Freeze-thaw cycles and their interrelation with microplastic contamination affecting the spread and propagation of antibiotic resistant genes in black soil of Northeast China

Do manure-borne or indigenous soil microorganisms influence the spread of antibiotic resistance genes in manured soil?

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

The objective of this study was to clarify and quantify the impact of fertilizer applications on antibiotic resistance genes (ARGs) in cropland soil. The target was to provide scientific basis for a better understanding of the source and accumulation and transportation characteristics of ARGs in soil and adaptive management strategy-making to secure the ecological environment and human health safety. By collecting data from literature published within the last 20 years (2000-2020), we established a database with 215 and 201 groups of a paired data-set consisting of the quantity and relative abundance of ARGs under independent experimental conditions. Compared to that with no fertilizer, the combined application of organic fertilizer significantly increased the quantity and relative abundance of soil ARGs by 110.0% and 91.0%, respectively. However, chemical fertilization had no significant effect on soil ARGs. The increment of relative abundance of soil ARGs by the combined application of organic fertilizer in the subtropical region was equivalent to 2.6 times that in the warm temperate zone. Compared with that in black soil and dark brown soil, the combined application of organic fertilizer significantly increased the relative abundance of ARGs in red soil and paddy soil in the subtropical region. The increment for the quantity of ARGs (147.6%) by the combined application of organic fertilizer in soil with pH<7 was significantly higher than that in soil with pH>7(110.4%). Compared to poultry manure, livestock manure application significantly increased the quantity and relative abundance of ARGs. The increment of the relative abundance of organic fertilizer to sulfonamide, multidrug, and macrolide ARGs (170.5%-201.2%) was significantly higher than that of quinolone, tetracycline, and aminoglycoside ARGs (61.5%-115.6%). After more than 10 years of applying organic fertilizer, the quantity of soil ARGs significantly increased by 104.2%-112.3%, whereas the effect on the relative abundance was uncertain. Climate, soil spatial properties, and source and amount of organic fertilizer were the main factors affecting the accumulation of ARGs in farmland soil. Management strategies and solutions should pay more attention to effectively minimizing the accumulation and spread of ARGs in agro-ecosystems for high-quality agricultural development in the future.

The objective of this study was to investigate the impact of various chemical nitrogen fertilizers on the profile of antibiotic resistance genes (ARGs) in soil. A microcosm experiment was conducted with four treatments, including CK (control with no nitrogen), AN (ammonium nitrogen), NN (nitrate nitrogen), and ON (urea nitrogen), and the abundance of ARGs was assessed over a 30-day period using a metagenomic sequencing approach. The levels of core ARGs varied between 0.16 and 0.22 copies per cell across different treatments over time. The abundance of core ARGs in the ON treatment closely resembled that of the CK treatment, suggesting that environmentally friendly nitrogen fertilizers, particularly those in controlled release formulations, may be preferable. The core ARG abundance in the AN and NN treatments exhibited noticeable fluctuations over time. Overall, chemical nitrogen fertilizers had minimal effects on the core ARG profile as determined by principal component analysis and clustering analyses. Conversely, distinct and significant changes in bacterial communities were observed with the use of different nitrogen fertilizers. However, the influence of nitrogen fertilizers on the core ARGs is limited due to the unaffected potential bacterial hosts. Nitrogen-cycling-related genes (NCRGs), such as those involved in nitrogen-fixing (nifK, nifD, nifH) and denitrification (narG, napA, nirK, norB, nosZ) processes, exhibit a positive correlation with ARGs (rosA, mexF, bacA, vanS), indicating a potential risk of ARG proliferation during intense denitrification activities. This study indicates that the application of chemical nitrogen has a minimal effect on the abundance of ARGs in soil, thereby alleviating concerns regarding the potential accumulation of ARGs due to the use of chemical nitrogen fertilizers.

The high production of animal protein results in a large production of manure, which has the soil as its final destination. The use of antibiotics in livestock stimulates the proliferation of antibiotic resistance genes (ARGs). This is problematic given the rise in antimicrobial resistance in human and animal pathogens. Composting is an established and viable method for the stabilization and satinization of manure. Therefore, it is important to evaluate the effect of composting on the set of ARGs in manure, the resistome. Furthermore, it is important to compare the effect of the application of the composts in soils, in order to assess their effectiveness on the reduction of the risk of ARGs proliferation in this environment. Chapter 1 examined the effect of composting on the reduction of the diversity and abundance of ARGs in the poultry manures, poultry litter and layer manure. Therefore, the objective of this step was to investigate whether two different composting strategies are effective on the reduction of ARGs in poultry litter. Additionally, it was evaluated which type of poultry manure is more effective in the attenuation of ARGs through composting. The two composting strategies evaluated in poultry litter proved to be effective in the reduction of the diversity of ARGs, although without effect on abundance. Composting proved to be even more effective in the attenuation of ARGs in layer manure, reducing the abundance and diversity of these genes. In chapter 2, the effect of the poultry manures, chicken litter and layer manure and their composts was evaluated on the ARGs of soils cultivated with the fresh vegetable salad rocket, Eruca sativa. It was observed that both types of composts do not increase the total abundance of ARGs, although both increase the richness and Shannon diversity in soils, to the same extent as fresh manures. Furthermore, it was observed that all organic fertilizers applied increased some specific ARGs classes. Finally, the application of manure and compost caused an increase in the abundance of plasmids, indicating a possible increase in horizontal transmission of ARGs. Thus, both fresh poultry manure and its composts can increase the dissemination of ARGs in soils. Chapter 3 explored the effect of successive applications of poultry or swine manure composts on ARGs in commercial field agricultural soils. Additionally, the interaction of physicochemical properties and heavy metals in the modulation of the resistome in these soils was evaluated. Soils with heavy metals in high concentrations are the dominant factor in the modulation of the resistome, attenuating the effect of compost application. The main factors modulating ARGs in soils with normal levels of heavy metals are available P and pH, which present elevated levels due to the successive application of composts. The heavy metal resistance genes for Cu and Zn are the most related to different classes of ARGs. The genus Bradyrhizobium stands out as a host for a large number of ARGs. This thesis highlights that composting reduces the diversity of ARGs in the manures. However, the application of composts to the soil increases the diversity of ARGs, whether with the successive application of composts in field soils or a single application in soils in a pot experiment. Physicochemical factors involved in fertility and heavy metals play an important role on the modulation of ARGs in soils.

Manure, which contains large amounts of antibiotics and antibiotic resistance genes (ARGs), is widely used in agricultural soils and may lead to the evolution and dispersal of ARGs in the soil environment. In the present study, soils that received manure or chemical fertilizers for 15 years were sampled on the North China Plain (NCP), which is one of the primary areas of intensive agriculture in China. High-throughput quantitative PCR and sequencing technologies were employed to assess the effects of long-term manure or chemical fertilizer application on the distribution of ARGs and microbial communities. A total of 114 unique ARGs were successfully amplified from all soil samples. Manure application markedly increased the relative abundance and detectable numbers of ARGs, with up to 0.23 copies/16S rRNA gene and 81 unique ARGs. The increased abundance of ARGs in manure-fertilized soil was mainly due to the manure increasing the abundance of indigenous soil ARGs. In contrast, chemical fertilizers only moderately affected the diversity of ARGs and had no significant effect on the relative abundance of the total ARGs. In addition, manure application increased the abundance of mobile genetic elements (MGEs), which were significantly and positively correlated with most types of ARGs, indicating that horizontal gene transfer via MGEs may play an important role in the spread of ARGs. Furthermore, the application of manure and chemical fertilizers significantly affected microbial community structure, and variation partitioning analysis showed that microbial community shifts represented the major driver shaping the antibiotic resistome. Taken together, our results provide insight into the long-term effects of manure and chemical fertilization on the dissemination of ARGs in intensive agricultural ecosystems.

Uncovering the prevalence and drivers of antibiotic resistance genes in soils across different land-use types

Varying characteristics and driving mechanisms of antibiotic resistance genes in farmland soil amended with high-density polyethylene microplastics

Soil is an important environmental reservoir of antibiotic resistance genes (ARGs), which are increasingly recognized as environmental contaminants. Methods to assess the risks associated with the acquisition or transfer of resistance mechanisms are still underdeveloped. Quantification of background levels of antibiotic resistance genes and what alters those is a first step in understanding our environmental resistome. Toward this goal, 62 samples were collected over 3 years from soils near the 30-year old Gondwana Research Station and for 4 years before and during development of the new Jang Bogo Research Station, both at Terra Nova Bay in Antarctica. These sites reflect limited and more extensive human impact, respectively. A qPCR array with 384 primer sets targeting antibiotic resistance genes and mobile genetic elements (MGEs) was used to detect and quantify these genes. A total of 73 ARGs and MGEs encompassing eight major antibiotic resistance gene categories were detected, but most at very low levels. Antarctic soil appeared to be a common reservoir for seven ARGs since they were present in most samples (42%-88%). If the seven widespread genes were removed, there was a correlation between the relative abundance of MGEs and ARGs, more typical of contaminated sites. There was a relationship between ARG content and distance from both research stations, with a significant effect at the Jang Bogo Station especially when excluding the seven widespread genes; however, the relative abundance of ARGs did not increase over the 4 year period. Silt, clay, total organic carbon, and SiO2 were the top edaphic factors that correlated with ARG abundance. Overall, this study identifies that human activity and certain soil characteristics correlate with antibiotic resistance genes in these oligotrophic Antarctic soils and provides a baseline of ARGs and MGEs for future comparisons.

Manure application is the primary input route for antibiotic resistance genes (ARGs) in farmland soil. This study investigated the effects of varying the rates of five chicken manure applications on the accumulation and distribution of ARGs across different soil depths (0–20, 20–40, and 40–60 cm) using metagenomic sequencing. The results revealed that the distribution of ARGs in farmland soil was closely linked to soil depth and influenced to some extent by the fertilizer quantity after 30 days of fertilization. ARGs were predominantly concentrated in the surface soil and exhibited a significant decrease in type and abundance with an increased soil depth. Compared with soil treated with chemical fertilizers alone, chicken manure-treated surface soil presented a higher diversity and abundance of ARGs. However, the diversity and abundance of ARGs did not increase proportionally with the increasing ratios of chicken manure application (0, 25, 50, 75, and 100%). ARGs in soil primarily conferred resistance to host bacteria through antibiotic efflux pumps (~33%), antibiotic target alteration (~31%), antibiotic inactivation (~20%), and antibiotic target protection (~8%). Correlation analysis involving ARGs and soil microorganisms revealed widespread multidrug resistance among soil microorganisms. Furthermore, two genera of human pathogenic bacteria (Pseudomonas sp. and Listeria sp.) were identified as potential microbial hosts of ARGs in all treatments. Correlation analysis involving ARGs and environmental factors indicated that soil ARGs are predominantly influenced by heavy metals and microorganisms. This paper offers valuable insights for environmental risk assessments regarding the utilization of livestock manure resources. Additionally, it furnishes a scientific foundation for farmland application strategies pertaining to livestock manure.

Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil

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.

Does nano silver promote the selection of antibiotic resistance genes in soil and plant?

Antibiotic resistance genes in layer farms and their correlation with environmental samples

Microplastics enhance the enrichment and dissemination of antibiotic resistance genes in aquatic environments: A meta-analysis.