[High-throughput qPCR and Amplicon Sequencing as Complementary Methods for Profiling Antibiotic Resistance Genes in Urban Wetland Parks].

Urban wetland parks are an important practice for urban wetland protection and utilization due to the vast ecosystem service value. As emerging contaminants, antibiotic resistance genes (ARGs) are great attractions for environmental research and public concerns. Based on high-throughput qPCR and high-throughput amplicon sequencing techniques, we investigated the occurrence, abundance, and distribution profiles of antibiotic resistance genes in the aquatic environment of Xiamen urban wetland parks (five sites). The influencing factors and driving mechanisms of antibiotic resistance genes were deciphered on the basis of microbial community structure and water quality. Diverse and abundant ARGs were observed and coexisted in urban wet parks. A total of 217 ARGs were detected in the water body of urban wetland parks, with an abundance up to 6.48×109 copies·L-1. Urban wetland parks were important hotspots and repositories of the antibiotic resistome. A total of nine bacterial genera, including Marivivens, NS5_marine_group, and Planktomarina, were identified as the potential carriers of diverse resistance genes (41 ARGs). The microbial communities could alone explain 51% of alterations in the antibiotic resistome in the aquatic environment of the urban wetland parks. Therefore, the microbial community was the key driving force for the occurrence and evolution of ARGs in urban wetland parks. Based on the results, with the presence of ARGs and antibiotic resistance bacteria, it is suggested that the water environments of urban wetland parks have potential risks of water ecological security and human health, and it is necessary to further enhance the research and control of microbial contaminants in the aquatic environment of urban wetland parks.

Mapping the profiles and underlying driving mechanisms of the antibiotic resistome and microbiome within a subtropical complex river watershed.

Antibiotic resistance is an escalating global concern, leading to millions of annual deaths worldwide. Human activities can impact antibiotic resistance gene (ARG) prevalence in aquatic ecosystems, but the intricate interplay between anthropogenic disturbances and river system resilience, and their respective contributions to the dynamics of different river segments, remains poorly understood. In this study, we investigate the antibiotic resistome and microbiome in water and sediment samples from two distinct sub-watersheds within a specific watershed. Results show a decrease in the number of core ARGs downstream in water, while sediments near densely populated areas exhibit an increase. PCoA ordination reveals clear geographic clustering of resistome and microbiome among samples from strong anthropogenic disturbed areas, reservoir areas, and estuary area. Co-occurrence networks highlight a higher connectivity of mobile genetic elements (MGEs) in disturbed areas compared to reservoir areas, presenting a threat to densely populated areas. Water quality parameters and antibiotics concentration were the key factors shaping the ARG profiles in sediment samples from urban regions. Overall, our study reveals distinct patterns of ARGs in sediment and water samples, emphasizing the importance of considering both anthropogenic and natural factors in comprehending and managing ARG distribution in river systems.

Long-term seawall barriers lead to the formation of an urban coastal lagoon with increased antibiotic resistome.

Urbanization has increased the spread of antibiotic resistance genes (ARGs) impacting urban aquatic ecosystems and threatening human health. However, an overview of the antibiotic resistome in artificial coastal lagoons formed by coastal seawall construction is unclear. This study investigated the resistome of sediment in a coastal lagoon, established for over 60 years and found that the composition of the resistome in the lagoon sediments associated with the seawall significantly differed from that of marine sediment external to the seawall. Moreover, the diversity, number, relative abundance, and absolute abundance of the antibiotic resistome in the lagoon sediments were significantly higher compared to marine sediment. Network analyses revealed that more co-occurrences were found in lagoon sediment between bacterial communities, ARGs and mobile genetic elements (MGEs) than in marine sediments, suggesting that bacteria in lagoon sediments may be associated with multiple antibiotic resistances. Random forest and structural equation models showed that an increase in the absolute abundance of MGEs had a concomitant effect on the absolute abundance and diversity of ARGs, whereas increasing salinity decreased the absolute abundance of ARGs. This study provides a basis to assess the risk of resistome diffusion and persistence in an artificial coastal lagoon.

Phagotrophic protists preserve antibiotic-resistant opportunistic human pathogens in the vegetable phyllosphere.

Food safety of leafy greens is an emerging public health issue as they can harbor opportunistic human pathogens (OHPs) and expose OHPs to consumers. Protists are an integral part of phyllosphere microbial ecosystems. However, our understanding of protist-pathogen associations in the phyllosphere and their consequences on public health remains poor. Here, we examined phyllosphere protists, human pathogen marker genes (HPMGs), and protist endosymbionts from four species of leafy greens from major supermarkets in Xiamen, China. Our results showed that Staphylococcus aureus and Klebsiella pneumoniae were the dominant human pathogens in the vegetable phyllosphere. The distribution of HPMGs and protistan communities differed between vegetable species, of which Chinese chive possessed the most diverse protists and highest abundance of HPMGs. HPMGs abundance positively correlated with the diversity and relative abundance of phagotrophic protists. Whole genome sequencing further uncovered that most isolated phyllosphere protists harbored multiple OHPs which carried antibiotic resistance genes, virulence factors, and metal resistance genes and had the potential to HGT. Colpoda were identified as key phagotrophic protists which positively linked to OHPs and carried diverse resistance and virulence potential endosymbiont OHPs including Pseudomonas nitroreducens, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia. We highlight that phyllosphere protists contribute to the transmission of resistant OHPs through internalization and thus pose risks to the food safety of leafy greens and human health. Our study provides insights into the protist-OHP interactions in the phyllosphere, which will help in food safety surveillance and human health.

Urban greenspace types influence the microbial community assembly and antibiotic resistome more in the phyllosphere than in the soil.

Urban greenspace (UGS) is recognized to confer significant societal benefits, but few studies explored the microbial communities and antibiotic resistance genes (ARGs) from different urban greenspace types. Here, we collected leaf and soil samples from forest, greenbelt, and parkland to analyze microbial community assembly and ARG profile. For phyllosphere fungal community, the α-diversity was higher in forest, compared to those in greenbelt and parkland. Moreover, urban greenspace types altered the community assembly. Stochastic processes had a greater effect on phyllosphere fungal community in greenbelt and parkland, while in forest they were dominated by deterministic processes. In contrast, no significant differences in bacterial community diversity, community assembly were observed between the samples collected from different urban greenspace types. A total of 153 ARGs and mobile genetic elements (MGEs) were detected in phyllosphere and soil with resistance to the majority classes of antibiotics commonly applied to humans and animals. Structural equation model further revealed that a direct association between greenspace type and ARGs in the phyllosphere even after considering the effects of all other factors simultaneously. Our findings provide new insights into the microbial communities and antibiotic resistome of urban greenspaces and the potential risk linked with human health.

Distinct patterns of the soil and phyllosphere antibiotic resistome in natural forest ecosystems under an altitudinal gradient.

Warming affects microbial functioning of soil and the phyllosphere across global ecosystems. However, little is known about the impact of increasing temperature on antibiotic resistome profiles in natural forests. To address this issue, we investigated antibiotic resistance genes (ARGs) in both soil and the plant phyllosphere using an experimental platform established in a forest ecosystem that delivers a temperature difference of 2.1 °C along an altitudinal gradient. Principal Coordinate Analysis (PCoA) showed that there were significant differences in the composition of soil and plant phyllosphere ARGs at different altitudes (P = 0.001). The relative abundance of phyllosphere ARGs and mobile genetic elements (MGEs) and soil MGEs increased with temperature. More resistance gene classes increased in abundance in the phyllosphere (10 classes) than soil (2 classes), and a Random Forest model analysis suggested that phyllosphere ARGs were more sensitive to temperature change than soil. Increasing temperature as a direct consequence of an altitudinal gradient, and the relative abundance of MGEs were the main drivers that shaped the profiles of ARGs in the phyllosphere and soil. Biotic and abiotic factors affected phyllosphere ARGs indirectly via MGEs. This study enhances our understanding of the influence of altitude gradients on resistance genes in natural environments.

Antibiotic resistome in groundwater and its association with mountain springs and river.

The distribution of antibiotic resistance genes (ARGs) in water sources potentially threatens drinking water safety. However, the sources of antibiotic resistome in groundwater are still under-investigated. Here, we evaluated the profiles of antibiotic resistome in peri-urban groundwater and its associated water sources (river and mountain spring) to characterize the antibiotic resistome from natural water sources on groundwater resistome. A total of 261 antibiotic resistome were detected in groundwater, mountain spring, and river samples. The relative abundances of ARGs and mobile genetic elements (MGEs) were significantly higher in the river samples than in spring water and groundwater samples. The resistome profiles were similar between groundwater and spring water but differed from the river samples. According to source tracking results, the groundwater resistome was likely to be derived from springs (28.0%-50.0%) and rivers (28.6%-48.6%), which share the same trend for the source tracking of bacterial communities. Bacterial α-diversity, bacterial ß-diversity, and MGEs directly or indirectly affected the ARGs in groundwater samples. Although the abundance of groundwater resistome was not elevated by river and spring water, groundwater resistomes were diverse and may be derived from both river and spring water. We highlight the importance of groundwater resistome and its association with potential water sources, providing a better understanding and basis for the effective control of the ARG proliferation and dissemination in groundwater from exogenous water bodies in the future.

Characterization of microbial community, ecological functions and antibiotic resistance in estuarine plastisphere.

The plastisphere is a new ecological niche. Compared to the surrounding water, microbial community composition associated with the plastisphere is known to differ with functional consequences. Here, this study characterized the bacterial and fungal communities associated with four types of plastisphere (polyethylene, polystyrene, polypropylene and polyvinyl chloride) in an estuarine habitat; assessed ecological functions including carbon, nitrogen, phosphorus and sulfur cycling, and determined the presence of antibiotic resistance genes (ARGs) and human pathogens. Stochastic processes dominated the community assembly of microorganisms on the plastisphere. Several functional genera related to nutrient cycling were enriched in the plastisphere. Compared to surrounding water and other plastisphere, the abundances of carbon, nitrogen and phosphorus cycling genes (cdaR, nosZ and chpy etc.) and ARGs (aadA2-1, cfa and catB8 etc.) were significantly increased in polyvinyl chloride plastisphere. In contrast, the polystyrene plastisphere was the preferred substrate for several pathogens being enriched with for example, Giardia lamblia 18S rRNA, Klebsiella pneumoniae phoE and Legionella spp. 23S rRNA. Overall, this study showed that different plastisphere had different effects on ecological functions and health risk in estuaries and emphasizes the importance of controlling plastic pollution in estuaries. Data from this study support global policy drivers that seek to reduce plastic pollution and offer insights into ecological functions in a new ecological niche of the Anthropocene.

Distinct patterns of soil bacterial and fungal community assemblages in subtropical forest ecosystems under warming.

Climate change globally affects soil microbial community assembly across ecosystems. However, little is known about the impact of warming on the structure of soil microbial communities or underlying mechanisms that shape microbial community composition in subtropical forest ecosystems. To address this gap, we utilized natural variation in temperature via an altitudinal gradient to simulate ecosystem warming. After 6 years, microbial co-occurrence network complexity increased with warming, and changes in their taxonomic composition were asynchronous, likely due to contrasting community assembly processes. We found that while stochastic processes were drivers of bacterial community composition, warming led to a shift from stochastic to deterministic drivers in dry season. Structural equation modelling highlighted that soil temperature and water content positively influenced soil microbial communities during dry season and negatively during wet season. These results facilitate our understanding of the response of soil microbial communities to climate warming and may improve predictions of ecosystem function of soil microbes in subtropical forests.

Manure and biochar have limited effect on lettuce leaf endophyte resistome.

Soil amendment with manure compost and biochar is widely adopted to improve soil fertility and promote plant growth, and their effects on soil microbial communities and resistome have been well documented. However, there is sparse information regarding their effects on vegetable endophytes, which represent a major source of human exposure to pathogens and antibiotic resistance genes (ARGs) when eaten raw. Here, we investigated the impacts of manure compost or biochar addition on the bacterial community compositions and ARGs in the soil-lettuce continuum including soil, seed, leaf, and root samples. A total of 137 ARGs and 31 mobile genetic elements (MGEs) were detected in all the samples after 60 days of cultivation. The relative abundance of ARGs and the diversity of bacteria communities presented a consistent decreasing trend from soil to root endophytes, then leaf endophytes. Manure compost addition increased the diversity and abundance of ARGs in soil, while significant changes in the ARG profiles and bacterial communities were not observed in leaf endophytes after manure compost or biochar addition, or both. Bipartite networks analysis suggested that seed microbiome was one of the major sources of plant endophytes and ARGs. Twenty potential human pathogens were isolated from lettuce, indicating potential exposure risk to pathogens via the consumption of raw lettuce. These results suggest limited impacts of manure compost and biochar addition on lettuce endophytes and highlight the contribution of seed microbiome to endophyte ARG profiles.

City-scale distribution of airborne antibiotic resistance genes.

Concerns around urban air quality have been increasing worldwide due to large-scale urbanization. A large volume of work has been focused on the chemical pollutants in the air and their impacts on human health. However, the profile of airborne microbial contaminants, especially antibiotic resistance genes (ARGs), is largely understudied. Here, high-throughput quantitative PCR (HT-qPCR) was employed to explore the temporal and spatial distribution of airborne ARGs from 11 sites with various functional zones and different urbanization levels within Xiamen, China. A total of 104 unique ARGs and 23 mobile genetic elements (MGEs) were detected across all samples. Temporal shift was observed in the distribution of ARG profiles, with significantly higher relative abundance of ARGs detected in summer than that in spring. Temperature is the key predictor of the total relative abundance of ARGs and MGEs in summer, while PM2.5 and PM10 were the two most important factors affecting the abundance in spring. Our findings suggest that urban aerosols accommodate rich and dynamic ARGs and MGEs, and emphasize the role of temperature, air quality and anthropogenic activities in shaping the profile of ARGs.

Skin microbiota interact with microbes on office surfaces.

The indoor environment is recognized as a potential contributor to human health impacts through resident microbiomes. Indoor surface microbial communities are formed from several sources, environmental and anthropogenic. In this study, we characterized the bacterial and fungal communities from various sources typical of a working office environment including dust, fingers, and computer keyboards and mice. The composition of the dust bacterial community was significantly different from the other tested surfaces (P < 0.05), whereas the dust fungal community was only significantly different from fingers (P < 0.05). Bacterial and fungal communities were both shaped by deterministic processes, and bacterial communities had a higher migration rate. Results of a network analysis showed that the microbial community interactions of keyboards and mice were mainly competitive. Fast expectation-maximization microbial source tracking (FEAST) identified the sources of > 70 % of the keyboard and mouse microbial communities. Biomarkers for each sample types were identified by LDA Effect Size (LEfSE) analysis, some of which were soil-derived and potential anthropogenic pathogens, indicating the potential for exchange of microbes among outdoor, human and indoor surfaces. The current study shows that the source of microorganisms at the office interface is highly traceable and that their migration is linked to human activity. The migration of potentially pathogenic microbes were identified, emphasising the importance of personal hygiene.

Dissemination of antibiotic resistance genes from landfill leachate to groundwater.

Landfill leachate, a highly concentrated organic wastewater containing diverse microorganisms and various heavy metals, has become an important reservoir of antibiotic resistance genes (ARGs). In this study, a total of 203 unique ARGs and 10 mobile genetic elements (MGEs) were identified from collected landfill leachate and groundwater. The number and abundance (normalized and absolute) of antibiotic resistome in effluent of leachate treatment plants decreased significantly compared to influent. The abundance of ARGs in groundwater increased as the distance from the leachate basin decreased. Fast expectation-maximization microbial source tracking (FEAST) showed that up to 96 % of ARGs in groundwater (GW3) may originate from nearby leachate, suggesting that ARGs in leachate can penetrate and spread into the groundwater environment. A significant correlation between ARGs and bacterial communities was identified. Together with network analysis showing the 12 bacterial taxa co-occurring with seven classes of antibiotic-associated ARGs, our results revealed the diverse potential microbial hosts of ARGs in water samples around the landfill sites. Heavy metals, bacterial community and MGEs were the driving factors shaping the ARGs patterns in the water samples, with their interactions explaining 57 % of ARGs variations. Our results provide an understanding of the distribution and dissemination of ARGs from landfill leachate to the nearby groundwater and suggest a comprehensive impact assessment of ARGs in aquatic environments of landfills.

HiLi-chip: A high-throughput library construction chip for comprehensive profiling of environmental microbial communities.

Investigating the contribution and associations of environmental microbes to ecological health and human well-being is in great demand with the goal of One Health proposed. To achieve the goal, there is an urgent need for accurate approaches to obtaining a large amount of high-resolution molecular information from various microbes. In this study, we developed a high-throughput library construction chip (HiLi-Chip) for profiling environmental microbial communities and evaluated its performance. The HiLi-Chip showed high conformity with the conventional Pacbio method in terms of α-diversity, community composition of abundant bacteria (>83%), as well as rare taxa (>84%) and human pathogens detection (>67%), indicating its advantages of accuracy, high-throughput, cost-efficiency, and broad practicability. It is suggested that the optimal strategy of the HiLi-Chip was a 2.4 µL PCR mixture per sample (∼2.4 ng DNA) with a 216-sample × 24-replicate format. We have successfully applied the HiLi-Chip to the Jiulongjiang River and identified 51 potential human bacterial pathogens with a total relative abundance of 0.22%. Additionally, under limited nutrients and similar upstream environments, bacteria tended to impose competitive pressures, resulting in a more connected network at the downstream river confluence (RC). Whereas narrow niche breadth of bacteria and upstream environmental heterogeneity probably promoted niche complementary and environment selection leading to fewer links at RC in the midsection of the river. Core bacteria might represent the entire bacterial community and enhance network stability through synergistic interactions with other core bacteria. Collectively, our results demonstrate that the HiLi-Chip is a robust tool for rapid comprehensive profiling of microbial communities in environmental samples and has significant implications for a profound understanding of environmental microbial interactions.

Conurbation size drives antibiotic resistance along the river.

With growing concerns about antibiotic resistance, the tracking of antibiotic resistance genes (ARGs) in urban waterways will facilitate our increased understanding of the impact of urbanization on ARGs dissemination. In the current study, we assessed the ARGs profiles and antibiotic resistome in water samples along the Jiulong River basin, a distance of 250 km, to better understand the impact of anthropogenic activities. A total of 244 ARGs and 12 MGEs were detected from 21 sampling sites. Both relative and absolute abundance of the observed resistome decreased with increasing distance from urban areas. Ordinary least-squares (OLS) regression revealed that both the relative and absolute resistome abundance were positively correlated with city size. The resistome had several inputs and outputs and Fast Expectation Maximization Microbial Source Tracking (FEAST), suggested that the majority of the antibiotic resistome originated from anthropogenic activities. A total of 8 ARGs and 20 microbial OTUs were considered as biomarkers that differentiated the location of sampling sites. Bacterial communities were significantly correlated with ARGs according to Procrustes analysis and Mantel test, which was also supported by a co-occurrence network. Variation partitioning analysis revealed that ARG profiles were driven by multiple factors. Although antibiotic resistome abundance significantly increased near urban conurbations, overall resistome abundance decreased as the river flowed downstream. Our study highlights the effect of conurbation size on antibiotic resistance profiles within the river basin and the potential resilience of rivers to recover from ARGs contamination.

Effects of biofertilizer on soil microbial diversity and antibiotic resistance genes.

Spread of antibiotic resistance or the presence of antibiotic resistance genes (ARGs) in pathogens is a globally recognized threat to human health. Numerous studies have shown that application of organic fertilizers may increase the risk of ARGs, however, the risk of resistance genes associated with biofertilizers is largely unknown. To investigate whether biofertilizer application introduces ARGs to the soil, we used high-throughput quantitative polymerization chain reaction (HT-qPCR) to explore the effect of biofertilizer application over three years on soil ARGs in three orchards with different locations in China. Redundancy analysis showed specific and significant differences in the beta diversity of soil bacteria and fungi between treatments (fertilizer vs. no fertilizer). One-way ANOVA analysis revealed findings of the main driver of the significant difference in microbial community structure between fertilizer and control treatment was the change in soil properties following the application of biofertilizer. A total of 139 ARGs and 27 MGEs (mobile genetic elements), and 46 ARGs and 6 MGEs from 11 major taxa were detected in biofertilizer and soil samples, respectively. Only the samples from Guangxi had significant differences in the detected number of ARGs and MGEs between fertilization and control. Through structural equation modeling (SEM), we found that soil properties indirectly affected ARGs by shaping bacterial diversity, while bacterial abundance directly affected ARGs. Biofertilizer application did not significantly alter the relative abundance of ARGs in soil due to the complexity of the soil environment and competition between exogenous and native microorganisms. This study provided new insights into the spread of the antibiotic resistome of the soil through biofertilizer applications.

Impacts of global change on the phyllosphere microbiome.

Plants form complex interaction networks with diverse microbiomes in the environment, and the intricate interplay between plants and their associated microbiomes can greatly influence ecosystem processes and functions. The phyllosphere, the aerial part of the plant, provides a unique habitat for diverse microbes, and in return the phyllosphere microbiome greatly affects plant performance. As an open system, the phyllosphere is subjected to environmental perturbations, including global change, which will impact the crosstalk between plants and their microbiomes. In this review, we aim to provide a synthesis of current knowledge of the complex interactions between plants and the phyllosphere microbiome under global changes and to identify future priority areas of research on this topic.

Discarded masks as hotspots of antibiotic resistance genes during COVID-19 pandemic.

The demand for facial masks remains high. However, little is known about discarded masks as a potential refuge for contaminants and to facilitate enrichment and spread of antibiotic resistance genes (ARG) in the environment. We address this issue by conducting an in-situ time-series experiment to investigate the dynamic changes of ARGs, bacteria and protozoa associated with discarded masks. Masks were incubated in an estuary for 30 days. The relative abundance of ARGs in masks increased after day 7 but levelled off after 14 days. The absolute abundance of ARGs at 30 days was 1.29 × 1012 and 1.07 × 1012 copies for carbon and surgical masks, respectively. According to normalized stochasticity ratio analysis, the assembly of bacterial and protistan communities was determined by stochastic (NST = 62%) and deterministic (NST = 40%) processes respectively. A network analysis highlighted potential interactions between bacteria and protozoa, which was further confirmed by culture-dependent assays, that showed masks shelter and enrich microbial communities. An antibiotic susceptibility test suggested that antibiotic resistant pathogens co-exist within protozoa. This study provides an insight into the spread of ARGs through discarded masks and highlights the importance of managing discarded masks with the potential ecological risk of mask contamination.

[Microplastic-Induced Alterations to Antibiotic Resistance Genes in Seawater].

The increasing and combined pollution of microplastics (MPs) and antibiotic resistance genes (ARGs) in aquatic environments is a great ecological and health concern. However, MP-induced alterations to ARGs in seawater is poorly understood, impeding risk assessment of plastics. We profiled the diversity and abundance of ARGs and mobile genetic elements (MGEs) in seawater after the addition of three different MPs (PE, PVC, and PVA) and 49-day aerated incubation.A total of 20, 35, 42, and 64 ARGs were detected in BLK, PE, PVC, and PVA, with 2, 4, 2, and 3 MGEs, respectively. The absolute abundance of ARGs in the seawater aerated with MPs ranged from 4.01×106 copies ·L-1 to 1.05×108 copies ·L-1. Additionally, the variety and richness of ARGs and MGEs in PVA were significantly higher than in the original seawater, or the seawater aerated with the other two MPs. This indicates that PVA, which is water soluble, could induce more diverse and abundant ARGs in seawater. Significant correlations among ARGs, MGEs, and 16S rRNA genes were observed, implying that the occurrence of MGEs in seawater may accelerate the transmission of ARGs through horizontal gene transfer, and bacterial microorganisms could directly affect the propagation and dissemination of ARGs.

Dynamics of antibiotic resistance and its association with bacterial community in a drinking water treatment plant and the residential area.

Drinking water treatment techniques are used globally in the context of water security and public health, yet they are not applicable to antibiotic resistance gene (ARG) contamination. Using high-throughput quantitative PCR, we analyzed the prevalence and diversity of ARGs and mobile genetic elements (MGEs) in water supplies. A total of 224 ARGs and MGEs were detected in all sampling sites. Absolute abundance and detected number of ARGs decreased significantly (P < 0.05) in sand filter water after drinking water treatment and increased thereafter at point-of-use (household tap water). Changes in the composition and diversity of the bacterial community were observed in water samples at different steps. A significant correlation (P < 0.001) between microbial communities and ARG profiles was observed, and variance in ARG profiles could be primarily attributed to community composition (11.9%), and interaction between community composition, environmental factors and MGEs (30.7%). A network analysis was performed, and the results showed eight bacterial phyla were significantly correlated with nine different classes of ARGs, suggesting the potential bacterial host for ARGs. This study suggested that although the absolute abundance of ARGs decreased after treatment of drinking water treatment plants (DWTPs), the rebounded of ARGs in the water distribution system should not be neglected.