Vertical distribution of antibiotic resistance genes in an urban green facade.

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.

Fates of Antibiotic Resistance Genes in the Gut Microbiome from Different Soil Fauna under Long-Term Fertilization.

Applying organic fertilizers has been well documented to facilitate the dissemination of antibiotic resistance genes (ARGs) in soil ecosystems. However, the role of soil fauna in this process has been seldom addressed, which hampers our ability to predict the fate of and to manage the spread of ARGs. Here, using high-throughput quantitative polymerase chain reaction (HT-qPCR), we examined the effect of long-term (5-, 8-, and 10-year) fertilization treatments (control, inorganic fertilizers, and mixed fertilizers) on the transfer of ARGs between soil, nematodes, and earthworms. We found distinct fates for ARGs in the nematodes and earthworms, with the former having higher enriched levels of ARGs than the latter. Fertilization impacted the number and abundance of ARGs in soil, and fertilization duration altered the composition of ARGs. Shared ARGs among soil, nematodes, and earthworm guts supported by a fast expectation-maximization microbial source tracking analysis demonstrated the trophic transfer potential of ARGs through this short soil food chain. The transfer of ARGs was reduced by fertilization duration, which was mainly ascribed to the reduction of ARGs in the earthworm gut microbiota. This study identified the transfer of ARGs in the soil-nematode-earthworm food chain as a potential mechanism for a wider dissemination of ARGs in the soil ecosystem.

Seasonal change is a major driver of soil resistomes at a watershed scale.

Soils harbor the most diverse naturally evolved antibiotic resistomes on Earth that threaten human health, ecosystem processes, and food security. Yet the importance of spatial and temporal variability in shaping the distribution of soil resistomes is not well explored. Here, a total of 319 topsoil samples were collected at a watershed scale during four seasons (spring to winter) and high-throughput quantitative PCR (HT-qPCR) was used to characterize the profiles of soil antibiotic resistance genes (ARGs). A significant and negative correlation was observed between soil ARG profiles and seasonal dissimilarity, which along with seasonally dependent distance-decay relationships highlight the importance of seasonal variability in shaping soil antibiotic resistomes. Significant, though weak, distance-decay relationships were identified in spring, summer and winter, for ARG similarities with geographic distances. There were also strong interactions between specific soil ARGs and Actinobacteria, Firmicutes and Proteobacteria. Moreover, we found that the relative abundance of soil Actinobacteria, Firmicutes and Proteobacteria correlated significantly with annual mean temperature and annual mean precipitation at a watershed scale. A random forest model showed that seasonal change rather than spatial variation was the most important predictor of the composition of soil ARGs. Together, these results constitute an advance in our understanding of the relative importance of spatial and temporal variability in shaping soil ARG profiles, which will provide novel insights allowing us to forecast their distribution under a changing environment.

Prevalence of Antibiotic Resistome in Ready-to-Eat Salad.

Ready-to-eat salad harbors microorganisms that may carry various antibiotic resistance genes (ARGs). However, few studies have focused on the prevalence of ARGs on salad, thus underestimating the risk of ARGs transferring from salad to consumers. In this small-scale study, high-throughput quantitative PCR was used to explore the presence, prevalence and abundance of ARGs associated with serving salad sourced from two restaurant types, fast-food chain and independent casual dining. A total of 156 unique ARGs and nine mobile genetic elements (MGEs) were detected on the salad items assessed. The abundance of ARGs and MGEs were significantly higher in independent casual dining than fast-food chain restaurants. Absolute copies of ARGs in salad were 1.34 × 107 to 2.71 × 108 and 1.90 × 108 to 4.87 × 108 copies per g salad in fast-food and casual dining restaurants, respectively. Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes were the dominant bacterial phyla detected from salad samples. Pseudomonas, Acinetobacter, Exiguobacterium, Weissella, Enterobacter, Leuconostoc, Pantoea, Serratia, Erwinia, and Ewingella were the 10 most dominant bacterial genera found in salad samples. A significant positive correlation between ARGs and MGEs was detected. These results integrate knowledge about the ARGs in ready-to-eat salad and highlight the potential impact of ARGs transfer to consumers.

Does reduced usage of antibiotics in livestock production mitigate the spread of antibiotic resistance in soil, earthworm guts, and the phyllosphere?

The overuse of antibiotics in animal husbandry is widespread and believed to significantly contribute to the selection of antibiotic resistance genes (ARGs) in animals. Thus, there is a global drive to reduce antibiotic use in the agricultural sector. However, it has not been established whether a reduction in the use of antibiotics in livestock production would be effective in reducing the spread of ARGs. A microcosm approach was used to determine how the addition of manure with either reduced antibiotic levels or with typical antibiotic levels could affect the spread of antibiotic resistance genes between soil, earthworms and the phyllosphere. When compared to the control soil, earthworm and phyllosphere samples had the greater increase in ARG abundance in conventional manure treatments (P < 0.05). Reduced antibiotic manure also enriched the abundance of ARGs in the phyllosphere and soil but not earthworm guts when compared to the control (P < 0.05). In both soil and earthworm guts, the enrichment of ARGs was lower in reduced antibiotic manure than in conventional manure. This study has identified bacterial transfer through the soil-earthworm-phyllosphere system as a potential means to spread ARGs between habitats after fertilization with livestock derived manures.

Microbial Flow Within an Air-Phyllosphere-Soil Continuum.

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.

Agricultural activities affect the pattern of the resistome within the phyllosphere microbiome in peri-urban environments.

The plant microbiome represents a crucial pathway for human exposure to environmental antibiotic resistance. However, little information is available regarding the plant associated resistome in human-related environments at a larger scale. Here, by high-throughput quantitative-PCR chip-based array and amplicon sequencing, we characterized antibiotic resistance genes (ARGs) and bacterial communities in plant and soil microbiomes from human highly disturbed peri-urban farmland and less disturbed forest at a watershed scale. A total of 71 ARGs were detected in the phyllosphere, which covered almost all the major recognized classes of antibiotics that are administered commonly to humans and animals. The overall pattern of the plant associated resistome in intensive anthropogenic influenced farmland was significantly different from that of forest environments (PERMANOVA, P < 0.01), indicating that agricultural activities might be important drivers in shaping the plant resistome. A bipartite network analysis suggested that all ARGs detected in the plant microbiome were also present in the soil microbiome. Together, our findings provide a better understanding of the plant resistome and suggest that land use is a key contributor to the composition of ARG profiles in the plant phyllosphere, and that the soil resistome may represent a critical reservoir of plant associated ARGs.

Phyllosphere of staple crops under pig manure fertilization, a reservoir of antibiotic resistance genes.

In China, the common use of antibiotics in agriculture is recognized as a potential public health risk through the increasing use of livestock derived manure as a means of fertilization. By doing so this may increase the transfer of antibiotic resistance genes (ARGs) from animals, to soils and plants. In this study two staple crops (rice and wheat) were investigated for ARG enrichment under differing fertilization regimes. Here, we applied 4 treatments, no fertilizer, mineral fertilizer, clean (reduced antibiotic practice) and dirty (current antibiotic practice) pig manure, to soil microcosms planted with either rice or wheat, to investigate fertilization effects on the abundance of ARGs in the respective phyllospheres. For both rice and wheat, samples were collected after two separate fertilization periods. In total, 162 unique ARGs and 5 mobile genetic elements (MGEs) were detected from all rice and wheat samples. The addition of both clean and dirty manure, enhanced ARG abundance significantly when compared to no fertilizer treatments (P < 0.001), though clean manure enriched ARGs to a lesser extent than dirty manure, in all rice and wheat samples (P < 0.001). The classes of ARGs recorded were different between crops, with wheat samples having a higher ARG diversity than rice. These results revealed that staple crops in China such as rice and wheat may be a reservoir for ARGs when clean and dirty pig manure is used for fertilization.

Mineral and organic fertilization alters the microbiome of a soil nematode Dorylaimus stagnalis and its resistome.

Although the effects of fertilization on the abundance and diversity of soil nematodes have been widely studied, the impact of fertilization on soil nematode microbiomes remains largely unknown. Here, we investigated how different fertilizers: no fertilizer, mineral fertilizer, clean slurry (pig manure with a reduced antibiotic burden) and dirty slurry (pig manure with antibiotics) affect the microbiome of a dominant soil nematode and its associated antibiotic resistance genes (ARGs). The results of 16S rRNA gene high throughput sequencing showed that the microbiome of the soil nematode Dorylaimus stagnalis is diverse (Shannon index: 9.95) and dominated by Proteobacteria (40.3%). Application of mineral fertilizers significantly reduced the diversity of the nematode microbiome (by 28.2%; P < 0.05) but increased the abundance of Proteobacteria (by 70.1%; P = 0.001). Microbial community analysis, using a null hypothesis model, indicated that microbiomes associated with the nematode are not neutrally assembled. Organic fertilizers also altered the diversity of the nematode microbiome, but had no impact on its composition as illustrated by principal coordinates analysis (PCoA). Interestingly, although no change of total ARGs was observed in the nematode microbiome and no significant relationship existed between nematode microbiome and resistome, the abundance of 48 out of a total of 75 ARGs was enriched in the organic fertilizer treatments. Thus, the data suggests that ARGs in the nematode microbiome still had a risk of horizontal gene transfer under fertilization and nematodes might be a potential refuge for ARGs.

Variable response of nirK and nirS containing denitrifier communities to long-term pH manipulation and cultivation.

Denitrification is a key process responsible for the majority of soil nitrous oxide (N2O) emissions but the influences of pH and cultivation on the soil denitrifier community remain poorly understood. We hypothesised that the abundance and community structure of the total bacterial community and bacterial denitrifiers would be pH sensitive and that nirK and nirS containing denitrifiers would differ in their responses to change in pH and cultivation. We investigated the effect of long-term pH-adjusted soils (ranging from pH 4.2 to 6.6) under different lengths of grass cultivation (one, two and three years of ley grass) on the general bacterial and denitrifier functional communities using 16S rRNA, nirK and nirS genes as markers. Denitrifier abundance increased with pH, and at pH below 4.7 there was a greater loss in nirS abundance per unit drop in pH than soils above this threshold pH. All community structures responded to changes in soil pH, while cultivation only influenced the community structure of nirK. These differences in denitrifier responses highlight the importance of considering both nirK and nirS gene markers for estimating denitrifier activity. Identifying such thresholds in response of the microbial community to changes in pH is essential to understanding impacts of management or environmental change.

Symbiotic relationships between soil fungi and plants reduce N2O emissions from soil.

N2O is a potent greenhouse gas involved in the destruction of the protective ozone layer in the stratosphere and contributing to global warming. The ecological processes regulating its emissions from soil are still poorly understood. Here, we show that the presence of arbuscular mycorrhizal fungi (AMF), a dominant group of soil fungi, which form symbiotic associations with the majority of land plants and which influence a range of important ecosystem functions, can induce a reduction in N2O emissions from soil. To test for a functional relationship between AMF and N2O emissions, we manipulated the abundance of AMF in two independent greenhouse experiments using two different approaches (sterilized and re-inoculated soil and non-mycorrhizal tomato mutants) and two different soils. N2O emissions were increased by 42 and 33% in microcosms with reduced AMF abundance compared to microcosms with a well-established AMF community, suggesting that AMF regulate N2O emissions. This could partly be explained by increased N immobilization into microbial or plant biomass, reduced concentrations of mineral soil N as a substrate for N2O emission and altered water relations. Moreover, the abundance of key genes responsible for N2O production (nirK) was negatively and for N2O consumption (nosZ) positively correlated to AMF abundance, indicating that the regulation of N2O emissions is transmitted by AMF-induced changes in the soil microbial community. Our results suggest that the disruption of the AMF symbiosis through intensification of agricultural practices may further contribute to increased N2O emissions.

Soil nitrate reducing processes - drivers, mechanisms for spatial variation, and significance for nitrous oxide production.

The microbial processes of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are two important nitrate reducing mechanisms in soil, which are responsible for the loss of nitrate ([Formula: see text]) and production of the potent greenhouse gas, nitrous oxide (N(2)O). A number of factors are known to control these processes, including O(2) concentrations and moisture content, N, C, pH, and the size and community structure of nitrate reducing organisms responsible for the processes. There is an increasing understanding associated with many of these controls on flux through the nitrogen cycle in soil systems. However, there remains uncertainty about how the nitrate reducing communities are linked to environmental variables and the flux of products from these processes. The high spatial variability of environmental controls and microbial communities across small sub centimeter areas of soil may prove to be critical in determining why an understanding of the links between biotic and abiotic controls has proved elusive. This spatial effect is often overlooked as a driver of nitrate reducing processes. An increased knowledge of the effects of spatial heterogeneity in soil on nitrate reduction processes will be fundamental in understanding the drivers, location, and potential for N(2)O production from soils.