalability IH of the human accessible ARG according to formula (2); IH = absolute abundance ARG x inhalable fraction x retention rate x breathing rate of exposed population x exposure time of exposed population... (2); wherein absolute abundance is the absolute abundance of the human accessible ARG in copies / m ARG 3 空气 ; inhalable proportion is the inhalable proportion of the human accessible ARG in %; retention is the retention of aerosol inhalation in human environment, taking 0.5; exposure population breathing rate is the breathing rate of the exposure population in m 3 / d; exposure population exposure time is the annual exposure time of the exposure population in h; IH is dimensionless;​ S3, number MGE represents the mobility MB of ARGs in the air medium of the target site by the carriage of MGEs between different host microorganisms, i.e. MB = number MGE ; the number MGE is the number of MGEs species significantly associated (p<0.05, r>0.7) with each human accessible ARG in the air medium of the target site; MB is dimensionless; S4, screening all host bacteria of each human accessible ARG through the correlation network of ARGs and bacteria in the air medium of the target place, screening pathogenic hosts, calculating the proportion of pathogenic hosts in all host bacteria, calculating the host pathogenicity HP of the human accessible ARG according to formula (3); ……（3）； wherein is the pathogenicity of the pathogenic host bacteria of the human accessible ARG among all host bacteria; is the pathogenicity of the pathogenic host bacteria of the species. HP is dimensionless; S5, statistically analyzing the amount of antibiotic resistant to each human accessible ARG in the air medium of the target place in clinic, calculating the clinical availability CA of the human accessible ARG according to formula (4); ……（4）； wherein the amount of antibiotic used i is the sum of global medical consumption of all antibiotic classes to which the human accessible ARG is resistant; CA is dimensionless; S6, comprehensively analyzing the human accessibility, inhalability, mobility, host pathogenicity and clinical availability, calculating the potential health risk value HR of each human accessible ARG in the air medium of the target place according to formula (5); ……（5）； S7、calculating the total value of health risk HR of ARGs in the air medium of the target site according to formula (6) S ; … (6); where HR S HR is the total value of health risk for ARGs in the air medium i HR is the potential health risk value for each human accessible ARG in the air medium.

2. The method for assessing potential health risks of ARGs in air media according to claim 1, characterized in that, In S1, the ARGs in the air medium of the target place are collected by a sampler for analysis and detection of species and abundance; and / or In S1, after the collected sample is pretreated, the ARGs type co-occurring in the air medium of the target place and the human environment is screened out by comparing with the data of ARGs species and abundance in the human environment recorded in literature.

3. The method for assessing potential health risks of ARGs in air media according to claim 1, wherein, In S2, the parameters for calculating the IH value are all subjected to data hypothesis, fitting and testing by Monte Carlo simulation, the Monte Carlo simulation of data is completed by Oracle Crystal Ball risk management software, all input variables are randomly selected from their probability distribution, and the output variables are iterated for 10000 times to make the parameter distribution reach a stable state; and / or The inhalable proportion in formula (2) of S2 is the ratio of inhalable bacterial aerosol concentration to total bacterial aerosol concentration.

4. The method for assessing potential health risks of ARGs in air media according to claim 3, characterized in that, The method for obtaining the inhalable bacterial aerosol concentration and the total bacterial aerosol concentration is as follows: collecting bacterial aerosols at each sampling point by using an impinger sampler, the cutting diameters of the impinger samplers I to VI are >7, 4.7-7.0, 3.3-4.7, 2.1-3.3, 1.1-2.1 and 0.65-1.1 μm respectively; separating and culturing the bacteria on each level of the sampler, revising the colony count results on each level of the culture dish of each sampler by using positive hole correction method, and calculating the bacterial aerosol concentration at each sampling point by using formula (7); … (7); where C is the bacterial aerosol concentration in CFU / m 3 空气 , Pr i is the number of viable bacteria on each stage sampler in CFU, Q is the sampling gas flow in L / min, and T is the sampling time in min. Pr = the number of colonies on the sampler i Pr = the number of colonies on the sampler i Pr = the number of colonies on the sampler 5. The method for assessing potential health risks of ARGs in air media according to claim 1, wherein, Spearman correlation between each human accessible ARG and MGEs was calculated using the cor () function in the igraph package in R language in S3, and the MGEs species significantly correlated with each human accessible ARG was screened, with the screening condition being p < 0.05 and r > 0.7; and / or The MGEs species included in the correlation calculation include insertion sequences and insertion sequence common regions, integrases, transposons, transposases and plasmids; and / or S3 uses Monte Carlo simulation for quantities MGE Assumption, fitting and testing of data.

6. The method for assessing potential health risks of ARGs in air media according to claim 1, wherein, The method for screening pathogenic hosts in S4 is: calculating the Spearman correlation of each human accessible ARG and bacteria by the cor() function in the igraph package of R language, screening the bacterial species significantly correlated with the human accessible ARG as the potential host bacteria of the human accessible ARG, and the screening condition is p<0.05 and r>0.7; screening the pathogenic hosts by comparing the obtained potential host bacteria with the Global Catalogue of Microorganisms database; and the proportion of the pathogenic host bacteria of the human accessible ARG in all host bacteria is the pathogenicity of the human accessible ARG. ; and / or the total number of VFs that the pathogenic host bacteria can possibly carry VF , the number of VF The method for obtaining is: the amino acid sequence of the non-redundant gene set obtained by Diamond macro genome is compared with Virulence Factorsof Pathogenic Bacteria Database, and the pathogenic VFs in the target site air medium are obtained by screening the annotation information, and the comparison parameter setting expectation value e-value is 1e-5; the correlation network of the pathogenic host bacteria and the pathogenic VFs related to the human accessible ARG is used to screen the pathogenic VFs that the pathogenic host bacteria can possibly carry, the correlation coefficient is calculated by the igraph package of R language, the Spearman correlation of the pathogenic host bacteria and the VFs is calculated by using the cor () function, and after calculation, the VFs significantly related to the pathogenic host bacteria are screened as the pathogenic VFs that the pathogenic host bacteria can possibly carry and counted, and the screening condition is p<0.05, r>0.7; and / or Monte Carlo simulations are used in S4 to and make assumptions, fit and test data.

7. The method for assessing potential health risk of ARGs in air media according to claim 1, wherein, The global medical consumption data in S5 is derived from the ResistanceMap database; and / or Monte Carlo simulation of antibiotic usage in S5 i Hypothesis, fitting and testing of data were performed.

8. Application of the air medium ARGs potential health risk assessment method in any one of claims 1-7 in assessing the air medium ARGs potential health risk in the area where sewage is located.

9. Use according to claim 8, characterized in that, The area where sewage is located is a sewage plant.

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