Antibiotic Resistance Genes In Soil Research Articles

Inorganic and organic fertilizers application enhanced antibiotic resistome in greenhouse soils growing vegetables

Antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in fertilizers pose risks to human health and their variation in soil after fertilization has been reported. However, some important questions, such as the origin of ARG and ARB observed in soil following fertilization, which are present in soil regardless of fertilizer type (i.e., core (shared) ARGs and ARB), and the contribution of various ARG subtypes to the soil antibiotic resistome, need to be addressed. In this study, the effects of a long-term (9-year) application of organic (manure) and inorganic (chemistry) fertilizers on ARGs in greenhouse soils growing vegetables were investigated using metagenomic sequencing. The results showed that both organic and inorganic fertilizers application increased the diversity and abundance of soil ARGs. The dominant ARG types in organic fertilizer (OF) were different from that in organic fertilizer treated soil (SO), inorganic fertilizer treated soil (SI) and no fertilizer control plots (SC). The difference of core ARGs abundance reflected the variation of ARG profiles among SC, SI and SO. The OF is likely a source of the elevated ARG subtypes in soil and almost all the soil core ARG subtypes can be detected in organic fertilizer. Fifteen ARG types were enriched in the soil with OF, and some ARG subtypes such as sul1, sul2, tetX and tetL might derived from OF while others including as vanR, tcmA, rosB, and mexF might be from indigenous microbes in soil. The nutrition factors were found to influence the ARG profiles in fertilized soil. In summary, this study revealed the possible reason for the soil total ARG numbers and their relative abundance increase after fertilization, which will facilitate the control of ARGs and ARB dissemination.

Metagenomic characterization of antibiotic resistance genes in Antarctic soils

Antibiotic resistance genes (ARGs) are considered environmental pollutants. Comprehensive characterization of the ARGs in pristine environments is essential towards understanding the evolution of antibiotic resistance. Here, we analyzed ARGs in soil samples collected from relatively pristine Antarctica using metagenomic approaches. We identified 79 subtypes related to 12 antibiotic classes in Antarctic soils, in which ARGs related to multidrug and polypeptide were dominant. The characteristics of ARGs in Antarctic soils were significantly different from those in active sludge, chicken feces and swine feces, in terms of composition, abundance and potential transferability. ARG subtypes (e.g., bacA, ceoB, dfrE, mdtB, amrB, and acrB) were more abundant than others in Antarctic soils. Approximately 60% of the ARGs conferred antibiotic resistance via an efflux mechanism, and a low fraction of ARGs (∼16%) might be present on plasmids. Culturable bacterial consortiums isolated from Antarctic soils were consistently susceptible to most of the tested antibiotics frequently used in clinical therapies. The amrB and ceoB carried by culturable species did not express the resistance to aminoglycoside and fluoroquinolone at the levels of clinical concern. Our results suggest that the wide use of antibiotics may have contributed to developing higher antibiotic resistance and mobility.

Antibiotics in the Soil Environment-Degradation and Their Impact on Microbial Activity and Diversity.

Antibiotics play a key role in the management of infectious diseases in humans, animals, livestock, and aquacultures all over the world. The release of increasing amount of antibiotics into waters and soils creates a potential threat to all microorganisms in these environments. This review addresses issues related to the fate and degradation of antibiotics in soils and the impact of antibiotics on the structural, genetic and functional diversity of microbial communities. Due to the emergence of bacterial resistance to antibiotics, which is considered a worldwide public health problem, the abundance and diversity of antibiotic resistance genes (ARGs) in soils are also discussed. When antibiotic residues enter the soil, the main processes determining their persistence are sorption to organic particles and degradation/transformation. The wide range of DT50 values for antibiotic residues in soils shows that the processes governing persistence depend on a number of different factors, e.g., physico-chemical properties of the residue, characteristics of the soil, and climatic factors (temperature, rainfall, and humidity). The results presented in this review show that antibiotics affect soil microorganisms by changing their enzyme activity and ability to metabolize different carbon sources, as well as by altering the overall microbial biomass and the relative abundance of different groups (i.e., Gram-negative bacteria, Gram-positive bacteria, and fungi) in microbial communities. Studies using methods based on analyses of nucleic acids prove that antibiotics alter the biodiversity of microbial communities and the presence of many types of ARGs in soil are affected by agricultural and human activities. It is worth emphasizing that studies on ARGs in soil have resulted in the discovery of new genes and enzymes responsible for bacterial resistance to antibiotics. However, many ambiguous results indicate that precise estimation of the impact of antibiotics on the activity and diversity of soil microbial communities is a great challenge.

Fate of Antibiotic Resistant Pseudomonas putida and Broad Host Range Plasmid in Natural Soil Microcosms.

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.

Salinity as a predominant factor modulating the distribution patterns of antibiotic resistance genes in ocean and river beach soils

Growing evidence points to the pivotal role of the environmental factors in influencing the transmission of antibiotic resistance genes (ARGs) and the propagation of resistant human pathogens. However, our understanding of the ecological and evolutionary environmental factors that contribute to development and dissemination of antibiotic resistance is lacking. Here, we profiled a wide variety of ARGs using the high-throughput quantitative PCR analysis in 61 soil samples collected from ocean and river beaches, which are hotspots for human activities and platforms for potential transmission of environmental ARGs to human pathogens. We identified the dominant abiotic and biotic factors influencing the diversity, abundance and composition of ARGs in these ecosystems. A total of 110 ARGs conferring resistance to eight major categories of antibiotics were detected. The core resistome was mainly affiliated into β-lactam and multidrug resistance, accounting for 66.9% of the total abundance of ARGs. The oprJ gene conferring resistance to multidrug was the most widespread ARG subtype detected in all the samples. The relative abundances of total ARGs and core resistome were significantly correlated with salinity-related properties including electrical conductivity and concentrations of sodium and chloride. Random forest analysis and structural equation modelling revealed that salinity was the most important factor modulating the distribution patterns of beach soil ARGs after accounting for multiple drivers. These findings suggest that beach soil is a rich reservoir of ARGs and that salinity is a predominant factor shaping the distribution patterns of soil resistome.

RefSoil+: a Reference Database for Genes and Traits of Soil Plasmids.

Plasmids harbor transferable genes that contribute to the functional repertoire of microbial communities, yet their contributions to metagenomes are often overlooked. Environmental plasmids have the potential to spread antibiotic resistance to clinical microbial strains. In soils, high microbiome diversity and high variability in plasmid characteristics present a challenge for studying plasmids. To improve the understanding of soil plasmids, we present RefSoil+, a database containing plasmid sequences from 922 soil microorganisms. Soil plasmids were larger than other described plasmids, which is a trait associated with plasmid mobility. There was a weak relationship between chromosome size and plasmid size and no relationship between chromosome size and plasmid number, suggesting that these genomic traits are independent in soil. We used RefSoil+ to inform the distributions of antibiotic resistance genes among soil microorganisms compared to those among nonsoil microorganisms. Soil-associated plasmids, but not chromosomes, had fewer antibiotic resistance genes than other microorganisms. These data suggest that soils may offer limited opportunity for plasmid-mediated transfer of described antibiotic resistance genes. RefSoil+ can serve as a reference for the diversity, composition, and host associations of plasmid-borne functional genes in soil, a utility that will be enhanced as the database expands. Our study improves the understanding of soil plasmids and provides a resource for assessing the dynamics of the genes that they carry, especially genes conferring antibiotic resistances. IMPORTANCE Soil-associated plasmids have the potential to transfer antibiotic resistance genes from environmental to clinical microbial strains, which is a public health concern. A specific resource is needed to aggregate the knowledge of soil plasmid characteristics so that the content, host associations, and dynamics of antibiotic resistance genes can be assessed and then tracked between the environment and the clinic. Here, we present RefSoil+, a database of soil-associated plasmids. RefSoil+ presents a contemporary snapshot of antibiotic resistance genes in soil that can serve as a reference as novel plasmids and transferred antibiotic resistances are discovered. Our study broadens our understanding of plasmids in soil and provides a community resource of important plasmid-associated genes, including antibiotic resistance genes.

Long-term application of organic fertilization causes the accumulation of antibiotic resistome in earthworm gut microbiota

Antibiotic resistance genes (ARGs), prevalent across multiple environmental media, threaten human health worldwide and are considered emerging environmental contaminants. Earthworm gut, a niche for bacteria to survive, represents a potential reservoir for ARGs in soil. However, the compositions of ARGs in the earthworm gut microbiota remain elusive, especially under field conditions. In this study, we applied high-throughput quantitative PCR to profile the ARGs in the gut microbiota of earthworms after chronic exposure to fertilizers. To elucidate the factors that impact the ARGs composition, the bacterial community of gut microbiota, mobile genetic elements (MGEs), soil (nutrients, heavy metals, and antibiotics) and the properties of gut content (pH and nutrients) were analyzed. A total of 98 subtypes among 9 major types of ARGs, and 3 different MGEs were detected in the gut microbiota of earthworms. Organic fertilizer (sewage sludge and chicken manure) application significantly increased the diversity and abundance of ARGs. Of the 1123 identified operational taxonomic units (OTUs) at 97% similarity cutoff, most of them were assigned to Firmicutes (55.5%) and Proteobacteria (33.6%) in earthworm gut microbiota. Long-term organic fertilization slightly changed the microbiota composition, but did not impact the diversity. Partial redundancy analysis (pRDA) revealed that bacterial community, combined with environmental factors (soil and gut content properties) and MGEs, explained 72% of the variations of ARGs in the earthworm gut. Furthermore, the co-occurrence pattern between ARGs and MGEs indicated that horizontal gene transfer via MGEs may occur in the earthworm gut. These findings improve the current understanding of the dynamics of soil fauna-associated ARGs and the gut microbiota of earthworms may be an underappreciated hotspot for ARGs in the environment.

Distribution of antibiotic resistance genes in soils and crops. A field study in legume plants (Vicia faba L.) grown under different watering regimes

Social concern has raised during the last years due to the development of antibiotic resistance hotspots in different environmental compartments, including the edible parts of crops. To assess the influence of the water quality used for watering, we collected samples from soil, roots, leaves and beans from the legume plant Vicia faba (broad beans) in three agricultural peri-urban plots (Barcelona, NE Spain), irrigated with either groundwater, river water, or reclaimed water. Antibiotic resistance genes (ARGs) sul1, tetM, qnrS1, blaCTX-M-32,blaOXA-58, mecA, and blaTEM were quantified by real-time PCR, along with 16S rDNA and intl1 sequences, as proxies for bacterial abundance and integron prevalence, respectively. Microbiome composition of all samples were analyzed by high-throughput DNA sequencing. Results show a gradient of bacterial species diversity and of ARG prevalence from highly diverse soil samples to microbially-poor beans and leaves, in which Rhizobiales essentially displaced all other groups, and that presented very small loads of ARGs and integron sequences. The data suggest that the microbiome and the associated resistome were likely influenced by agricultural practices and water quality, and that future irrigation water legal standards should consider the specific Physiology of the different crop plants.

Fate of tetracycline and sulfonamide resistance genes in a grassland soil amended with different organic fertilizers

This study provided an assessment of the environmental fate of antibiotic resistance genes (ARGs) in a Scottish grassland field repeatedly treated with different organic fertilizers. The impacts of manure, biosolids and municipal food-derived compost on the relative abundances of tetracycline ARGs (tetA, tetB, tetC, tetG and tetW), sulfonamide ARGs (sul1 and sul2) and class 1 integron-integrase gene (IntI1) in soils were investigated, with inorganic fertilizer (NPK) as a comparison. The background soil with a history of low intensity farming showed a higher total relative abundance of tet ARGs over sul ARGs, with tetracycline efflux genes occurring in a higher frequency. In all treatments, the relative abundances of most ARGs detected in soils decreased over time, especially IntI1 and tet ARGs. This general attenuation of soil ARGs is a reflection of changes in the soil microbial community, which is supported by the result that almost all the soils at the end of the experiment had different bacterial communities from the untreated soil at the beginning of the experiment. Multiple applications of organic fertilizers to some extent counteracted the decreasing trend of soil ARGs relative abundances, which resulted in higher ARGs relative abundances in comparison to NPK, either by a lesser decrease of IntI1 and tet ARGs or an increase of sul ARGs. The enhancement of existing soil ARG prevalence by organic fertilizers was strongly dependent on the organic fertilizer type and the particular ARG. Compost contained the lowest relative abundance of inherent ARGs and had the least effect on the soil ARG decrease after application. The relative increase of tet ARGs caused by biosolids was larger than that of sul ARGs, while manure caused the opposite effect. Fertilization practices did not exert effective impacts on the soil bacterial community, although it caused significant changes in the profile of the ARG pool. Organic fertilization may thus accelerate the dissemination of ARGs in soil mainly through horizontal gene transfer (HGT), consistent with the enrichment of IntI1 in organic fertilized soils.

Field-based evidence for enrichment of antibiotic resistance genes and mobile genetic elements in manure-amended vegetable soils

The increasing prevalence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the soil environment represents a serious threat to public health. In this study, the diversity and abundance of ARGs and mobile genetic elements (MGEs) in different years of manure-amended vegetable soils were investigated. A total of eight genes, including four tetracycline resistance genes: tetW, tetM, tetO and tetT; two sulfonamide resistance genes: sul1 and sul2; and two MGEs: intI1 and intI2; were quantified in ten vegetable soils. The relative abundance of ARGs in soils amended with manure was significantly higher than that in soils without manure application. The relative abundance of the intI1 and intI2 genes had significantly positive correlations with the relative abundance of the tetW, tetO, sul1 and sul2 genes. Under different concentrations of antibiotics, the resistant bacteria rates of manure-amended soil were much higher than the control soil. Bacillus and Chryseobacterium, more likely to be multi-drug-resistant bacteria, were detected in both two antibiotics. Moreover, the significant correlation was found between the concentrations of Cu and Zn and the ARGs. Our findings provide empirical evidence that the dissemination risk of ARGs and ARB in long-term manure-amended vegetable soils, which might promote to the development of effective strategies to reduce the spread of ARGs in agro-ecosystems.

The potential impact of naturally produced antibiotics, environmental factors, and anthropogenic pressure on the occurrence of erm genes in urban soils

The occurrence of environmental antibiotic resistance genes (ARGs) are often attributed to selective pressure from antibiotics from point source pollution. However, the potential effects of natural production of antibiotics, environmental factors, and anthropogenic pressure on the development and spread of ARGs have not been fully investigated. This study evaluated the occurrence and distribution of erythromycin resistance methylase (erm) genes in urban soils. The ermA, ermB, ermC, ermD, ermF, ermG, ermT, and ermY genes were detected with detection frequencies ranging from 20% to 80% and abundances ranging between 5.95 × 101 and 6.94 × 106 copies g−1 dw soil. Both polyketide synthase (PKS) type I and type II biosynthesis genes—which are responsible for biosynthesis of polyketides, such as erythromycin—were detected in all soil samples with a range between 5.77 × 102 and 9.39 × 106 copies g−1 dw soil. The abundances of PKS genes were significantly correlated with 16S rRNA genes (r = 0.487 to 0.741, p < 0.001) and absolute abundances of ermB, ermC, ermD, ermG, and ermY (r = 0.302–0.490, p < 0.05), suggesting that the wide occurrence of ARGs in soils could be potentially driven by naturally produced antibiotics. Erythromycin was strongly correlated with ermB, ermC, ermF and ermY genes (r = 0.462 to 0.667, p < 0.05), but no significant correlation was observed between macrolides and PKS genes, suggesting other environmental factors may have contributed to detected macrolides. The fact that erm gene presented higher extent of variability than PKS genes in different land use types suggests that anthropogenic activity might also influence the occurrence of erm genes in urban soils.

A rationale for the high limits of quantification of antibiotic resistance genes in soil

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR.The limits of quantification (LOQ) of the ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Short-term copper exposure as a selection pressure for antibiotic resistance and metal resistance in an agricultural soil

Owing to the similar mechanisms of antibiotic and metal resistance, there is a growing concern that metal contamination may select for antibiotic resistance genes (ARGs) in the environment. Here, we constructed short-term laboratory microcosms to investigate the dynamics of a wide range of ARGs and two copper (Cu) resistance genes in an agricultural soil amended with a gradient of Cu concentrations (0~1000mgkg-1). Mobile genetic elements (MGEs) were also quantified as a proxy for the horizontal gene transfer potential of ARGs. We detected 126 unique ARGs across all the soil samples using the high-capacity quantitative PCR array, and multidrug and β-lactam resistance were the most abundant ARG categories. The copper amendments significantly enhanced the absolute and relative abundances of ARGs and MGEs, which gradually increased along the gradient of increasing Cu concentrations. The two Cu resistance genes (copA and pcoR) were highly enriched in low-level Cu treatment (50 and 100mgkg-1), and their abundances decreased with the increasing Cu concentrations. The level of metal and antibiotic resistance gradually declined over time in all Cu-amended treatments but was still considerably higher in contaminated soils than untreated soils after 56days' incubation. Significant associations among ARGs and MGEs were revealed by the network analysis, suggesting the mobility potential of antibiotic resistance in Cu-amended soils. No significant positive correlations were found between ARGs and copper resistance genes, suggesting that these genes are not located in the same bacterial hosts. Taken together, our results provide empirical evidence that short-term copper stress can cause evolution of high-level antibiotic and metal resistance and significantly change the diversity, abundance, and horizontal transfer potential of soil ARGs.

Reducing water use by alternate-furrow irrigation with livestock wastewater reduces antibiotic resistance gene abundance in the rhizosphere but not in the non-rhizosphere

Livestock wastewater is rich in nutrients but may contain antibiotics and antibiotic resistance genes (ARGs). Their discharge to watercourses or soil may result in proliferation of ARGs. Irrigation with wastewater appears to be the most feasible option of disposing of it. One efficient irrigation technology used in arid regions is alternate-furrow irrigation (AFI) by alternately drying part of the plant roots for a prolonged period to physiologically reduce transpiration without compromising yield. However, the extent to which AFI with wastewater influences the concentration of antibiotics and spread of ARGs in soil is poorly understood. The purpose of this paper is to investigate how AFI using swine wastewater alters antibiotic kinetics and ARGs abundance under different irrigation rates, using pepper as the model plant. We examined three AFI treatments using 50%, 65% and 80% of the amount of water employed in sufficient conventional furrow irrigation. Each treatment had a groundwater irrigation control. The results showed that antibiotic concentrations and relative ARGs abundance in the top 20 cm of soil did not increase with the irrigation amount, although they were higher than those in the groundwater-irrigated soils. The relative ARGs abundance in the soil was modulated by irrigation amount and reducing the irrigation amount in AFI reduced ARGs dispersion only in rhizosphere. When the soil moisture was close to field capacity, ARGs were more abundant in rhizosphere than in non-rhizosphere, possibly because the rhizosphere is rich in microbes and increasing antibiotic concentrations due to an increase in irrigation rate favors antibiotic-resistant microbiome in competing for substrates. These, however, were not mirrored in the relative ARGs abundance in the roots. These results have important implications as it revealed that reducing the input of antibiotics and ARGs into soil with AFI does not necessarily reduce ARGs proliferation.

Effect of long-term manure slurry application on the occurrence of antibiotic resistance genes in arable purple soil (entisol)

The application of animal manure is a highly recommended traditional agricultural practice for soils of relatively low fertility. However, for the farmland purple soils that are widely distributed in the upper Yangtze River region, little knowledge has been established in previous studies about the changes in the antibiotic resistome upon manure amendment. In the present study, the impact of long-term pig manure slurry application on the occurrence of antibiotic resistance genes (ARGs) and bacterial community was assessed in arable calcareous purple soil using high-throughput quantitative polymerase chain reaction and Illumina sequencing. Four treatments, including a non-fertilization control (CK) and pig manure (OM), OM plus mineral N fertilizer (OMN) and OM plus mineral NPK fertilizer (OMNPK) treatments were investigated. Across all the soil samples receiving different treatments, a total of 139 unique ARGs and 6 mobile genetic element genes were detected, with multidrug and beta-lactam the two most dominant types of ARGs. The results of the principal coordinate analysis (PCoA) suggest that the profiles of soil ARGs in the two treatments of OM combined with mineral fertilizer(s) (i.e., OMN and OMNPK) were similar to those in the control treatment, while the soil receiving only pig manure application had a different pattern of ARGs from the soils in the other three treatments. A clear reduction of soil ARGs was observed in the OM treatment. Significant and positive relationships were found not only among ARGs but also between mobile genetic elements (MGEs) and ARGs. However, no significant relationships were detected between ARG patterns and bacterial community composition. These results imply that the long-term application of pig manure slurry to purple soil does not lead to the prevalence of ARGs; however, the potential for the horizontal transfer of ARGs in calcareous purple soil should not be ignored.

Investigating the environmental risks from the use of spray-dried cephalosporin mycelial dreg (CMD) as a soil amendment

Cephalosporin mycelial dreg (CMD) is a by-product of the pharmaceutical industry. Spray-drying is widely used for the dewatering process prior to the application of CMD as a soil amendment. However, the potential environmental behaviors and risks of spray-dried CMD amendment remain unclear. Here, a lab-scale incubation experiment was conducted to investigate the salinity, phytotoxicity, introduced antibiotics, heavy metals and the potential impacts of resistance genes in CMD-amended soil. Spray-dried CMD amendment generally increased soil salinity and only high dosed soils showed phytotoxic effects at the end of the incubation period, implying the physiological damage to plant growth. The introduced antibiotics quickly degraded over time, indicating a relatively low environmental persistence. Heavy metal slightly increased in soil receiving spray-dried CMD, and regulations should be developed to avoid metal accumulation. A decreased diversity and distinct patterns of β-lactam resistance genes as well as a dose-effect of their enrichment were observed in CMD-amended soil, which might be partially explained by the specific metals and introduced antibiotics. Antibiotic resistance genes in soil may be a valuable tool for evaluating the environmental risk associated with use of CMD as a soil amendment.

Tetracycline and Sulfonamide Antibiotic Resistance Genes in Soils From Nebraska Organic Farming Operations.

There is widespread agreement that agricultural antibiotic resistance should be reduced, however, it is unclear from the available literature what an appropriate target for reduction would be. Organic farms provide a unique opportunity to disentangle questions of agricultural antibiotic drug use from questions of antibiotic resistance in the soil. In this study, soil was collected from 12 certified organic farms in Nebraska, evaluated for the presence of tetracycline and sulfonamide resistance genes (n = 15 targets), and correlated to soil physical, chemical, and biological parameters. Tetracycline and sulfonamide antibiotic resistance genes (ARGs) were found in soils from all 12 farms, and 182 of the 196 soil samples (93%). The most frequently detected gene was tetG (55% of samples), followed by tet(Q) (49%), tet(S) (46%), tet(X) (30%), and tetA(P) (29%). Soil was collected from two depths. No differences in ARGs were observed based on soil depth. Positive correlations were noted between ARG presence and soil electrical conductivity, and concentrations of Ca, Na, and Mehlich-3 phosphorus. Data from this study point to possible relationships between selected soil properties and individual tetracycline resistance genes, including tet(O) which is a common target for environmental samples. We compared organic farm results to previously published data from prairie soils and found significant differences in detection frequency for 12 genes, eight of which were more commonly detected in prairie soils. Of interest, when tetracycline ARG results were sorted by gene mechanism, the efflux genes were generally present in higher frequency in the prairie soils, while the ribosomal protection and enzymatic genes were more frequently detected in organic farm soils, suggesting a possible ecological role for specific tetracycline resistance mechanisms. By comparing soil from organic farms with prairie soils, we can start to determine baseline effects of low-chemical input agricultural production practices on multiple measures of resistance.

Diversity of herbaceous plants and bacterial communities regulates soil resistome across forest biomes.

Antibiotic resistance is ancient and prevalent in natural ecosystems and evolved long before the utilization of synthetic antibiotics started, but factors influencing the large-scale distribution patterns of natural antibiotic resistance genes (ARGs) remain largely unknown. Here, a large-scale investigation over 4000 km was performed to profile soil ARGs, plant communities and bacterial communities from 300 quadrats across five forest biomes with minimal human impact. We detected diverse and abundant ARGs in forests, including over 160 genes conferring resistance to eight major categories of antibiotics. The diversity of ARGs was strongly and positively correlated with the diversity of bacteria, herbaceous plants and mobile genetic elements (MGEs). The ARG composition was strongly correlated with the taxonomic structure of bacteria and herbs. Consistent with this strong correlation, structural equation modelling demonstrated that the positive effects of bacterial and herb communities on ARG patterns were maintained even when simultaneously accounting for multiple drivers (climate, spatial predictors and edaphic factors). These findings suggest a paradigm that the interactions between aboveground and belowground communities shape the large-scale distribution of soil resistomes, providing new knowledge for tackling the emerging environmental antibiotic resistance.

Application of biosolids drives the diversity of antibiotic resistance genes in soil and lettuce at harvest

High-throughput quantitative polymerase chain reaction (PCR) of antibiotic resistance genes (ARGs) was used to profile the composition and diversity of ARGs in biosolid-amended soil and in lettuce roots and shoots. Biosolid application significantly increased the ARGs in soil and influenced the soil antibiotics resistome mainly through exogenous introduction. Four dominant ARGs occurred in sterilized and water-washed leaves, namely catB8, php, vanB and str, which accumulated to >50% of total abundance. Nine subclasses of ARGs were determined in both sterilized and water-washed leaves, and they were shared ARGs among soil, roots and shoots. This suggests that the ARGs in ready-to-eat lettuce were mainly located in endophytes and the internal route may contribute more to the ARGs in routinely consumed raw lettuce. Moreover, a previously unsuspected shift in multridrug ARGs to root and shoot compartments due to biosolid application was discovered. These results reveal that biosolid application drives the evolution and dissemination of ARGs in the soil-plant system.

Amendment soil with biochar to control antibiotic resistance genes under unconventional water resources irrigation: Proceed with caution

The spread of antibiotic resistance genes (ARGs) has become a cause for serious concern because of its potential risk to public health. The use of unconventional water resources (e.g., reclaimed water or piggery wastewater) in agriculture to relieve groundwater shortages may result in an accumulation of ARGs in soil. Biochar addition has been proven to be a beneficial method to alleviate the pollution of ARGs in manure-amended soil. However, the role of biochar on ARGs in soil-plant systems repeatedly irrigated with unconventional water resources is unknown. Under reclaimed water or piggery wastewater irrigation, rhizobox experiments using maize plants in soil amended with biochar were conducted to investigate the variation of typical ARGs (tet and sul genes) in soil-plant systems during a 60-day cultivation, and ARGs was characterized by high-throughput qPCR with a 48 (assays) × 108 (samples) array. Only piggery wastewater irrigation significantly increased the abundance of ARGs in rhizosphere and bulk soils and root endophytes. Following 30-day cultivation, the abundance of ARGs in soil was significantly lower due to biochar addition. However, by day 60, the abundance of ARGs in soil supplemented with biochar was significantly higher than in the control soils. Antibiotics, bio-available heavy metals, nutrients, bacterial community, and mobile gene elements (MGEs) were detected and analyzed to find factors shaping ARGs dynamics. The behavior of ARGs were associated with antibiotics but not with bio-available heavy metals. The correlation between ARGs and available phosphorus was stronger than that of ARGs with total phosphorus. MGEs had good relationship with ARGs, and MGEs shifts contributed most to ARGs variation in soil and root samples. In summary, this study provides insights into potential options for biochar use in agricultural activities.