Co-driving factors of tidal effect on the abundance and distribution of antibiotic resistance genes in the Yongjiang Estuary, China.

The unreasonable use of antibiotics and the transmission of antibiotic resistance genes (ARGs) induced by antibiotics have led to a large number of ARGs entered the water environment, which seriously threatened human health and environmental safety. The estuarine aquatic environment connects with inland rivers and sea and is frequently influenced by human activities. This study aims to reveal the occurrences and abundances of ARGs and bacterial community composition by high-throughput quantitative PCR including 296 primers and high-throughput sequencing in the tide rising and ebbing of surface water in the Yongjiang Estuary, China. The results showed that there were a large number of ARGs and mobile genetic elements (MGEs) detected in the rising tide and ebb tide water bodies. The numbers of detected ARGs in each sample at rising and ebb tide ranged from 16 to 77 and 61 to 88, respectively, and the absolute abundance ranges were 1.69 × 104-1.69 × 109 copies/L and 3.18 × 103-2.57 × 109 copies/L, respectively. Obvious tidal distribution characteristics of ARGs were showed. Most of ARGs conferred resistance to multidrug, aminoglycosides and sulfanilamides. Proteobacteria, Actinobacteria and Bacteroidetes were the dominantly bacterial phylum in the Yongjiang Estuary. Network analysis results indicated that multi-genera were identified as possible ARGs hosts, and they carried more than two types of ARGs genes. Partial least squares path modeling further revealed that MGEs and bacterial community composition were the most important driving factors. The results of the study can provide the corresponding scientific basis for the diffusion and control of ARGs in estuaries.

Influence of anthropogenic disturbances on antibiotic resistance gene distributions along the Minjiang River in Southeast China.

River-reservoir systems have become ubiquitous among modern global aquatic environments due to the widespread construction of dams. However, little is known of antibiotic resistance gene (ARG) distributions in reservoir-river systems experiencing varying degrees of anthropogenic impacts. Here, the diversity, abundance, and spatial distribution of ARGs were comprehensively characterized along the main stem of the Minjiang River, a typical subtropic reservoir-river system in Southeast China using high-throughput quantitative PCR. A total of 252 ARG subtypes were detected from twelve sampling sites that were dominated by aac(3)-Via, followed by czcA, blaTEM, and sul1. Urban river waters (sites S9-S12) harbored more diverse ARGs than did the reservoir waters (sites S1-S7), indicating more serious antibiotic resistance pollution in areas with larger population densities. Dam construction could reduce the richness and absolute abundance of ARGs from upstream (site S7) to downstream (site S8). Urban river waters also harbored a higher proportion of mobile genetic elements (MGEs), suggesting that intensive human activities may promote ARG horizontal gene transfers. The mean relative abundance of Proteobacteria that could promote antibiotic resistance within microbial communities was also highest in urban river waters. Variance partitioning analysis indicated that MGEs and bacterial communities could explain 67.33%, 44.7%, and 90.29% of variation in selected ARGs for the entire watershed, aquaculture waters, and urban river waters, respectively. These results further suggest that urban rivers are ideal media for the acquisition and spread of ARGs. These findings provide new insights into the occurrence and potential mechanisms determining the distributions of ARGs in a reservoir-river system experiencing various anthropogenic disturbances at the watershed scale.

The fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) from livestock wastewater (dominated by quinolone antibiotics) treated by microbial fuel cell (MFC).

The removal characteristics of antibiotic resistance genes and mobile genetic elements from livestock wastewater (dominated by quinolone antibiotics) treated with MFC were evaluated by High-throughput quantitative (HT-qPCR). The results showed that 144 ARGs and 8 MEGs were detected in the livestock wastewater. After MFC treatment, the number of AGRs decreased as a whole, and the relative abundance of macrolide-lincosamide-streptogramin group B (MLSB) and aminoglycosider decreased by 62.7% and 92.9%, respectively. MGEs decreased by 57.3% and multidrug genes decreased by 90%. After MFC treatment, the absolute abundance of tetracycline in raw sewage decreased by two orders of magnitude from 5.8 × 105 copies L-1 to 5.1.× 103 copies L-1. However, MFC was less efficient in the removal of vancomycin and beta-lactamase genes. It was also found that chloramphenicol resistance genes slightly increased. Illumina sequencing showed that Syntrophobacterales and Synergistales were predominant in MFCs. Desulfovibrio was resistant to high concentration of moxifloxacin hydrochloride. The removal efficiency of MFC for moxifloxacin hydrochloride at a concentration of 5 mg L-1 was 86.55%. The maximum power density and coulomb efficiency were 109.3 mV·cm-3 and 41.97%, respectively. With the increase of antibiotic concentration, the sewage treatment efficiency and electrical performance were inhibited. This study shows that untreated livestock wastewater had a great risk of gene horizontal transfer. Although MFC had limited treatment capacity for high-concentration quinolone wastewater, it is an effective method to reduce ARGs and the risk of horizontal gene transfer.

Horizontal and vertical gene transfer drive sediment antibiotic resistome in an urban lagoon system.

Rapid urbanization has resulted in pervasive occurrence of antibiotic resistance genes (ARGs) in urban aquatic ecosystems. However, limited information is available concerning the ARG profiles and the forces responsible for their assembly in urban landscape lagoon systems. Here, we employed high-throughput quantitative PCR (HT-qPCR) to characterize the spatial variations of ARGs in surface and core sediments of Yundang Lagoon, China. The results indicated that the average richness and absolute abundance of ARGs were 11 and 53 times higher in the lagoon sediments as compared to pristine reference Tibetan lake sediments, highlighting the role of anthropogenic activities in ARG pollution. Co-occurrence network analysis indicated that various anaerobic prokaryotic genera belonging to Alpha-, Deltaproteobacteria, Bacteroidetes, Euryarchaeota, Firmicutes and Synergistetes were the potential hosts of ARGs. The partial least squares-path modeling (PLS-PM) analysis revealed positive and negative indirect effects of physicochemical factors and heavy metals on the lagoon ARG profiles, via biotic factors, respectively. The horizontal (mediated by mobile genetic elements) and vertical (mediated by prokaryotic communities) gene transfer may directly contribute the most to drive the abundance and composition of ARGs, respectively. Furthermore, the neutral community model demonstrated that the assembly of sediment ARG communities was jointly governed by deterministic and stochastic processes. Overall, this study provides novel insights into the diversity and distribution of ARGs in the benthic habitat of urban lagoon systems and underlying mechanisms for the spread and proliferation of ARGs.

High-throughput characterization of the expressed antibiotic resistance genes in sewage sludge with transcriptional analysis.

Antibiotic resistance genes (ARGs) are emerging micro-pollutants that pose potential threats to environments and humans. Sewage sludge from wastewater is an important source for ARGs and current studies mainly focus on their existence in microbial genomes. However, little is known about which ARGs are expressed even though ARGs expression remains a better proxy for functional activity. In this study, the expressed ARGs in sewage sludge were characterized by high-throughput quantitative PCR (296 primer sets) combined with transcriptional analysis. A total of 202 ARG transcripts were detected and their abundances ranged from 3.1 × 109 to 1.2 × 1010 copies/g dry weight. The sum abundance of five most abundant ARG transcripts (qacEdelta1-02, sul2, qacEdelta1-01, aadA2-03, tetX) exhibited a linear correlation with the total abundance of ARG transcripts (R2 = 0.88, p < 10-4), suggesting that these genes could be regarded as indicators to quantitatively predict the total abundance of expressed ARGs. Dynamics of expressed ARGs were observed with lower abundances in summer and winter than those in other seasons (p < 0.05, Kruskal-Wallis test). Variation partitioning analysis indicated that the shift in bacterial community structures induced by changes in environmental attributes might be the main driver for the dynamics of expressed ARGs. Results of this study provided new insights into the ARGs in sewage sludge.

Higher Temperatures Do Not Always Achieve Better Antibiotic Resistance Gene Removal in Anaerobic Digestion of Swine Manure.

This study employed high-throughput quantitative PCR and 16S rRNA sequencing to evaluate the effect of temperature and residual antibiotics on the dynamics of antibiotic resistance genes (ARGs) and microbial communities during anaerobic digestion of swine manure. The abundances of total ARGs and 16S rRNA genes significantly decreased in all of four treatments (25°C, 37°C, and 37°C with 50 mg of wet weight antibiotics of body weight, and 55°C). The abundances of most ARG types were significantly correlated with those of the 16S rRNA gene and transposase gene (P < 0.01). However, the abundances of total ARGs at 55°C were much higher than those of other treatments. Meanwhile, the microbial communities at 55°C, where the Streptococcus pathogen remained at a relatively high abundance and cellulose degraders and hydrogen producers, such as Ethanoligenens and Coprococcus bacteria, increased, were markedly different from those of other treatments. Redundancy analysis indicates that temperature, pH, and the genus Streptococcus had the highest explanation for ARG variation among experimental factors, chemical properties, and representative genera, respectively. Network analysis further showed that the genus Streptococcus contributed greatly to the higher ARG abundance at 55°C. The moderate antibiotic residue only caused a slight and transitory inhibition for microbially diverse populations and promotion for ARG abundance, probably due to the degradation of antibiotics and microbial adaptability. Our results clarify the cooperativity of gene transfer-related items on ARG variation and intensively prove that higher temperature cannot always achieve better ARG removal in anaerobic digestion unless pathogens and gene transfer elements are more efficiently inhibited.IMPORTANCE Antibiotic resistance genes (ARGs) are frequently detected with high abundance in manure-applied soils. Anaerobic digestion is one of widely used processes for animal waste treatment. Thus, it is critical to understand the potential of anaerobic digestion to attenuate ARGs. Although some previous studies recommended thermophilic digestion for ARG removal, they did not get sufficient evidence to support this view. The antibiotics applied to animals are mostly excreted through feces and urine because of incomplete metabolism. It is indispensable to know whether residual antibiotics in manure will hinder ARG attenuation in anaerobic digesters. The significance of our research is in comprehensively understanding the evolution and mechanism of ARGs in anaerobic digestion of swine manure affected by temperature and residual antibiotics, which will allow the development of an ARG elimination strategy before their release into the environment.

Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese.

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Differential impact of swine, bovine and poultry manure on the microbiome and resistome of agricultural grassland.

Land spreading of animal manure is an essential process in agriculture. Despite the importance of grassland in global food security the potential of the grass phyllosphere as a reservoir of antimicrobial resistance (AMR) is unknown. Additionally, the comparative risk associated with different manure sources is unclear. Due to the One Health nature of AMR there is an urgent need to fully understand the risk associated with AMR at the agriculture - environmental nexus. We performed a grassland field study to assess and compare the relative and temporal impact of bovine, swine and poultry manure application on the grass phyllosphere and soil microbiome and resistome over a period of four months, using 16S rRNA amplicon sequencing and high-throughput quantitative PCR (HT-qPCR). The soil and grass phyllosphere contained a diverse range of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). Manure treatment was found to introduce ARGs belonging to clinically important antimicrobial classes, such as aminoglycoside and sulphonamide into grass and soil. Temporal analysis of ARGs and MGEs associated with manure treatment indicated ARGs patterns were similar across the different manure types in the manure treated soil and grass phyllosphere. Manure treatment resulted in the enrichment in members of the indigenous microbiota and the introduction of manure associated bacteria, with this impact extending past the recommended six-week exclusion period. However, these bacteria were in low relative abundance and manure treatment was not found to significantly impact the overall composition of the microbiome or resistome. This provides evidence that the current guidelines facilitate reduction of biological risk to livestock. Additionally, in soil and grass samples MGEs correlated with ARGs from clinically important antimicrobial classes, indicating the key role MGEs play in horizontal gene transfer in agricultural grassland. These results demonstrate the role of the grass phyllosphere as an under-studied sink of AMR.

Composting reduces the risks of antibiotic resistance genes in maize seeds posed by gentamicin fermentation waste.

Using high-throughput quantitative PCR and next generation sequencing, the impact of land application of raw and composted gentamicin fermentation waste (GFW) on antibiotic resistance genes (ARGs) in maize seeds was studied in a three-year field trial. The raw and composted GFW changed both the bacterial community composition and the ARGs diversity in the maize seeds compared to non-amended controls and chemical fertilizer. The abundance of ARGs after raw GFW amendment was significantly higher than other treatments because of a high abundance of aadA1, qacEdeltal and aph(2')-Id-02; probably induced by gentamicin selection pressure in maize tissues. Meanwhile, the potential host of these three ARGs, pathogenic bacteria Tenacibaculum, also increased significantly in maize seeds after the application of raw GFW. But our result proved that composting could weaken the risk posed by GFW. We further reveal that the key biotic driver for shaping the ARG profiles in maize seeds is bacterial community followed by heavy metal resistance genes, and ARGs are more likely located on bacterial chromosomes. Our findings provide new insight into ARGs dispersal mechanism in maize seeds after long-term GFW application, demonstrate the potential benefits of composting the GFW to reduce risks as well as the potential efficient management method to GFW.

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.

Environmental filtering dominated the antibiotic resistome assembly in river networks.

River networks play important roles in dissemination of antibiotic resistance genes (ARGs). The occurrence, diversity, and abundance of ARGs in river networks have been widely investigated. However, the assembly processes that shaped ARGs profiles across space and time are largely unknown. Here, the dynamics of ARGs profiles in river networks (Taihu Basin) were revealed by high-throughput quantitative PCR followed by multiple statistical analyses to assess the underlying ecological processes. The results revealed clear variations for ARGs profiles across wet, normal, and dry seasons. Meanwhile, a significant negative correlation (p < 0.01) was observed between the similarity of ARGs profiles and geographic distance, indicating ARGs profiles exhibited distance-decay patterns. Null model analysis showed that ARGs profiles were mainly assembled via deterministic processes. Redundancy analysis followed by hierarchical partitioning revealed that environmental attributes (mainly pH and temperature) were the major factors affecting the dynamics of ARGs profiles. Together, these results indicated that environmental filtering was the dominant ecological process that shaped ARGs profiles. This study enhances our understanding how the antibiotic resistome is assembled in river networks and will be beneficial for the development of management strategies to control ARGs dissemination.

Biogeographical Patterns of Bacterial Communities and Their Antibiotic Resistomes in the Inland Waters of Southeast China.

Freshwater ecosystems are important sources of drinking water and provide natural settings for the proliferation and dissemination of bacteria and antibiotic resistance genes (ARGs). However, the biogeographical patterns of ARGs in natural freshwaters and their relationships with the bacterial community at large scales are largely understudied. This is of specific importance because data on ARGs in environments with low anthropogenic impact is still very limited. We characterized the biogeographical patterns of bacterial communities and their ARG profiles in 24 reservoirs across southeast China using 16S rRNA gene high-throughput sequencing and high-throughput-quantitative PCR, respectively. We found that the composition of both bacterial communities and ARG profiles exhibited a significant distance-decay pattern. However, ARG profiles displayed larger differences among different water bodies than bacterial communities, and the relationship between bacterial communities and ARG profiles was weak. The biogeographical patterns of bacterial communities were simultaneously driven by stochastic and deterministic processes, while ARG profiles were not explained by stochastic processes, indicating a decoupling of bacterial community composition and ARG profiles in inland waters under relatively low-human-impact at a large scale. Overall, this study provides an overview of the biogeographical patterns and driving mechanisms of bacterial community and ARG profiles and could offer guidance and reference for the control of ARGs in drinking water sources. IMPORTANCE Antibiotic resistance has been a serious global threat to environmental and human health. The "One Health" concept further emphasizes the importance of monitoring the large-scale dissemination of ARGs. However, knowledge about the geographical patterns and driving mechanisms of bacterial communities and ARGs in natural freshwater environments is limited. This study uncovered the distinct biogeographical patterns of bacterial communities and ARG profiles in inland waters of southeast China under low-anthropogenic impact at a large scale. This study improved our understanding of ARG distribution in inland waters with emphasis on drinking water supply reservoirs, therefore providing the much-needed baseline information for future monitoring and risk assessment of ARGs in drinking water resources.

Natural selenium stress influences the changes of antibiotic resistome in seleniferous forest soils.

BACKGROUND: Metal(loid)s can promote the spread and enrichment of antibiotic resistance genes (ARGs) in the environment through a co-selection effect. However, it remains unclear whether exposure of microorganisms to varying concentrations of selenium (Se), an essential but potentially deleterious metal(loid) to living organisms, can influence the migration and distribution of ARGs in forest soils. RESULTS: Precisely 235 ARGs conferring resistance to seven classes of antibiotics were detected along a Se gradient (0.06-20.65 mg kg-1) across 24 forest soils. (flor)/(chlor)/(am)phenicol resistance genes were the most abundant in all samples. The total abundance of ARGs first increased and then decreased with an elevated available Se content threshold of 0.034 mg kg-1 (P = 2E-05). A structural equation model revealed that the dominant mechanism through which Se indirectly influences the vertical migration of ARGs is by regulating the abundance of the bacterial community. In addition, the methylation of Se (mediated by tehB) and the repairing of DNA damages (mediated by ruvB and recG) were the dominant mechanisms involved in Se resistance in the forest soils. The co-occurrence network analysis revealed a significant correlated cluster between Se-resistance genes, MGEs and ARGs, suggesting the co-transfer potential. Lelliottia amnigena YTB01 isolated from the soil was able to tolerate 50 µg mL-1 ampicillin and 1000 mg kg-1 sodium selenite, and harbored both Se resistant genes and ARGs in the genome. CONCLUSIONS: Our study demonstrated that the spread and enrichment of ARGs are enhanced under moderate Se pressure but inhibited under severe Se pressure in the forest soil (threshold at 0.034 mg kg-1 available Se content). The data generated in this pilot study points to the potential health risk associated with Se contamination and its associated influence on ARGs distribution in soil.

The reduction and fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in microbial fuel cell (MFC) during treatment of livestock wastewater.

The fate and removal efficiency of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in livestock wastewater by microbial fuel cell (MFC) was evaluated by High-throughput quantitative PCR. The results showed that 137 ARGs and 9 MGEs were detected in untreated livestock wastewater. The ARG number of macrolide-lincosamide-streptogramin group B (MLSB), tetracycline and sulfonamide were relatively higher. Throughout the treatment process, the number and abundance of ARGs and MGEs significantly decreased. The relative abundance of tetracycline, sulfonamide and chloramphenicol resistance genes showed the most obvious decreasing trend, and the relative abundance of MGEs decreased by 75% (from 0.012 copies/16S rRNA copies to 0.003 copies/16S rRNA copies). However, the absolute abundance of beta-lactamase resistance genes slightly increased. The operation process of MFC produces selective pressure on microorganisms, and Actinobacteria were predominant and had the ability to decompose antibiotics. The COD removal rate and TN removal rate of livestock wastewater were 67.81% and 62.09%, and the maximum power density and coulomb efficiency (CE) reached 11.49% and 38.40% respectively. This study demonstrated that although the removal of COD and TN by MFC was limited, MFC was quite effective in reducing the risk of antibiotic toxicity and horizontal gene transfer.

Diversity, abundance and expression of the antibiotic resistance genes in a Chinese landfill: Effect of deposit age.

Landfills are the hotspots for the occurrence of antibiotic resistance genes (ARGs) in the environment. However, limited information is available on the profile of ARGs in response to the varying age of refuse in landfills. In this study, the diversity, abundance and expression of ARGs in a Chinese landfill were assessed by high-throughput quantitative PCR. A total of 154 ARGs were detected and 66% of them were transcriptionally active. The total abundance of ARG transcripts was one magnitude lower than that of ARGs. The ermT-01, tetX, sul2, aadA-02 and aadA2-03 genes were found to be the most abundant ARGs (ARG transcripts) and their sum abundance showed a linear relation with the total abundance of ARGs (ARG transcripts). The total abundance of ARGs (ARG transcripts) in young refuse was significantly higher than that in old refuse (p < 0.01) and the profile of ARGs (ARG transcripts) between the old and young refuse was distinct as revealed by the principal coordinates analysis. The variation partitioning analysis showed heavy metals (mainly Cr and Zn) were the major drivers that affect the profile of ARGs (ARG transcripts). These findings provided new insights into the ARGs in landfills and indicated their potential threats should not be neglected.

Urban ponds as hotspots of antibiotic resistome in the urban environment.

The occurrence, dissemination and assembly processes of antibiotic resistance genes (ARGs) in urban water ecosystems are far from being understood. Here, we examined the diversity and abundance of ARGs in urban water ecosystems including landscape ponds, drinking water reservoirs, influents (IFs) and effluents (EFs) of wastewater treatment plants of a coastal city, China through high-throughput quantitative PCR. A total of 237 ARGs were identified, where multidrug, aminoglycoside and beta-lactamase resistance genes were the most abundant. Urban ponds had a comparatively high diversity and large numbers of shared ARGs with IFs and EFs. The average absolute abundance of ARGs (1.38 × 107 copies/mL) and mobile genetic elements (MGEs) (4.19 × 106 copies/mL) in ponds were only one order of magnitude lower than those of IFs, but higher than those of EFs and reservoirs. Stochastic processes dominated the ARG community assembly in IFs and ponds due to the random horizontal gene transfer caused by MGEs. These results imply that urban ponds are hotspots of ARGs. We further identified 25, 3, and 11 indicator ARGs for tracing the ARG contamination from IFs, EFs and ponds, respectively. Our study represents the first to highlight the role of urban ponds in the dissemination of ARGs.

[Animal Manure Fertilization Promotes Antibiotic Resistance Gene Dissemination Among Manure, Soil, and Vegetables].

Increasing antibiotic resistant genes (ARG) are being detected in animal manure-amended soil, causing public concerns. However, the effects of animal manure fertilization on the ARG of vegetable endophytes remain unknown. In this study, high-throughput quantitative PCR (HT-qPCR) was used to explore the effects of poultry manure fertilization on the bacterial community and resistome in the soil, root endophytes, and leaf endophytes from Cichorium endivia L.. The results indicated that poultry manure fertilization not only enhanced the number of ARG in soil and root endophytes, but also increased the abundance of ARG in the soil, root endophytes, and leaf endophytes. The relationship analysis demonstrated that the ARG profile in the soil and vegetable endophytes was significantly correlated with the bacterial community, and the ARG abundance was highly correlated with Proteobacteria, Acidobacteria, Actinobacteria, and Cyanobacteria. In addition, the overlapping ARG between the Cichorium endivia L. endophyte and soil indicated the internal spread route of the ARG from the manure to Cichorium endivia L through the soil. In conclusion, poultry manure fertilization increased the diversity and abundance of the ARG in vegetables via the manure-soil-vegetable pathway.

Twenty-first century molecular methods for analyzing antimicrobial resistance in surface waters to support One Health assessments.

Antimicrobial resistance (AMR) in the environment is a growing global health concern, especially the dissemination of AMR into surface waters due to human and agricultural inputs. Within recent years, research has focused on trying to understand the impact of AMR in surface waters on human, agricultural and ecological health (One Health). While surface water quality assessments and surveillance of AMR have historically utilized culture-based methods, culturing bacteria has limitations due to difficulty in isolating environmental bacteria and the need for a priori information about the bacteria for selective isolation. The use of molecular techniques to analyze AMR at the genetic level has helped to overcome the difficulties with culture-based techniques since they do not require advance knowledge of the bacterial population and can analyze uncultivable environmental bacteria. The aim of this review is to provide an overview of common contemporary molecular methods available for analyzing AMR in surface waters, which include high throughput real-time polymerase chain reaction (HT-qPCR), metagenomics, and whole genome sequencing. This review will also feature how these methods may provide information on human and animal health risks. HT-qPCR works at the nanoliter scale, requires only a small amount of DNA, and can analyze numerous gene targets simultaneously, but may lack in analytical sensitivity and the ability to optimize individual assays compared to conventional qPCR. Metagenomics offers more detailed genomic information and taxonomic resolution than PCR by sequencing all the microbial genomes within a sample. Its open format allows for the discovery of new antibiotic resistance genes; however, the quantity of DNA necessary for this technique can be a limiting factor for surface water samples that typically have low numbers of bacteria per sample volume. Whole genome sequencing provides the complete genomic profile of a single environmental isolate and can identify all genetic elements that may confer AMR. However, a main disadvantage of this technique is that it only provides information about one bacterial isolate and is challenging to utilize for community analysis. While these contemporary techniques can quickly provide a vast array of information about AMR in surface waters, one technique does not fully characterize AMR nor its potential risks to human, animal, or ecological health. Rather, a combination of techniques (including both molecular- and culture-based) are necessary to fully understand AMR in surface waters from a One Health perspective.

Do combined nanoscale polystyrene and tetracycline impact on the incidence of resistance genes and microbial community disturbance in Enchytraeus crypticus?

It has been proved that nanoplastics can effectively adsorb pollutants and thus influence their behavior and availability. The combined toxic effects of nanoplastic and its adsorbed pollutant on the soil fauna are still not well known. We used high-throughput quantitative PCR to explore the effects of oral nanoscale polystyrene and tetracycline exposure on antibiotic resistance genes in the soil invertebrate Enchytraeus crypticus, and used bacterial 16S rRNA gene amplification sequencing to examine the response of the microbiome of E. crypticus. After 14 days of tetracycline and nanoscale polystyrene exposure, we terminated exposure and monitored the restoration of ARGs and microbiome in the E. crypticus. Results showed that the number of ARGs, especially macrolide-lincosamide-streptogramin B (MLSB), tetracycline ARGs, as well as multidrug ARGs, increased with exposure to nanoscale polystyrene and tetracycline. The abundance of Aminoglycoside and Beta_Lactamase ARGs in E. crypticus also significantly increased. The exposure significantly perturbed the abundance of families Microbacteriaceae, Streptococcaceae, Enterobacteriaceae, Rhodocyclaceae and Sphinomonadaceae. After terminating exposure for 14 days, the diversity and abundance of ARGs were not completely restored, while the microbiome was not permanently changed but reversibly impacted.