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Natural environments play a crucial role in transmission of antimicrobial resistance (AMR). Development of methods to manage antibiotic resistance genes (ARGs) in natural environments are usually limited to the laboratory or field scale, partially due to the complex dynamics of transmission between different environmental compartments. Here, we conducted a nine-year longitudinal profiling of ARGs at a watershed scale, and provide evidence that restrictions on livestock farms near water bodies significantly reduced riverine ARG abundance. Substantial reductions were revealed in the relative abundance of genes conferring resistance to aminoglycosides (42%), MLSB (36%), multidrug (55%), tetracyclines (53%), and other gene categories (59%). Additionally, improvements in water quality were observed, with distinct changes in concentrations of dissolved reactive phosphorus, ammonium, nitrite, pH, and dissolved oxygen. Antibiotic residues and other pharmaceuticals and personal care products (PPCPs) maintain at a similarly low level. Microbial source tracking demonstrates a significant decrease in swine fecal indicators, while human fecal pollution remains unchanged. These results suggest that the reduction in ARGs was due to a substantial reduction in input of antibiotic resistant bacteria and genes from animal excreta. Our findings highlight the watershed as a living laboratory for understanding the dynamics of AMR, and for evaluating the efficacy of environmental regulations, with implications for reducing environmental risks associated with AMR on a global scale.

Summary: Oyster reefs are hotspots of denitrification mediated removal of dissolved nitrogen (N), however, information on their denitrifier microbiota is scarce. Furthermore, in oyster aquaculture, triploids are often preferred over diploids, yet again, microbiome differences between oyster ploidies are unknown. To address these knowledge gaps, farmed diploid and triploid oysters were collected over an annual growth cycle and analyzed using shotgun metagenomics and quantitative microbial elemental cycling (QMEC) techniques. Regardless of ploidy, Psychrobacter genus was abundant, with positive correlations found for genes of central metabolism, DNA metabolism, and carbohydrate metabolism. MAGs (metagenome-assembled genomes) yielded multiple Psychrobacter genomes harboring norB, narH, narI, and nirK denitrification genes, indicating their functional relevance within the eastern oysters. QMEC analysis indicated the predominance of carbon (C) and nitrogen (N) cycling genes, with no discernable patterns between ploidies. Among the N-cycling genes, the nosZII clade was overrepresented, suggesting its role in the eastern oyster’s N removal processes.

Abstract 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.

Rapid urbanization in the Asia-Pacific region is expected to place two-thirds of its population in concrete-dominated urban landscapes by 2050. While diverse architectural facades define the unique appearance of these urban systems. There remains a significant gap in our understanding of the composition, assembly, and ecological potential of microbial communities on building exteriors. Here, we examined bacterial and protistan communities on building surfaces along an urbanization gradient (urban, suburban and rural regions), investigating their spatial patterns and the driving factors behind their presence. A total of 55 bacterial and protist phyla were identified. The bacterial community was predominantly composed of Proteobacteria (33.7% to 67.5%). The protistan community exhibited a prevalence of Opisthokonta and Archaeplastida (17.5% to 82.1% and 1.8% to 61.2%, respectively). The composition and functionality of bacterial communities exhibited spatial patterns correlated with urbanization. In urban buildings, factors such as facade type, light exposure, and building height had comparatively less impact on bacterial composition compared to suburban and rural areas. The highest bacterial diversity and lowest Weighted Average Community Identity (WACI) were observed on suburban buildings, followed by rural buildings. In contrast, protists did not show spatial distribution characteristics related to facade type, light exposure, building height and urbanization level. The distinct spatial patterns of protists were primarily shaped by community diffusion and the bottom-up regulation exerted by bacterial communities. Together, our findings suggest that building exteriors serve as attachment points for local microbial metacommunities, offering unique habitats where bacteria and protists exhibit independent adaptive strategies closely tied to the overall ecological potential of the community.

Abstract The environment disseminates antimicrobial-resistance genes; however, it remains challenging to distinguish whether human activities exacerbate antimicrobial resistance or what is natural. Here, we quantified ~300 resistance-related genes in 200+ Scottish soil samples. Location or land use does not explain gene differences, but nutrient levels reduce gene richness. Elevated levels of metals increased gene richness, and selenium increased transposase levels. Rainfall and persistent organic pollutants also increased transposase relative abundance, possibly promoting conditions conducive to the horizontal transfer of antimicrobial-resistance genes. Selenium and polychlorinated biphenyls were primary factors in gene abundance, while polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and pH influenced gene diversity. Polychlorinated biphenyls are derived from anthropogenic activities, highlighting human activities’ potential impact on gene prevalence. This is the first national-scale, high spatial resolution dataset of antimicrobial-resistance genes in Scottish soils and provides a novel resource on which to build future studies.

Abstract Airborne microorganisms, including pathogens, would change with surrounding environments and become issues of global concern due to their threats to human health. Microbial communities typically contain a few abundant but many rare species. However, how the airborne abundant and rare microbial communities respond to environmental changes is still unclear, especially at hour scale. Here, we used a sequencing approach based on bacterial 16S rRNA genes and fungal ITS2 regions to investigate the high time‐resolved dynamics of airborne bacteria and fungi and to explore the responses of abundant and rare microbes to the atmospheric changes. Our results showed that air pollutants and microbial communities were significantly affected by human activities related to the Chinese New Year (CNY). Before CNY, significant hour‐scale changes in both abundant and rare subcommunities were observed, while only abundant bacterial subcommunity changed with hour time series during CNY. Air pollutants and meteorological parameters explained 61.5%−74.2% variations of abundant community but only 13.3%−21.6% variations of rare communities. These results suggested that abundant species were more sensitive to environmental changes than rare taxa. Stochastic processes predominated in the assembly of abundant communities, but deterministic processes determined the assembly of rare communities. Potential bacterial pathogens during CNY were the highest, suggesting an increased health risk of airborne microbes during CNY. Overall, our findings highlighted the “holiday effect” of CNY on airborne microbes and expanded the current understanding of the ecological mechanisms and health risks of microbes in a changing atmosphere.

Soil viral communities remain understudied. Here, Liao et al. retrieve a catalogue of around sixty thousand vOTUs through a systematic viromic pipeline, and uncover the response of soil viral communities to anthropogenic land use changes.

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.

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

ABSTRACT 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.

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.

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.

Abstract 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.

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

Community and composition of dust-borne microbes would affect human health and are regulated by microbial community assembly. The dust in kindergarten is always collected to evaluate the microbial exposure of children, yet the microbial assembly, their interactions, and potential pathogens in kindergarten dust remain unclear. Here, we aim to investigate the microbial community assembly and structures, and potential bacterial pathogens in outdoor dust of kindergartens, and reveal the factors influencing the assembly and composition of microbial community. A total of 118 urban dust samples were collected on the outdoor impervious surfaces of 59 kindergartens from different districts of Xiamen in January and June 2020. We extracted microbial genomic DNA in these dusts and characterized the microbial (i.e., bacteria and fungi) community compositions and diversities using target gene-based (16S rRNA genes for bacterial community and ITS 2 regions for fungal community) high-throughput sequencing. Potential bacterial pathogens were identified and the interactions between microbes were determined through a co-occurrence network analysis. Our results showed the predominance of Actinobacteria and α-Proteobacteria in bacterial communities and Capnodiales in fungal communities. Season altered microbial assembly, composition, and interactions, with both bacterial and fungal communities exhibiting a higher heterogeneity in summer than those in winter. Although stochastic processes predominated in bacterial and fungal community assembly, the season-depended environmental factors (e.g., temperature) and interactions between microbes play important roles in dust microbial community assembly. Potential bacterial pathogens were detected in all urban dust, with significantly higher relative abundance in summer than that in winter. These results indicated that season exerted more profound effects on microbial community composition, assembly, and interactions, and suggested the seasonal changes of potential risk of microbes in urban dust. Our findings provide new insights into microbial community, community assembly, and interactions between microbes in the urban dust, and indicate that taxa containing opportunistic pathogens occur commonly in urban dust.

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.

Antibiotic-resistance genes (ARGs) and resistant bacteria (ARB) are abundant in stormwater that could cause serious infections, posing a potential threat to public health. However, there is no inference about how stormwater contributes to ARG profiles as well as the dynamic interplay between ARGs and bacteria via vertical gene transfer (VGT) or horizontal gene transfer (HGT) in urban water ecosystems. In this study, the distribution of ARGs, their host communities, and the source and community assembly process of ARGs were investigated in Yundang Lagoon (China) via high-throughput quantitative PCR, 16S rRNA gene amplicon sequencing, and application of SourceTracker before, after and recovering from an extreme precipitation event (132.1 mm). The abundance of ARGs and mobile genetic elements (MGEs) was the highest one day after precipitation and then decreased 2 days after precipitation and so on. Based on SourceTracker and NMDS analysis, the ARG and bacterial communities in lagoon surface water from one day after precipitation were mainly contributed by the wastewater treatment plant (WWTP) influent and effluent. However, the contribution of WWTP to ARG communities was minor 11 days after the precipitation, suggesting that the storm promoted the ARG levels by introducing the input of ARGs, MGEs, and ARB from point and non-point sources, such as sewer overflow and land-applied manure. Based on a novel microbial network analysis framework, the contribution of positive biological interactions between ARGs and MGEs or bacteria was the highest one day after precipitation, indicating a promoted VGT and HGT for ARG dissemination. The microbial networks deconstructed 11 days after precipitation, suggesting the stormwater practices (e.g., tide gate opening, diversion channels, and pumping) alleviated the spread of ARGs. These results advanced our understanding of the distribution and transport of ARGs associated with their source in urban stormwater runoff.

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 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.

Abstract Background Therapeutic and growth-promoting antibiotics are frequently used in broiler production. Indirect evidence indicates that these practices are linked to the proliferation of antimicrobial resistance (AMR), the spread of antibiotic-resistant bacteria from food animals to humans, and the environment, but there is a lack of comprehensive experimental data supporting this. We investigated the effects of growth promotor (bacitracin) and therapeutic (enrofloxacin) antibiotic administration on AMR in broilers for the duration of a production cycle, using a holistic approach that integrated both culture-dependent and culture-independent methods. We specifically focused on pathogen-harboring families (Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae). Results Antibiotic-resistant bacteria and antibiotic resistance genes were ubiquitous in chicken cloaca and litter regardless of antibiotic administration. Environment (cloaca vs. litter) and growth stage were the primary drivers of variation in the microbiomes and resistomes, with increased bacterial diversity and a general decrease in abundance of the pathogen-harboring families with age. Bacitracin-fed groups had higher levels of bacitracin resistance genes and of vancomycin-resistant Enterococcaceae (total Enterococcaceae counts were not higher). Although metagenomic analyses classified 28–76% of the Enterococcaceae as the commensal human pathogens E. faecalis and E. faecium, culture-based analysis suggested that approximately 98% of the vancomycin-resistant Enterococcaceae were avian and not human-associated, suggesting differences in the taxonomic profiles of the resistant and non-resistant strains. Enrofloxacin treatments had varying effects, but generally facilitated increased relative abundance of multidrug-resistant Enterobacteriaceae strains, which were primarily E. coli. Metagenomic approaches revealed a diverse array of Staphylococcus spp., but the opportunistic pathogen S. aureus and methicillin resistance genes were not detected in culture-based or metagenomic analyses. Camphylobacteriaceae were significantly more abundant in the cloacal samples, especially in enrofloxacin-treated chickens, where a metagenome-assembled C. jejuni genome harboring fluoroquinolone and β-lactam resistance genes was identified. Conclusions Within a “farm-to-fork, one health” perspective, considering the evidence that bacitracin and enrofloxacin used in poultry production can select for resistance, we recommend their use be regulated. Furthermore, we suggest routine surveillance of ESBL E. coli, vancomycin-resistant E. faecalis and E. faecium, and fluoroquinolone-resistant C. jejuni strains considering their pathogenic nature and capacity to disseminate AMR to the environment. Video Abstract

Farmers apply broiler chicken litter to soils to enrich organic matter and provide crops with nutrients, following varying periods of stockpiling. However, litter frequently harbors fecal-derived microbial pathogens and associated antibiotic resistance genes (ARGs), and may be a source of microbial contamination of produce. We coupled a cutting-edge Loop Genomics long-read 16S rRNA amplicon-sequencing platform with high-throughput qPCR that targeted a suite of ARGs, to assess temporal (five time points over a 60-day period) and spatial (top, middle and bottom layers) microbiome and resistome dynamics in a broiler litter stockpile. We focused on potentially pathogenic species from the Enterobacteriaceae, Enterococcaceae and Staphylococcaceae families associated with food-borne disease. Bacterial diversity was significantly lower in the middle of the stockpile, where targeted pathogens were lowest and Bacillaceae were abundant. E. coli was the most abundant Enterobacteriaceae species, and high levels of the opportunistic pathogen Enterococcus faecium were detected. Correlation analyses revealed that the latter was significantly associated with aminoglycoside (aac(6′)-Ib(aka aacA4), aadA5), tetracycline (tetG), vancomycin (vanC), phenicol (floR) and MLSB (mphB) resistance genes. Staphylococcaceae were primarily non-pathogenic, but extremely low levels of the opportunistic pathogen S. aureus were detected, as was the opportunistic pathogen S. saprophyticus, which was linked to vancomycin (vanSA, vanC1), MLSB (vatE, ermB) and tetracycline (tetK) resistance genes. Collectively, we found that stockpile microbiomes and resistomes are strongly dictated by temporal fluctuations and spatial heterogeneity. Insights from this study can be exploited to improve stockpile management practice to support sustainable antimicrobial resistance mitigation policies in the future.

Anaerobic ammonium oxidation coupled to nitrite reduction (termed as Anammox) was demonstrated as an efficient pathway to remove nitrogen from a wastewater treatment system. Recently, anaerobic ammonium oxidation was also identified to be linked to iron(III) reduction (termed Feammox) with dinitrogen, nitrite, or nitrate as end-product, reporting to enhance nitrogen removal from the wastewater treatment system. However, little is known about the role of Anammox bacteria in the Feammox process. Here, slurry from wastewater reactor amended with ferrihydrite was employed to investigate activity of Anammox bacteria in the Feammox process using the 15N isotopic tracing technique combined with 16S rRNA gene amplicon sequencing. A significantly positive relationship between rates of 15N2 production and iron(III) reduction indicated the occurrence of Feammox during incubation. Relative abundances of Anammox bacteria including Brocadia, Kuenenia, Jettenia, and unclassified Brocadiaceae were detected with low relative abundances, whereas Geobacteraceae dominated in the treatment throughout the incubation. 15N2 production rates significantly positively correlated with relative abundances of Geobacter, unclassified Geobacteraceae, and Anammox bacteria, revealing their contribution to nitrogen generation via Feammox. Overall, these findings suggested Anammox bacteria or cooperation between Anammox bacteria and iron(III) reducers serves a potential role in Feammox process.

Urban green space has been implicated in shaping airborne microbes, but there is an only rudimentary understanding of the key factors of urban green space affecting the composition and structures of airborne microbes. Here, we selected 40 urban sites based on stratified random sampling design and investigated the effects of multiple factors including landscapes, plant, soil, and anthropogenic factors on airborne microbial communities, especially bacterial and fungal pathogens. Bacterial and fungal communities in the control area with lower greenness were significantly (P 30% masson pine. Together, these results provide insights into the importance of green space for providing health benefits for city dwellers by reducing pathogens in air, as well as providing support for the inclusion of plant species in the management of urban green space to reduce exposure risk of airborne pathogens.

Conjunctive transfer of antibiotic resistance genes (ARGs) among bacteria driven by plasmids facilitated the evolution and spread of antibiotic resistance. Heavy metal exposure accelerated the plasmid-mediated conjunctive transfer of ARGs. Nanomaterials are well-known adsorbents for heavy metals removal, with the capability of combatting resistant bacteria/facilitating conjunctive transfer of ARGs. However, co-effect of heavy metals and nanomaterials on plasmid-mediated conjunctive transfer of ARGs was still unknown. In this study, we investigated the effect of the simultaneous exposure of Cd2+ and nano Fe2O3 on conjugative transfer of plasmid RP4 from Pseudomonas putida KT2442 to water microbial community. The permeability of bacterial cell membranes, antioxidant enzyme activities and conjugation gene expression were also investigated. The results suggested that the combination of Cd2+ and high concentration nano Fe2O3 (10 mg/L and 100 mg/L) significantly increased conjugative transfer frequencies of RP4 plasmid (p

The phyllosphere is considered a key site for the transfer of both naturally and anthropogenically selected antimicrobial resistance genes (ARGs) to humans. Consequently, the development of green building systems may pose an, as yet, unexplored pathway for ARGs and pathogens to transfer from the environment to outdoor plants. We collected leaves from plants climbing up buildings at 1, 2, 4 and 15 m above ground level and collected associated dust samples from adjacent windowsills to determine the diversity and relative abundance of microbiota and ARGs. Overall, a total of 143 ARGs from 11 major classes and 18 mobile genetic elements (MGEs) were detected. The relative abundance of ARGs within the phyllosphere decreased with increasing height above ground level. Fast expectation–maximization microbial source tracking (FEAST) suggested that the contribution of soil and aerosols to the phyllosphere microbiome was limited. A culture-dependent method to isolate bacteria from plant tissues identified a total of 91 genera from root, stem, and leaf samples as well as endophytes isolated from leaves. Of those bacteria, 20 isolates representing 9 genera were known human pathogenic members to humans. Shared bacterial from culture-dependent and culture-independent methods suggest microorganisms may move from soil to plant, potentially through an endophytic mechanism and thus, there is a clear potential for movement of ARGs and human pathogens from the outdoor environment.

Increasing introduction of antibiotic residues from humans and animal farming into the environment impacts the functioning of natural ecosystems and significantly contributes to the propagation of antibiotic resistance. Microbial degradation is the major sink of antibiotics in soil but the identification of in situ degrading populations is challenging. Here, we investigated sulfamethoxazole-degrading bacteria in soil microcosms by culture-independent DNA and protein stable isotope probing. 0.5% of the carbon from 13C6-labeled sulfamethoxazole amended to soil microcosms was transformed to 13CO2 demonstrating partial mineralization of the antibiotic. DNA stable isotope probing revealed incorporation of 13C from 13C6-labeled sulfamethoxazole into Actinobacteria and among them into the families Intrasporangiaceae, Nocardioidaceae, and Gaiellaceae and the order Solirubrobacterales. Protein stable isotope probing demonstrated the incorporation of 13C from 13C6-labeled sulfamethoxazole into proteins of bacteria of the families Intrasporangiaceae, Nocardioidaceae and the order Solirubrobacterales, which is consistent with the results of DNA stable isotope probing. The 13C abundance of 60 to 80% in several taxonomically relevant proteins indicated that Intrasporangiaceae directly acquired carbon from 13C6-labeled sulfamethoxazole. The results highlight the crucial role of yet-uncultivated indigenous bacteria for antibiotics degradation, and the potential of cultivation-independent stable isotope based molecular approaches to elucidate the structure of antibiotic-degrading populations in complex microbial communities under natural conditions. Keywords: Antibiotics, Degradation, Stable isotope probing (SIP), Actinobacteria, Intrasporangiaceae, Soil

Soils in tropical and temperate locations are known to be a sink for the genetic potential of anthropogenic-driven acquired antibiotic resistance (AR). In contrast, accumulation of acquired AR is less probable in most Polar soils, providing a platform for characterizing background resistance and establishing a benchmark for assessing AR spread. Here, high-throughput qPCR and geochemistry were used to quantify the abundance and diversity of both antibiotic resistance genes (ARGs) and selected mobile genetic elements (MGEs) across eight soil clusters in the Kongsfjorden region of Svalbard in the High Arctic. Relative ARG levels ranged by over two orders of magnitude (10−6 to 10−4 copies/16S rRNA gene copy), and showed a gradient of potential human and wildlife impacts across clusters as evidenced by altered geochemical conditions and increased “foreign” ARG abundances (i.e., allochthonous), including blaNDM-1. Impacted clusters exhibited 100× higher total ARGs and MGEs in tandem with elevated secondary nutrients, especially available P that is typically low and limiting in Arctic soils. In contrast, ARGs in less-impacted clusters correlated strongly to local soil lithology. The most plausible source of exogenous P and allochthonous ARGs in this region is bird and other wildlife guano, disseminated either by local human wastes or via direct carriage and deposition. Regardless of pathway, accumulation of apparent allochthonous ARGs and MGEs in High Arctic soils is concerning, highlighting the importance of characterizing Arctic sites now to establish benchmarks for tracking AR spread around the world. Keywords: High Arctic, Antibiotic resistance, International spread, Wildlife, Geochemistry, High throughput qPCR

The phyllosphere is populated by numerous microorganisms. Microbes from the wider environment, i.e., air and soil, are considered key contributors to phyllosphere microbial communities, but their contribution is unclear. This study seeks to address this knowledge gap by controlling the movement of microbes along the air-phyllosphere-soil continuum. Customized equipment with dual chambers was constructed that permitted airflow to enter the first chamber while the second chamber recruited filtered microbe-free air from the initial chamber. Allium schoenoprasum (chive) and Sonchus oleraceus (sow thistle) were cultivated in both chambers, and the microbial communities from air, phyllosphere, and soil samples were characterized. Shares of microbial OTUs in the equipment suggested a potential interconnection between the air, phyllosphere, and soil system. Fast expectation-maximization microbial source tracking (FEAST) suggested that soil was the major source of airborne microbial communities. In contrast, the contribution of airborne and soil microbes to phyllosphere microbial communities of either A. schoenoprasum or S. oleraceus was limited. Notably, the soilborne microbes were the only environmental sources to phyllosphere in the second chamber and could affect the composition of phyllosphere microbiota indirectly by air flow. The current study demonstrated the possible sources of phyllosphere microbes by controlling external airborne microbes in a designed microcosm system and provided a potential strategy for recruitment for phyllosphere recruitment.

Antibiotic resistance genes (ARGs) are widespread in aquatic environments, but we know little about their biogeographical distribution and occurrence at national scales. Here we analyzed the patterns of ARGs from 42 natural waterbodies (natural lakes and reservoirs) across China using high-throughput approaches. The major ARGs were multidrug genes and the main resistance mechanism was the efflux pump. Although the absolute abundance of ARGs (gene copies/L) in the south/central waterbodies was similar to the northern waterbodies, the normalized abundance of ARGs (ARGs/16S rRNA gene copy number) was higher in the south/central waterbodies than in the north (mainly because of the aminoglycoside and multidrug resistance genes). Human activities strongly correlated with the normalized abundance of ARGs. The composition of ARGs in the waterbodies of south/central China was different from that in the north, and ARGs showed a distance-decay relationship. Anthropogenic factors had the most significant effects on this spatial distribution of ARG composition, followed by the spatial, bacterial and physicochemical factors. These indicate that the ARGs exhibited biogeographical patterns and that multiple ecological mechanisms - such as environmental selection (human activities and local physicochemical parameters) and dispersal limitation - influence distribution of ARGs in these waters. In general, our results provide a valuable ecological insight to explain the large-scale dispersal patterns in ARGs, thereby having potential applications for both public health and environmental management. Keywords: Antibiotic resistance genes, Functional biogeography, Anthropogenic activities, Community ecology, Geographical distance, Multidrug resistance gene, Lake and reservoir

Abstract Background Antibiotic-resistant pathogens are challenging treatment of infections worldwide. Urban sewage is potentially a major conduit for dissemination of antibiotic resistance genes into various environmental compartments. However, the diversity and abundance of such genes in wastewater are not well known. Methods Here, seasonal and geographical distributions of antibiotic resistance genes and their host bacterial communities from Chinese urban sewage were characterized, using metagenomic analyses and 16S rRNA gene-based Illumina sequencing, respectively. Results In total, 381 different resistance genes were detected, and these genes were extensively shared across China, with no geographical clustering. Seasonal variation in abundance of resistance genes was observed, with average concentrations of 3.27 × 1011 and 1.79 × 1012 copies/L in summer and winter, respectively. Bacterial communities did not exhibit geographical clusters, but did show a significant distance-decay relationship (P

Abstract Poultry are an important source of fecal contamination in environments. However, tools for detecting and tracking this fecal contamination are in the early stages of development. In practice, we have found that source tracking methods targeting the 16S rRNA genes of poultry-specific microbiota are not sufficiently sensitive. We therefore developed two quantitative PCR assays for detection of poultry fecal contamination, by targeting chicken and duck mitochondrial genes: NADH dehydrogenase subunit 5 (ND5) and cytochrome b (cytb). The sensitivity of both assays was 100% when tested on 50 chicken and duck fecal samples from 10 provinces of China. These assays were also tested in field samples, including soil and water collected adjacent to duck farms, and soils fertilized with chicken manure. Poultry mitochondrial DNA was detected in most of these samples, indicating that the assays are a robust method for monitoring environmental contamination with poultry feces. Complemented with existing indicators of fecal contamination, these markers should improve the efficiency and accuracy of microbial source tracking.

Microorganisms capable of anaerobic nitrate-dependent Fe(II) (ferrous iron) oxidation (ANDFO) contribute significantly to iron and nitrogen cycling in various environments. However, lab efforts in continuous cultivation of ANDFO strains suffer from loss of activity when ferrous iron is used as sole electron donor. Here, we used a novel strain of nitrate-dependent Fe(II)-oxidizing bacterium Bacillus ferroxidians as a model and focused on the physiological activity of cells during ANDFO. It was shown that B. ferrooxidans entered a metabolically inactive state during ANDFO. B. ferrooxidans exhibited nitrate reduction coupled with Fe(II) oxidation, and the activity gradually declined and was hardly detected after 48-h incubation. Propidium monoazide (PMA) assisted 16S rRNA gene real-time PCR suggested that a large number of B. ferrooxidans cells were alive during incubation. However, 2H(D)-isotope based Raman analysis indicated that the cells were metabolically inactive after 120-h of ANDFO. These inactive cells re-awakened in R2A medium and were capable of growth and reproduction, which was consistent with results in Raman analysis. Scanning electron microscopy (SEM) observation and x-ray diffraction (XRD) revealed the formation of Fe minerals in close proximity of cells in the Fe(II)-oxidizing medium after Fe(II) oxidation. Overall, our results demonstrated that continued ANDFO can induce a metabolically inactive state in B. ferrooxidans, which was responsible for the loss of activity during ANDFO. This study provides an insight into the ANDFO process and its contribution to iron and nitrogen cycling in the environments.

Manure application to agricultural soil introduces antibiotic residues and increases the abundance of antibiotic-resistant bacteria (ARB) carrying antibiotic resistance genes (ARGs), often located on mobile genetic elements (MGEs). The rhizosphere is regarded as a hotspot of microbial activity and gene transfer, which can alter and prolong the effects of organic fertilizers containing antibiotics. However, not much is known about the influence of plants on the effects of doxycycline applied to soil via manure. In this study, the effects of manure spiked with or without doxycycline on the prokaryotic community composition as well as on the relative abundance of ARGs and MGEs in lettuce rhizosphere and bulk soil were investigated by means of a polyphasic cultivation-independent approach. Samples were taken 42 days after manure application, and total community DNA was extracted. Besides a pronounced manure effect, doxycycline spiking caused an additional enrichment of ARGs and MGEs. High-throughput quantitative PCR revealed an increase in tetracycline, aminoglycoside, and macrolide–lincosamide–streptogramin B (MLSB) resistance genes associated with the application of manure spiked with doxycycline. This effect was unexpectedly lower in the rhizosphere than in bulk soil, suggesting a faster dissipation of the antibiotic and a more resilient prokaryotic community in the rhizosphere. Interestingly, the tetracycline resistance gene tetA(P) was highly enriched in manure-treated bulk soil and rhizosphere, with highest values observed in doxycycline-treated bulk soil, concurring with an enrichment of Clostridia. Thus, the gene tetA(P) might be a suitable marker of soil contamination by ARB, ARGs, and antibiotics of manure origin. These findings illustrate that the effects of manure and doxycycline on ARGs and MGEs differ between rhizosphere and bulk soil, which needs to be considered when assessing risks for human health connected to the spread of ARGs in the environment.

Plasmid conjugation is one of the dominant mechanisms of horizontal gene transfer, playing a noticeable role in the rapid spread of antibiotic resistance genes (ARGs). Broad host range plasmids are known to transfer to diverse bacteria in extracted soil bacterial communities when evaluated by filter mating incubation. However, the persistence and dissemination of broad range plasmid in natural soil has not been well studied. In this study, Pseudomonas putida with a conjugative antibiotic resistance plasmid RP4 was inoculated into a soil microcosm, the fate and persistence of P. putida and RP4 were monitored by quantitative PCR. The concentrations of P. putida and RP4 both rapidly decreased within 15-day incubation. P. putida then decayed at a significantly lower rate during subsequent incubation, however, no further decay of RP4 was observed, resulting in an elevated RP4/P. putida ratio (up to 10) after 75-day incubation, which implied potential transfer of RP4 to soil microbiota. We further sorted RP4 recipient bacteria from the soil microcosms by fluorescence-activated cell sorting. Spread of RP4 increased during 75-day microcosm operation and was estimated at around 10-4 transconjugants per recipient at the end of incubation. Analysis of 16S rRNA gene sequences of transconjugants showed that host bacteria of RP4 were affiliated to more than 15 phyla, with increased diversity and shift in the composition of host bacteria. Proteobacteria was the most dominant phylum in the transconjugant pools. Transient transfer of RP4 to some host bacteria was observed. These results emphasize the prolonged persistence of P. putida and RP4 in natural soil microcosms, and highlight the potential risks of increased spread potential of plasmid and broader range of host bacteria in disseminating ARGs in soil.

Unfortunately, after publication of this article [1], it was noticed that the accession numbers for the study were missing. The correct numbers are PRJNA438554, SRP148953.