Trajectory of antibiotic resistome response to antibiotics gradients: A comparative study from pharmaceutical and associated wastewater treatment plants to receiving river.

Pharmaceutical wastewater often contains significant levels of antibiotic residues, which continuously induce and promote antibiotic resistance during the sewage treatment process. However, the specific impact of antibiotics on the emergence of antibiotic resistance genes (ARGs), microbiomes, and mobile genetic elements (MGEs), as well as the dose-response relationship remain unclear. Herein, through metagenomic sequencing and analysis, we investigated the fate, transmission, and associated risk of ARGs over a ten-year period of exposure to a gradient of sulfonamide antibiotics at a pharmaceutical wastewater treatment plant (PWWTP), an associated wastewater treatment plant (WWTP), and the receiving river. Through abundance comparison and principal co-ordinates analysis (PCoA), our results revealed distinct ARG, microbiome, and MGE profiles across different antibiotic concentrations. Notably, there was a decreasing trend in the abundance of ARGs and MGEs as the antibiotic concentrations were attenuated (p < 0.05). Further partial least squares path modeling analysis, Procrustes analysis and network analysis indicated that variation in MGEs and microbiomes were the driving forces behind the distribution of ARGs. Based on these findings, we proposed an antibiotic-microbiome-MGE-ARG dissemination paradigm, in which integrons as key drivers were closely associated with prevalent ARGs such as sul1, sul2, and aadA. With a focus on human pathogenic bacteria and the associated health risks of ARGs, we conducted pathogen source analysis and calculated the antibiotic resistome risk index (ARRI). Our findings highlighted potential risks associated with the transition from PWWTP to WWTP, raising concerns regarding risk amplification due to the mixed treatment of antibiotic-laden industrial wastewater and domestic sewage. Overall, the results of our study provide valuable information for optimizing wastewater treatment practices to better manage antibiotic resistance.

Biofilm enhanced the mitigations of antibiotics and resistome in sulfadiazine and trimethoprim co-contaminated soils.

Reducing antibiotic levels in soil ecosystems is vital to curb the dissemination of antimicrobial resistance genes (ARGs) and mitigate global health threats. However, gaps persist in understanding how antibiotic resistome can be suppressed during antibiotic degradation. Herein, we investigate the efficacy of a biochar biofilm incorporating antibiotics-degrading bacterial strain (Arthrobacter sp. D2) to mitigate antibiotic resistome in non-manured and manure-amended soils with sulfadiazine (SDZ) and trimethoprim (TMP) contamination. Results show that biofilm enhanced SDZ degradation by 83.0% within three days and increased TMP attenuation by 55.4% over 60 days in non-manured soils. In the non-manured black soil, the relative abundance of ARGs increased initially after biofilm inoculation. However, by day 30, it decreased by 20.5% compared to the controls. Moreover, after 7 days, biofilm reduced TMP by 38.5% in manured soils and decreased the total ARG abundance by 19.0%. Thus, while SDZ degradation did not increase sulfonamide resistance genes, TMP dissipation led to a proliferation of insertion sequences and related TMP resistance genes. This study underscores the importance of antibiotic degradation in reducing related ARGs while cautioning against the potential proliferation and various ARGs transfer by resistant microorganisms.

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.

Microbial metabolism affects the antibiotic resistome in the intestine of laying hens.

Intestinal microbial metabolism has an important impact on the health of laying hens, and microbes are also important hosts for ARGs. However, the relationship between intestinal microbes and antibiotic resistance in laying hens is unclear. In this study, a slaughtering experiment, an in vitro fermentation experiment and a single-bacteria culture experiment were carried out, and metagenomic and metabolomic analyses were used to investigate the relationships between microbial metabolism and the antibiotic resistome in the cecum of laying hens. The results showed that there were different types of ARGs in the intestines of laying hens, and the risk scores of the ARGs tended to decrease with growth stage. A total of 1142 metagenome-assembled genomes (MAGs) were obtained, and Escherichia coli was found to be the dominant ARG host, carrying 62 ARGs. Metabolomics revealed that indole and its derivatives, such as indole-3-lactic acid, were negatively correlated with a variety of ARGs. Moreover, in vitro fermentation experiment and single-bacteria culture experiment demonstrated that indole-3-lactic acid reduced the abundance and risk of multiple ARGs in the intestine and inhibited the growth of the ARG host Escherichia coli. In the context of high concern about intestinal microbial metabolism and antibiotic resistance, this is the first study to focus on the relationship between intestinal microbial metabolism and antibiotic resistance in laying hens. These findings have important implications for healthy farming and antibiotic resistance control.

Gut antibiotic resistome during pregnancy associates with the risk of gestational diabetes mellitus: New evidence from a prospective nested case-control study.

Antibiotic resistome has emerged as a global threat to public health. However, gestational antibiotic resistome and potential link with adverse pregnancy outcomes remains poorly understood. Our study reports for the first time an association between gut antibiotic resistome during early pregnancy and the risk of gestational diabetes mellitus (GDM) based on a prospective nested case-control cohort including 120 cases and 120 matched controls. A total of 214 antibiotic resistance gene (ARG) subtypes belonging to 17 ARG types were identified in > 10 % fecal samples collected during each trimester. The data revealed dynamic profiles of gut antibiotic resistome through pregnancy, and significant positive associations between selected features (i.e., ARG abundances and a GDM-ARG score which is a new feature characterizing the association between ARGs and GDM) of gut antibiotic resistome during early pregnancy and GDM risk as well as selected endogenous metabolites. The findings demonstrate ubiquitous presence of ARGs in pregnant women and suggest it could constitute an important risk factor for the development of GDM.

The hidden threat: Comprehensive assessment of antibiotic and disinfectant resistance in commercial pig slaughterhouses.

The presence of antibiotic resistance genes (ARGs), disinfectant resistance genes (DRGs), and pathogens in animal food processing environments (FAPE) poses a significant risk to human health. However, knowledge of the contamination and risk profiles of a typical commercial pig slaughterhouse with periodic disinfectant applications is limited. By creating the overall metagenomics-based behavior and risk profiles of ARGs, DRGs, and microbiomes in a nine-section pig slaughterhouse, an important FAPE in China. A total of 454 ARGs and 84 DRGs were detected in the slaughterhouse with resistance genes for aminoglycosides and quaternary ammonium compounds, respectively. The entire slaughtering chain is a hotspot for pathogens, including 83 human pathogenic bacteria (HPB), with 47 core HPB. In addition, 68 high-risk ARGs were significantly correlated with 55 HPB, 30 of which were recognized as potential bacteria co-resistant to antibiotics and disinfectants, confirm a three-fold risk of ARGs, DRGs, and pathogens prevailing throughout the chain. Pre-slaughter pig house (PSPH) was the major risk source for ARGs, DRGs, and HPB. Moreover, 75 Escherichia coli and 47 Proteus mirabilis isolates showed sensitivity to potassium monopersulfate and sodium hypochlorite, suggesting that slaughterhouses should use such related disinfectants. By using whole genome multi-locus sequence typing and single nucleotide polymorphism analyses, genetically closely related bacteria were identified across distinct slaughter sections, suggesting bacterial transmission across the slaughter chain. Overall, this study underscores the critical role of the PSPH section as a major source of HPB, ARGs, and DRGs contamination in commercial pig slaughterhouses. Moreover, it highlights the importance of addressing clonal transmission and cross-contamination of antibiotic- and disinfectant-resistant bacteria within and between slaughter sections. These issues are primarily attributed to the microbial load carried by animals before slaughter, carcass handling, and content exposure during visceral treatment. Our findings provide valuable insights for One Health-oriented slaughterhouse management practices.

Daily occupational exposure in swine farm alters human skin microbiota and antibiotic resistome.

Antimicrobial resistance (AMR) is a major threat to global public health, and antibiotic resistance genes (ARGs) are widely distributed across humans, animals, and environment. Farming environments are emerging as a key research area for ARGs and antibiotic resistant bacteria (ARB). While the skin is an important reservoir of ARGs and ARB, transmission mechanisms between farming environments and human skin remain unclear. Previous studies confirmed that swine farm environmental exposures alter skin microbiome, but the timeline of these changes is ill defined. To improve understanding of these changes and to determine the specific time, we designed a cohort study of swine farm workers and students through collected skin and environmental samples to explore the impact of daily occupational exposure in swine farm on human skin microbiome. Results indicated that exposure to livestock-associated environments where microorganisms are richer than school environment can reshape the human skin microbiome and antibiotic resistome. Exposure of 5 h was sufficient to modify the microbiome and ARG structure in workers' skin by enriching microorganisms and ARGs. These changes were preserved once formed. Further analysis indicated that ARGs carried by host microorganisms may transfer between the environment with workers' skin and have the potential to expand to the general population using farm workers as an ARG vector. These results raised concerns about potential transmission of ARGs to the broader community. Therefore, it is necessary to take corresponding intervention measures in the production process to reduce the possibility of ARGs and ARB transmission.

Critical insights into the Hormesis of antibiotic resistome in saline soil: Implications from salinity regulation.

Soil is recognized as an important reservoir of antibiotic resistance genes (ARGs). However, the effect of salinity on the antibiotic resistome in saline soils remains largely misunderstood. In this study, high-throughput qPCR was used to investigate the impact of low-variable salinity levels on the occurrence, health risks, driving factors, and assembly processes of the antibiotic resistome. The results revealed 206 subtype ARGs across 10 categories, with medium-salinity soil exhibiting the highest abundance and number of ARGs. Among them, high-risk ARGs were enriched in medium-salinity soil. Further exploration showed that bacterial interaction favored the proliferation of ARGs. Meanwhile, functional genes related to reactive oxygen species production, membrane permeability, and adenosine triphosphate synthesis were upregulated by 6.9%, 2.9%, and 18.0%, respectively, at medium salinity compared to those at low salinity. With increasing salinity, the driver of ARGs in saline soils shifts from bacterial community to mobile gene elements, and energy supply contributed 28.2% to the ARGs at extreme salinity. As indicated by the neutral community model, stochastic processes shaped the assembly of ARGs communities in saline soils. This work emphasizes the importance of salinity on antibiotic resistome, and provides advanced insights into the fate and dissemination of ARGs in saline soils.

The mystery of rich human gut antibiotic resistome in the Yellow River with hyper-concentrated sediment-laden flow.

Human gut antibiotic resistome widely occur in anoxic environments characterized by high density of bacterial cells and frequent transmission of antibiotic resistance genes (ARGs). Such resistome is greatly diluted, degraded, and restrained in the aerobic habitats within most natural rivers (regarded as "terrestrial guts") connecting continents and the oceans. Here we implemented a large-scale monitoring campaign extending 5,200 km along the Yellow River, and provide the first integral biogeographic pattern for both ARGs and their hosts. We identified plentiful ARGs (24 types and 809 subtypes) and their hosts (24 phyla and 757 MAGs) in three media (water, suspended particulate matter (SPM), and sediment). Unexpectedly, we found diverse human gut bacteria (HGB) acting as supercarriers of ARGs in this oxygen-rich river. We further discovered that numerous microhabitats were created within stratified biofilms that surround SPMs, particularly regarding the aggregation of anaerobic HGB. These microhabitats provide numerous ideal sinks for anaerobic bacteria and facilitate horizontal transfer of ARGs within the stratified biofilms, Furthermore, the stratification of biofilms surrounding SPMs has facilitated synergy between human gut flora and denitrifiers for propagation of ARGs in the anoxic atmospheres, leading to high occurrence of human gut antibiotic resistome. SPMs play active roles in the dynamic interactions of river water and sediment, thus accelerating the evolution of riverine resistome and transmission of human gut antibiotic resistome. This study revealed the special contribution of SPMs to the propagation of ARGs, and highlighted the necessity of making alternative strategies for sustainable management of large rivers with hyper-concentrated sediment-laden flows.

Distinct Assembly Patterns of Soil Antibiotic Resistome Revealed by Land-Use Changes over 30 Years.

Compared with the ever-growing information about the anthropogenic discharge of nutrients, metals, and antibiotics on the disturbance of antibiotic resistance genes (ARGs), less is known about how the potential natural stressors drive the evolutionary processes of antibiotic resistance. This study examined how soil resistomes evolved and differentiated over 30 years in various land use settings with spatiotemporal homogeneity and minimal human impact. We found that the contents of soil organic carbon, nitrogen, soil microbial biomass, and bioavailable heavy metals, as well as related changes in the antibiotic resistome prevalence including diversity and abundance, declined in the order of grassland > cropland > bareland. Sixty-nine remaining ARGs and 14 mobile genetic elements (MGEs) were shared among three land uses. Multiple factors (i.e., soil properties, heavy metals, bacterial community, and MGEs) contributed to the evolutionary changes of the antibiotic resistome, wherein the resistome profile was dominantly driven by MGEs from both direct and indirect pathways, supported by a partial least-squares path model analysis. Our results suggest that pathways to mitigate ARGs in soils can coincide with land degradation processes, posing a challenge to the common goal of managing our environment sustainably.

Reducing transmission of high-risk antibiotic resistance genes in whole-crop corn silage through lactic acid bacteria inoculation and increasing ensiling temperature.

The microbial hosts of antibiotic resistance genes (ARGs) found epiphytically on plant materials could grow and flourish during silage fermentation. This study employed metagenomic analysis and elucidated the occurrence and transmission mechanisms of ARGs and their microbial hosts in whole-crop corn silage inoculated with homofermentative strain Lactiplantibacillus plantarum or heterofermentative strain Lentilactobacillus buchneri ensiled under different temperature (20 and 30 °C). The results revealed that the corn silage was dominated by Lactobacillus, Leuconostoc, Lentilactobacillus, and Latilactobacillus. Both the ensiling temperature and inoculation had greatly modified the silage microbiota. However, regardless of the ensiling temperature, L. buchneri had significantly higher ARGs, while it only exhibited significantly higher mobile genetic elements (MGEs) in low temperature treatments. The microbial community of the corn silage hosted highly diverse form of ARGs, which were primarily MacB, RanA, bcrA, msbA, TetA (58), and TetT and mainly corresponded to macrolides and tetracyclines drug classes. Plasmids were identified as the most abundant MGEs with significant correlation with some high-risk ARGs (tetM, TolC, mdtH, and NorA), and their abundances have been reduced by ensiling process. Furthermore, higher temperature and L. buchneri reduced abundances of high-risk ARGs by modifying their hosts and reduced their transmission in the silage. Therefore, ensiling, L. buchneri inoculation and higher storage temperature could improve the biosafety of corn silage.

Insight into environmental adaptability of antibiotic resistome from surface water to deep sediments in anthropogenic lakes by metagenomics.

The escalating antibiotic resistance threatens the long-term global health. Lake sediment is a vital hotpot in transmitting antibiotic resistance genes (ARGs); however, their vertical distribution pattern and driving mechanisms in sediment cores remain unclear. This study first utilized metagenomics to reveal how resistome is distributed from surface water to 45 cm sediments in four representative lakes, central China. Significant vertical variations in ARG profiles were observed (R2 = 0.421, p < 0.001), with significant reductions in numbers, abundance, and Shannon index from the surface water to deep sediment (all p-values < 0.05). ARGs also has interconnections within the vertical profile of the lakes: twelve ARGs persistently exist all sites and depths, and shared ARGs (e.g., vanS and mexF) were assembled by diverse hosts at varying depths. The 0-18 cm sediment had the highest mobility and health risk of ARGs, followed by the 18-45 cm sediment and water. The drivers of ARGs transformed along the profile of lakes: microbial communities and mobile genetic elements (MGEs) dominated in water, whereas environmental variables gradually become the primary through regulating microbial communities and MGEs with increasing sediment depth. Interestingly, the stochastic process governed ARG assembly, while the stochasticity diminished under the mediation of Chloroflexi, Candidatus Bathyarcaeota and oxidation-reduction potential with increasing depth. Overall, we formulated a conceptual framework to elucidate the vertical environmental adaptability of resistome in anthropogenic lakes. This study shed on the resistance risks and their environmental adaptability from sediment cores, which could reinforce the governance of public health issues.

Linking bacterial life strategies with the distribution pattern of antibiotic resistance genes in soil aggregates after straw addition.

Straw addition markedly affects the soil aggregates and microbial community structure. However, its influence on the profile of antibiotic resistance genes (ARGs), which are likely associated with changes in bacterial life strategies, remains unclear. To clarify this issue, a soil microcosm experiment was incubated under aerobic (WS) or anaerobic (AnWS) conditions after straw addition, and metagenomic sequencing was used to characterise ARGs and bacterial communities in soil aggregates. The results showed that straw addition shifted the bacterial life strategies from K- to r-strategists in all aggregates, and the aerobic and anaerobic conditions stimulated the growth of aerobic and anaerobic r-strategist bacteria, respectively. The WS decreased the relative abundances of dominant ARGs such as QnrS5, whereas the AnWS increased their abundance. After straw addition, the macroaggregates consistently exhibited a higher number of significantly altered bacteria and ARGs than the silt+clay fractions. Network analysis revealed that the WS increased the number of aerobic r-strategist bacterial nodes and fostered more interactions between r-and K-strategist bacteria, thus promoting ARGs prevalence, whereas AnWS exhibited an opposite trend. These findings provide a new perspective for understanding the fate of ARGs and their controlling factors in soil ecosystems after straw addition. ENVIRONMENTAL IMPLICATIONS: Straw soil amendment has been recommended to mitigate soil fertility degradation, improve soil structure, and ultimately increase crop yields. However, our findings highlight the importance of the elevated prevalence of ARGs associated with r-strategist bacteria in macroaggregates following the addition of organic matter, particularly fresh substrates. In addition, when assessing the environmental risk posed by ARGs in soil that receives crop straw, it is essential to account for the soil moisture content. This is because the species of r-strategist bacteria that thrive under aerobic and anaerobic conditions play a dominant role in the dissemination and accumulation of ARG.

Deciphering the removal of antibiotics and the antibiotic resistome from typical hospital wastewater treatment systems.

Hospital wastewater treatment systems (HWTSs) are a significant source and reservoir of antibiotic resistance genes (ARGs) and a crucial hub for transmitting ARGs from clinical to natural environments. However, there is a lack of research on the antibiotic resistome of clinical wastewater in HWTSs. In this study, we used metagenomics to analyze the prevalence and abundance of ARGs in five typical HWTSs. A total of 17 antibiotics from six categories were detected in the five HWTSs; ß-lactam antibiotics were found at the highest concentrations, with up to 4074.08 ng·L-1. We further found a total of 21 ARG types and 1106 subtypes of ARGs with the highest percentage of multi-drug resistance genes (evgS, msbA, arlS, and baeS). The most abundant last-resort ARGs were mcr, which were detected in 100 % of the samples. HWTSs effluent is a major pathway for the transmission of last-resort ARGs into urban wastewater networks. The removal of antibiotics, antibiotic-resistant bacteria, and ARGs from HWTSs was mainly achieved by tertiary treatment, i.e., chlorine disinfection, but antibiotics and ARGs were still present in the HWTSs effluent or even increased after treatment. Moreover, antibiotics and heavy metals (especially mercury) in hospital effluents can exert selective pressure for antibiotic resistance, even at low concentrations. Qualitative analyses based on metagenome-assembled genome analysis revealed that the putative hosts of the identified ARGs are widely distributed among Pseudomonas, Acidovorax, Flavobacterium, Polaromonas, and Arcobacter. Moreover, we further assessed the clinical availability of ARGs and found that multidrug ARGs had the highest clinical relevance values. This study provides new impulses for monitoring and removing antibiotics and ARGs in the hospital sewage treatment process.

Daily fluctuation of Lactobacillus species and their antibiotic resistome in the colon of growing pigs.

There are various types of bacteria inhabiting the intestine that help maintain the balance of the intestinal microbiota. Lactobacillus is one of the important beneficial bacteria and is widely used as a food starter and probiotic. In this study, we investigated the daily fluctuation of the colonic Lactobacillus species and their distribution of antibiotic resistance genes (ARGs) as well as antibiotic susceptibility in pigs. Metagenomic analysis revealed that genus Lactobacillus was one of the most dominant genera in the colon of growing pigs. Rhythmicity analysis revealed that 84 out of 285 Lactobacillus species exhibited rhythmic patterns. Lactobacillus johnsonii and Lactobacillus reuteri were the two most abundant lactobacilli with circadian oscillation, which increased during the day and decreased at night. The profile of the antibiotic resistome was modified over time within 24-h period. Elfamycin resistance genes were the most enriched class found in Lactobacillus. Furthermore, the seven strains of Lactobacillus isolated from the pig intestine mainly exhibited resistance to gentamicin, erythromycin, and lincomycin. The whole genome annotation of four Lactobacillus strains indicated the presence of multiple ARGs, including elfamycin resistance genes, however, the most abundant ARG was optrA in genome of four strains. These results indicate the presence of various Lactobacillus species harboring a large number of ARGs in the swine intestine. This implies that when using animal-derived lactobacilli, it is essential to assess antibiotic resistance to prevent further transmission between animals and the environment.

Long-term application of organic fertilizer prompting the dispersal of antibiotic resistance genes and their health risks in the soil plastisphere.

Microplastic (MP) pollution is a rapidly growing global environmental concern that has led to the emergence of a new environmental compartment, the plastisphere, which is a hotspot for the accumulation of antibiotic resistance genes (ARGs) and human bacterial pathogens (HBPs). However, studies on the effects of long-term organic fertilizer application on the dispersal of ARGs and virulence factor genes (VFGs) in the plastisphere of farmland soil have been limited. Here, we performed a field culture experiment by burying nylon bags filled with MPs in paddy soil that had been treated with different fertilizers for over 30 years to explore the changes of ARGs and VFGs in soil plastisphere. Our results show that the soil plastisphere amplified the ARG and VFG pollution caused by organic fertilization by 1.5 and 1.4 times, respectively. And it also led to a 2.7-fold increase in the risk of horizontal gene transfer. Meanwhile, the plastisphere tended to promote deterministic process in the community assembly of HBPs, with an increase of 1.4 times. Network analysis found a significant correlation between ARGs, VFGs, and bacteria in plastisphere. Correlation analysis highlight the important role of mobile genetic elements (MGEs) and bacterial communities in shaping the abundance of ARGs and VFGs, respectively. Our findings provide new insights into the health risk associated with the soil plastisphere due ARGs and VFGs derived from organic fertilizers.

Meta-analysis addressing the characterization of antibiotic resistome in global hospital wastewater.

Hospital wastewater (HWW) is a significant environmental reservoir of antibiotic resistance genes (ARGs). However, currently, no comprehensive understanding exists of the antibiotic resistome in global HWW. In this study, we attempted to address this knowledge gap through an in silico reanalysis of publicly accessible global HWW metagenomic data. We reanalyzed ARGs in 338 HWW samples from 13 countries in Africa, Asia, and Europe. In total, 2420 ARG subtypes belonging to 30 ARG types were detected, dominated by multidrug, beta-lactam, and aminoglycoside resistance genes. ARG composition in Europe differed from that in Asia and Africa. Notably, the ARGs presented co-occurrence with mobile genetic elements (MGEs), metal resistance genes (MRGs), and human bacterial pathogens (HBP), indicating a potential dissemination risk of ARGs in the HWW. Multidrug resistance genes presented co-occurrence with MGEs, MRGs, and HBP, is particularly pronounced. The abundance of contigs that contained ARG, contigs that contained ARG and HBP, contigs that contained ARG and MGE, contigs that contained ARG and MRG were used for health and transmission risk assessment of antibiotic resistome and screened out 40 high risk ARGs in the global HWW. This study first provides a comprehensive characterization and risk of the antibiotic resistome in global HWW.

Insights into the circular: The cryptic plasmidome and its derived antibiotic resistome in the urban water systems.

Plasmids have been a concern in the dissemination and evolution of antibiotic resistance in the environment. In this study, we investigated the total pool of plasmids (plasmidome) and its derived antibiotic resistance genes (ARGs) in different compartments of urban water systems (UWSs) in three European countries representing different antibiotic usage regimes. We applied a direct plasmidome approach using wet-lab methods to enrich circular DNA in the samples, followed by shotgun sequencing and in silico contig circularisation. We identified 9538 novel sequences in a total of 10,942 recovered circular plasmids. Of these, 66 were identified as conjugative, 1896 mobilisable and 8970 non-mobilisable plasmids. The UWSs' plasmidome was dominated by small plasmids (≤10 Kbp) representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups. A shared collection of plasmids from different countries was detected in all treatment compartments, and plasmids could be source-tracked in the UWSs. More than half of the ARGs-encoding plasmids carried mobility genes for mobilisation/conjugation. The richness and abundance of ARGs-encoding plasmids generally decreased with the flow, while we observed that non-mobilisable ARGs-harbouring plasmids maintained their abundance in the Spanish wastewater treatment plant. Overall, our work unravels that the UWS plasmidome is dominated by cryptic (i.e., non-mobilisable, non-typeable and previously unknown) plasmids. Considering that some of these plasmids carried ARGs, were prevalent across three countries and could persist throughout the UWSs compartments, these results should alarm and call for attention.

Swine farming shifted the gut antibiotic resistome of local people.

Antibiotic resistance genes (ARGs) are prevalent in the livestock environment, but little is known about impacts of animal farming on the gut antibiotic resistome of local people. Here we conducted metagenomic sequencing to investigate gut microbiome and resistome of residents in a swine farming village as well as environmental relevance by comparing with a nearby non-farming village. Results showed a shift of gut microbiome towards unhealthy status in the residents of swine farming village, with an increased abundance and diversity in pathogens and ARGs. The resistome composition in human guts was more similar with that in swine feces and air than that in soil and water. Mobile gene elements were closely associated with the prevalence of gut resistome. Some plasmid-borne ARGs were colocalized in similar genetic contexts in gut and environmental samples. Metagenomic binning obtained 47 ARGs-carrying families in human guts, and therein Enterobacteriaceae posed the highest threats in antibiotic resistance and virulence. Several ARGs-carrying families were shared by gut and environmental samples (mainly in swine feces and air), and the ARGs were evolutionarily conservative within genera. The findings highlight that swine farming can shape gut resistome of local people with close linkage to farm environmental exposures.

Microbial-influenced pesticide removal co-occurs with antibiotic resistance gene variation in soil-earthworm-maize system.

Within human-influenced landscapes, pesticides cooccur with a variety of antibiotic stressors. However, the relationship between pesticides removal process and antibiotic resistance gene variation are not well understood. This study explored pesticide (topramezone, TPZ) and antibiotic (polymyxin E, PME) co-contamination using liquid chromatography-tandem mass spectrometry (LC-MS/MS), bacterial-16 S rRNA sequencing and high-throughput quantitative polymerase chain reaction (HT-qPCR) in a soil-earthworm-maize system. After incubating soil for 28 days with TPZ and PME (10 mg kg-1 dry weight), earthworm weight-gain, mortality rates, and maize plant weight-gain only differed slightly, but height-gain significantly decreased. PME significantly increased TPZ-removal in the soil. Accumulation of TPZ in earthworm's tissues may pose potential risks in the food chain. Combined pollution altered the microbial community structure and increased the abundance of functional microorganisms involved in aromatic compound degradation. Furthermore, maize rhizosphere can raise resistance genes, however earthworms can reduce resistance genes. Co-contamination increased absolute abundance of mobile genetic elements (MGEs) in bulk-soil samples, antibiotic resistance genes (ARGs) in skin samples and number of ARGs in bulk-soil samples, while decreased absolute abundance of transposase gene in bulk-soil samples and number of ARGs in rhizosphere-soil samples. Potential hosts harbouring ARGs may be associated with the antagonistic effect during resistance and detoxification of TPZ and PMB co-occurrence. These findings provide insights into the mechanism underlining pesticide removal regarding occurrence of ARGs in maize agroecosystem.