No abstract

In cow farms, the interaction between animal and environmental microbiomes creates hotspots for antibiotic resistance dissemination. A shotgun metagenomic approach was used to survey the resistome risk in five dairy cow farms. To this purpose, 10 environmental compartments were sampled: 3 of them linked to productive cows (fresh slurry, stored slurry, slurry-amended pasture soil); 6 of them to nonproductive heifers and dry cows (faeces, fresh manure, aged manure, aged manure-amended orchard soil, vegetables-lettuces and grazed soil); and, finally, unamended control soil. The resistome risk was assessed using MetaCompare, a computational pipeline which scores the resistome risk according to possible links between antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and human pathogens. The resistome risk decreased from slurry and manure microbiomes to soil and vegetable microbiomes. In total (sum of all the compartments), 18,157 ARGs were detected: 24% related to ansamycins, 21% to multidrugs, 14% to aminoglycosides, 12% to tetracyclines, 9% to β-lactams, and 9% to macrolide-lincosamide-streptogramin B. All but two of the MGE-associated ARGs were only found in the animal dejections (not in soil or vegetable samples). Several ARGs with potential as resistome risk markers (based on their presence in hubs of cooccurrence networks and high dissemination potential) were identified. As a precautionary principle, improved management of livestock dejections is necessary to minimize the risk of antibiotic resistance.

The application of organic amendments to agricultural soil can enhance crop yield, while improving the physicochemical and biological properties of the recipient soils. However, the use of manure-derived amendments as fertilizers entails environmental risks, such as the contamination of soil and crops with antibiotic residues, antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In order to delve into these risks, we applied dairy cow manure-derived amendments (slurry, fresh manure, aged manure), obtained from a conventional and an organic farm, to soil. Subsequently, lettuce and wheat plants were grown in the amended soils. After harvest, the abundance of 95 ARGs and MGE-genes from the amended soils and plants were determined by high-throughput qPCR. The structure of soil prokaryotic communities was determined by 16S rRNA amplicon sequencing and qPCR. The absolute abundance of ARGs and MGE-genes differed between treatments (amended vs. unamended), origins of amendment (conventional vs. organic), and types of amendment (slurry vs. fresh manure vs. aged manure). Regarding ARG-absolute abundances in the amendments themselves, higher values were usually found in slurry vs. fresh or aged manure. These abundances were generally higher in soil than in plant samples, and higher in wheat grain than in lettuce plants. Lettuce plants fertilized with conventional amendments showed higher absolute abundances of tetracycline resistance genes, compared to those amended with organic amendments. No single treatment could be identified as the best or worst treatment regarding the risk of antibiotic resistance in soil and plant samples. Within the same treatment, the resistome risk differed between the amendment, the amended soil and, finally, the crop. In other words, according to our data, the resistome risk in manure-amended crops cannot be directly inferred from the analysis of the amendments themselves. We concluded that, depending on the specific question under study, the analysis of the resistome risk should specifically focus on the amendment, the amended soil or the crop.

The application of sewage sludge (SS) to agricultural soil can help meet crop nutrient requirements and enhance soil properties, while reusing an organic by-product. However, SS can be a source of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), resulting in an increased risk of antibiotic resistance dissemination. We studied the effect of the application of thermally-dried anaerobically-digested SS on (i) soil physicochemical and microbial properties, and (ii) the relative abundance of 85 ARGs and 10 MGE-genes in soil. Soil samples were taken from a variety of SS-amended agricultural fields differing in three factors: dose of application, dosage of application, and elapsed time after the last application. The relative abundance of both ARGs and MGE-genes was higher in SS-amended soils, compared to non-amended soils, particularly in those with a more recent SS application. Some physicochemical parameters (i.e., cation exchange capacity, copper concentration, phosphorus content) were positively correlated with the relative abundance of ARGs and MGE-genes. Sewage sludge application was the key factor to explain the distribution pattern of ARGs and MGE-genes. The 30 most abundant families within the soil prokaryotic community accounted for 66% of the total variation of ARG and MGE-gene relative abundances. Soil prokaryotic α-diversity was negatively correlated with the relative abundance of ARGs and MGE-genes. We concluded that agricultural soils amended with thermally-dried anaerobically-digested sewage sludge showed increased risk of antibiotic resistance dissemination.

The use of manure as a fertilizer is a common agricultural practice that can improve soil physicochemical and biological properties. However, antibiotics and their metabolites are often present, leading to the adaptation of soil bacterial communities to their presence. The aim of this study was to assess the effects of the extensively used, broad-spectrum antibiotic oxytetracycline on soil microbial community adaptation using a pollution-induced community tolerance assay. Manure-amended soil was spiked with oxytetracycline (0, 2, 20, 60, 150, and 500 mg kg−1) three times every ten days in the selection phase. The detection phase was conducted in Biolog EcoPlates with a second oxytetracycline exposure (0, 5, 20, 40, 60, and 100 mg L−1). All treatments demonstrated decreased metabolic activity after exposure to ≥ 5 mg L−1 oxytetracycline during the detection phase. Meanwhile, a significant increase in tolerance was observed following exposure to ≥ 20 mg oxytetracycline per kg soil during the selection phase. Therefore, the pollution-induced community tolerance approach with Biolog EcoPlates was a useful system for the detection of antibiotic selection pressures on soil bacterial communities. It is important to properly manage animal waste before their application to the soil to reduce the occurrence of antibiotic-resistance in the environment.

No abstract

Agricultural fertilization with organic amendments of animal origin often leads to antibiotic resistance dissemination. In this study, we evaluated the effect of different treatments (anaerobic digestion, biochar application, ozonation, zerovalent iron nanoparticle application, and spent mushroom substrate addition) on the resistome in dairy cow manure-derived amendments (slurry, manure, and compost). Anaerobic digestion and biochar application resulted in the highest reduction in antibiotic resistance gene (ARG) and mobile genetic element (MGE) gene abundance. These two treatments were applied to cow manure compost, which was then used to fertilize the soil for lettuce growth. After crop harvest, ARG and MGE gene absolute and relative abundances in the soil and lettuce samples were determined by droplet digital PCR and high-throughput qPCR, respectively. Prokaryotic diversity in cow manure-amended soils was determined using 16S rRNA metabarcoding. Compared to untreated compost, anaerobic digestion led to a 38% and 83% reduction in sul2 and intl1 absolute abundances in the soil, respectively, while biochar led to a 60% reduction in intl1 absolute abundance. No differences in lettuce gene abundances were observed among treatments. We conclude that amendment treatments can minimize the risk of antibiotic resistance in agroecosystems.