A database on the abundance of environmental antibiotic resistance genes.

Antimicrobial resistance (AMR) poses a severe threat to global health. The wide distribution of environmental antibiotic resistance genes (ARGs), which can be transferred between microbiota, especially clinical pathogens and human commensals, contributed significantly to AMR. However, few databases on the spatiotemporal distribution, abundance, and health risk of ARGs from multiple environments have been developed, especially on the absolute level. In this study, we compiled the ARG occurrence data generated by a high-throughput quantitative PCR platform from 1,403 samples in 653 sampling sites across 18 provinces in China. The database possessed 291,870 records from five types of habitats on the abundance of 290 ARGs, as well as 8,057 records on the abundance of 30 mobile genetic elements (MGEs) from 2013 to 2020. These ARGs conferred resistance to major common types of antibiotics (a total of 15 types) and represented five major resistance mechanisms, as well as four risk ranks. The database can provide information for studies on the dynamics of ARGs and is useful for the health risk assessment of AMR.

Movement of protistan trophic groups in soil-plant continuums.

Protists, functionally divided into consumers, phototrophs, and parasites act as integral components and vital regulators of microbiomes in soil-plant continuums. However, the drivers of community structure, assembly mechanisms, co-occurrence patterns, and the associations with human pathogens and different protistan trophic groups remain unknown. Here, we characterized the phyllosphere and soil protistan communities associated with three vegetables under different fertilization treatments (none and organic fertilization) at five growth stages. In this study, consumers were the most diverse soil protist group, had the role of inter-kingdom connector, and were the primary biomarker for rhizosphere soils which were subjected to decreasing deterministic processes during plant growth. In contrast, phototrophs had the greatest niche breadth and formed soil protistan hubs, and were the primary biomarkers for both bulk soils and the phyllosphere. Parasites had minimal input to microbial co-occurrence networks. Organic fertilization increased the relative abundance (RA) of pathogenic protists and the number of pathogen-consumer connections in rhizosphere soils but decreased protistan richness and the number of internal protistan links. This study advances our understanding of the ecological roles and potential links between human pathogens and protistan trophic groups associated with soil-plant continuums, which is fundamental to the regulation of soil-plant microbiomes and maintenance of environmental and human health.

Local environment, surface characteristics and stochastic processes shape the dynamics of urban dustbin surface microbiome.

Dustbins function as critical infrastructures for urban sanitation, creating a distinct breeding ground for microbial assemblages. However, there is no information regarding the dynamics of microbial communities and the underlying mechanism for community assembly on dustbin surfaces. Here, surface samples were collected from three sampling zones (business building, commercial street and residential community) with different types (kitchen waste, harmful waste, recyclables, and others) and materials (metallic and plastic); and distribution pattern and assembly of microbial communities were investigated by high-throughput sequencing. Bacterial and fungal communities showed the distinct community variations across sampling zones and waste sorting. Core community and biomarker species were significantly correlated with the spatial distribution of overall community. The detection of pathogens highlighted the potential risk of surface microbiome. Human skin, human feces and soil biomes were the potential source environments of the surface microbiomes. Neutral model prediction suggested that microbial community assembly was significantly driven by stochastic processes. Co-association patterns varied with sampling zones and waste types, and neutral amplicon sequence variants (ASVs) that fall within the 95 % confidence intervals of neutral model were largely involved in the stability of microbial networks. These findings improve our understanding of the distribution pattern and the underlying assembly of microbial community on the dustbin surface, thus enabling prospective prediction and assessment of urban microbiomes and their impacts on human health.

VFG-Chip: A high-throughput qPCR microarray for profiling virulence factor genes from the environment.

As zoonotic pathogens are threatening public health globally, the virulence factor genes (VFGs) they carry underlie latent risk in the environment. However, profiling VFGs in the environment is still in its infancy due to lack of efficient and reliable quantification tools. Here, we developed a novel high-throughput qPCR (HT-qPCR) chip, termed as VFG-Chip, to comprehensively quantify the abundances of targeted VFGs in the environment. A total of 96 VFGs from four bacterial pathogens including Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Salmonella enterica were targeted by 120 primer pairs, which were involved in encoding five types of virulence factors (VFs) like toxin, adherence, secretion system, immune evasion/invasion, and iron uptake. The specificity of VFG-Chip was both verified computationally and experimentally, with high identity of amplicon sequencing and melting curves analysis proving its robust capability. The VFG-Chip also displayed high sensitivity (by plasmid serial dilution test) and amplification efficiency averaging 97.7%. We successfully applied the VFG-Chip to profile the distribution of VFGs along a wastewater treatment system with 69 VFGs detected in total. Overall, the VFG-Chip provides a robust tool for comprehensively quantifying VFGs in the environment, and thus provides novel information in assessing the health risks of zoonotic pathogens in the environment.

Dynamics of Microbial Community and Potential Microbial Pollutants in Shopping Malls.

Shopping malls offer various niches for microbial populations, potentially serving as sources and reservoirs for the spread of microorganisms of public health concern. However, knowledge about the microbiome and the distribution of human pathogens in malls is largely unknown. Here, we examine the microbial community dynamics and genotypes of potential pathogens from floor and escalator surfaces in shopping malls and adjacent road dusts and greenbelt soils. The distribution pattern of microbial communities is driven primarily by habitats and seasons. A significant enrichment of human-associated microbiota in the indoor environment indicates that human interactions with surfaces might be another strong driver for mall microbiomes. Neutral community models suggest that the microbial community assembly is strongly driven by stochastic processes. Distinct performances of microbial taxonomic signatures for environmental classifications indicate the consistent differences of microbial communities of different seasons/habitats and the strong anthropogenic effect on homogenizing microbial communities of shopping malls. Indoor environments harbored higher concentrations of human pathogens than outdoor samples, also carrying a high proportion of antimicrobial resistance-associated multidrug efflux genes and virulence genes. These findings enhanced the understanding of the microbiome in the built environment and the interactions between humans and the built environment, providing a basis for tracking biothreats and communicable diseases and developing sophisticated early warning systems. IMPORTANCE Shopping malls are distinct microbial environments which can facilitate a constant transmission of microorganisms of public health concern between humans and the built environment or between human and human. Despite extensive investigation of the natural environmental microbiome, no comprehensive profile of microbial ecology has been reported in malls. Characterizing microbial distribution, potential pathogens, and antimicrobial resistance will enhance our understanding of how these microbial communities are formed, maintained, and transferred and help establish a baseline for biosurveillance of potential public health threats in malls.

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.

Characterizing potential pathogens from intracellular bacterial community of protists in wastewater treatment plants.

Protists are a trophically diverse and biogeochemically significant component of water environments and are widely reported as hosts of bacteria. However, the potential role of protists in wastewater treatment plants (WWTPs) as reservoirs for human pathogens does not appear to have received adequate attention. Here, a combination of fluorescence-activated cell sorting and Illumina sequencing was applied to characterize the dynamics of the internalized bacterial community of the enriched protists from the influents and effluents of five WWTPs. The results showed that Proteobacteria (mainly Betaproteobacteria) dominate the intracellular bacterial communities of protists in both influents and effluents of WWTPs, accounting for 72.6% of the total intracellular bacterial communities. The most frequently detected genus was Sulfuricurvum in the influent samples, Chryseobacterium and Pseudomonas were most prevalent in the effluent samples. Compared with the influents, a more diverse and abundant intracellular bacterial community was observed in the effluents. Moreover, the potential intracellular bacterial pathogens were 26 times higher in effluents than in influents, with Pseudomonas fluorescens and Pseudomonas putida significantly enriched in effluents. This work provides insights into the dynamics of bacterial communities and potential pathogens harbored by protists in the influents and effluents from WWTPs, contributing to the improved evaluation of biosafety in WWTPs.

Evaluation of rice straw and its transformation products on norfloxacin degradation and antibiotic resistome attenuation during soil incorporation.

Straw incorporation into reclaimed soils has been demonstrated to increase soil nutrients and has the potential to efficiently increase crop production. However, which incorporation mode is more helpful in the control of antibiotic resistance genes (ARGs) remains unknown. In this study, we systematically compared the occurrence of antibiotic resistome in norfloxacin contaminated soils amended with rice straw (RS) and the transformation products, biochar (RSB) and ash (RSA). RS significantly promoted the degradation of norfloxacin (0.0648 d-1, 3 times faster than control), whereas RSB had little effect and RSA hindered the degradation. Based on metagenomic analysis, RS and RSB significantly reduced the ARGs relative abundance (0.1421 and 0.1991 compared to 0.2540 in control) at the end of soil incubation. Adonis test indicated that all of amendment treatments significantly affect the microbial communities in soils, whereas only RS and RSB significantly affect the variation of antibiotic resistome. Procrustes analysis confirmed the association of microbial communities and ARGs. Network analysis further revealed that the reduction in Actinobacteria was the main reason for the general decrease of ARGs relative abundance during soil incorporation, whereas Proteobacteria and Bacteroidetes were responsible for temporary promotion of ARGs in RS and RSB at the early stage. Finally, scientifically setting up the usage of rice straw and optimizing the preparation process of biochar are suggested for the synchronous control of the risk of antibiotics and ARGs during soil incorporation.

Diverse functional genes harboured in extracellular vesicles from environmental and human microbiota.

Exchange of mobile functional genes within microbiota benefits the microbial community. However, the status of the mobile gene pool in environment is still largely unclear, impeding the understanding on the process of gene transfer in natural microbial communities. The release of extracellular vesicles (EVs) by diverse organisms has been proposed to be a vital way in the complex networks of interactions between microbes and their habitats. In this study, we hypothesized that microbial EVs encapsulating functional DNA are widely distributed in the environmental matrix. The prevalence, source and DNA cargoes of EVs in three types of typical microbial habitats were studied. High abundance of EVs comparable to the bacterial concentration was found in human faeces, wastewater and soil. Metagenomic analysis showed the diverse and differential taxonomy of EVs-associated DNA compared to source microbiome. An array of efficient EVs producing species was identified. A wide variety of mobile genes including glycoside hydrolase family 25 were enriched. Antibiotic resistance genes co-localizing with mobile genetic elements were abundant in the EVs. This study provides novel insights into the prevalent EVs as a reservoir for the mobile functional genes in the natural environment.

Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables.

Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1-2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and blaTEM-157 and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.

Response of soil viral communities to land use changes.

Soil viruses remain understudied when compared to virus found in aquatic ecosystems. Here, we investigate the ecological patterns of soil viral communities across various land use types encompassing forest, agricultural, and urban soil in Xiamen, China. We recovered 59,626 viral operational taxonomic units (vOTUs) via size-fractioned viromic approach with additional mitomycin C treatment to induce virus release from bacterial fraction. Our results show that viral communities are significantly different amongst the land use types considered. A microdiversity analysis indicates that selection act on soil vOTUs, resulting in disparities between land use associated viral communities. Soil pH is one of the major determinants of viral community structure, associated with changes of in-silico predicted host compositions of soil vOTUs. Habitat disturbance and variation of soil moisture potentially contribute to the dynamics of putative lysogenic vOTUs. These findings provide mechanistic understandings of the ecology and evolution of soil viral communities in changing environments.

Effects of manure fertilization on human pathogens in endosphere of three vegetable plants.

Pathogens can colonize plant endosphere and, be transferred into human beings through the food chain. However, our understanding of the influences of agricultural activities, such as fertilization, on endophytic microbial communities and human pathogens is still limited. Here, we conducted a microcosm experiment using the combination of 16 S rRNA gene amplicon sequencing and high-throughput qPCR array to reveal the effects of manure fertilization on microbiomes of soils and plants and how such impact is translated into endophytic pathogens. Our results showed that manure fertilization significantly altered soil microbiomes, whereas with less influence on endophytic microbial communities. Soil is a vital source of both bacterial communities and human pathogens for the plant endosphere. The abundance of pathogens was increased both in soils and endosphere under manure fertilization. These findings provide an integrated understanding of the impact of manure fertilization on endophytic pathogens.

Metagenomic and viromic analysis reveal the anthropogenic impacts on the plasmid and phage borne transferable resistome in soil.

Anthropogenic land use changes have been recognized with significant effects on the abundance and diversity of antibiotic resistance genes (ARGs) in soil, but their impacts on ARGs with potential health risk remained poorly understood. In this study, paired metagenomes and viromes were obtained from soils (Anthrosols and Nitisols) with different land uses including urban parks, road verge, forests, vegetable and paddy in a subtropical city, Xiamen, and soils (Anthrosols) with various long-term fertilization treatments in Dezhou located in temperate region, respectively, to explore the influence of anthropogenic activity on soil resistome. The diversity and abundance of antibiotic resistance genes (ARGs) were profiled, and the risk associated factors of ARGs, i.e., genetic location, host, and co-existence with virulence factors (VFs), were systematically investigated at reads and contigs level. We observed that agricultural areas significantly enriched human-related ARGs and viruses, and positively related with clinical ARGs. Most of the ARG-carrying contigs were chromosomes (∼85 %), while, human-related ARGs presented a higher odds ratio to locate on plasmids. Soil VFs exhibited land use pattern and distinct distribution between chromosome and plasmids, but less mobile than ARGs. Analysis of 131,014 soil viral genomes indicated that they barely encoded ARGs, nevertheless, transduction of VLPs was implicated in the spread of ARGs. The results can be mutually verified in Xiamen and Dezhou datasets. Overall, the agricultural soils with dry-farming are hotspots for the clinical ARGs, and the transmission of clinical ARGs between human dominated environments and soil is primarily mediated by plasmids, rather than bacterial chromosomes, and the transduction of human-gut related viruses could participate the process. These results highlight the importance of tracking the fate of clinical ARGs for better evaluating the impacts of human activities on soil resistome.

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.

Nanopore sequencing analysis of integron gene cassettes in sewages and soils.

Integrons are genetic elements that can facilitate rapid spread of antibiotic resistance by insertion and removal of genes. However, knowledge about the diversity and distribution of gene cassettes embedded in class 1 integron is still limited. In this study, we sequenced integron gene cassettes using nanopore sequencing and quantified antibiotic resistance genes (ARGs) and integrase genes in the manured soils and sewages of a bioreactor. The results showed that class 1 integron integrase genes were the most abundant in soils and sewages compared with class 2 and class 3 integrase genes. Long-term manure application exacerbated the enrichment of total ARGs, integrase genes and antibiotic resistance-associated gene cassettes, while antibiotics and heavy metals showed no impact on the overall resistome profile. Sewage treatment could efficiently remove the absolute abundance of integrase genes (~3 orders of magnitude, copies/L) and antibiotic resistance gene cassettes. The resistance gene cassettes mainly carried the ARGs conferring resistance to aminoglycoside and beta-lactams in soils and sewages, some of which were persistent during the sewage treatment. This study underlined that soil and sewage were potential reservoirs for integron-mediated ARGs transfer, indicating that anthropogenic activity played a vital role in the prevalence and diversity of resistance gene cassettes in integrons.

Mite gut microbiome and resistome exhibited species-specific and dose-dependent effect in response to oxytetracycline exposure.

The importance of the gut microbiome to host health is well recognized, but the effects of environmental pressures on the gut microbiome of soil fauna are poorly understood. Here, Illumina sequencing and high-throughput qPCR were applied to characterize the gut microbiomes and resistomes of two mites, Nenteria moseri and Chiropturopoda sp. AL5866, exposed to different concentrations of oxytetracycline (0, 0.01, 0.1 and 1 µg mg-1). Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the dominant phyla in the gut microbiomes of both studied mite species, but the relative abundance of them was different between mites. After exposure to oxytetracycline, there was no variation in the gut microbiome and resistome of C. sp. AL5866, whereas the gut microbiome and resistome of N. moseri were altered significantly. The relative abundance of Proteobacteria significantly decreased, and those of Bacteroidetes and Firmicutes significantly increased at the high-concentration antibiotic treatments. Excepting the 0.01 µg mg-1 treatment, gut microbial diversity increased with ascending concentrations. A significant resistome enrichment of relative abundance in N. moseri gut microbiome at low-dose antibiotic treatment was noted. These results indicated that the gut microbiome in N. moseri was potentially more sensitive to antibiotics than C. sp. AL5866, which was supported by the greater relative abundance of key tetracycline-resistant genes in the gut microbiome of C. sp. AL5866 compared to N. moseri. Mite gut microbiomes were correlated with their associated resistomes, demonstrating the consistent changes between microbiome and resistome. Thus, this study showed that oxytetracycline amendment resulted in a dose-dependent and species-specific effect on the gut microbiomes and resistomes of two mite species. It will contribute to understanding the relationship between the soil mite gut microbiome and resistome under antibiotic exposure, and extend our knowledge regarding the emergence and transfer of resistomes in soil food webs.

Earthworms reduce the dissemination potential of antibiotic resistance genes by changing bacterial co-occurrence patterns in soil.

Globally distributed earthworms affect compositions of soil compounds, microbial community structures, as well as antibiotic resistance genes (ARGs). Compared to their surroundings, earthworm gut is a simpler environment which could filter out microbes when soil passes through it. However, little is known about how earthworms affect the dissemination of ARGs in soil, and the understanding of the relationship between microbe-microbe interactions and ARGs is still lacking. Here, we designed a microcosm experiment with earthworm addition, and determined bacterial and fungal community compositions based on amplicon sequencing. We also examined mobile genetic elements (MGEs) and ARGs in earthworm gut and soils using high-throughput qPCR. The results showed significant differences of bacterial, fungal and ARG patterns between gut and soil. Earthworms indirectly impacted the patterns of ARGs in soils by affecting bacterial communities and soil properties, which play key roles in the distribution of ARGs and MGEs. The absolute abundances of MGEs in earthworm gut were significantly lower than those in soils, and earthworms reduce the absolute abundance of MGEs in soils. Earthworms changed the microbial co-occurrence patterns, and reduced bacterial connectivity, which were significantly and positively correlated with MGE abundance. These results highlight the importance of earthworm on the distribution and dissemination of ARGs in soils.

Viral Community and Virus-Associated Antibiotic Resistance Genes in Soils Amended with Organic Fertilizers.

Antibiotic resistance is a global health concern. Long-term organic fertilization can influence the antibiotic resistome of agricultural soils, posing potential risks to human health. However, little is known about the contribution of viruses to the dissemination of antibiotic resistance genes (ARGs) in this context. Here, we profiled the viral communities and virus-associated ARGs in a long-term (over 10 years) organic fertilized field by viral metagenomic analysis. A total of 61,520 viral populations (viral operational taxonomic units, vOTUs) were retrieved, of which 21,308 were assigned at the family level. The viral community structures were significantly correlated with the bacterial community structures (P < 0.001) and the dosage of applied sewage sludge (r2 = 0.782). A total of 16 unique ARGs were detected in soil viromes, and the number of virus-associated ARG subtypes was higher in sewage sludge treatments (except for 1 SS) than others. The network analysis showed that the application of the organic fertilizer increased the bacteria-virus interactions, suggesting that the chances of ARG exchange between viruses and their hosts may increase. Overall, our results provide a novel understanding about virus-associated ARGs and factors affecting the profile of viral community in fertilized soil.

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The spread of antibiotic resistance in soil is a global threat to public health and food safety, challenging the prevention and treatment of human infectious disease. The horizontal transfer of ARGs mediated by bacteriophages (phages) is an important pathway for the spread of antibiotic resistance genes (ARGs). However, the knowledge on the contribution of phages to ARGs transmission in soil is elusive. Here, we reviewed the distribution characteristics of phages in soil and its driving factors. We summarized the main methods for purification and enrichment of soil phage, reviewed recent achievements in the mechanism of phage-mediated horizontal transfer of ARGs in soil and proposed some outstanding questions. This review would contribute to understanding the important ecological role of phages in driving the horizontal transfer of ARGs, and provide a basis for developing management strategies to mitigate ARGs pollution.