Antibiotic Resistance in Agricultural Soil and Crops Associated to the Application of Cow Manure-Derived Amendments from Conventional and Organic Livestock Farms

he 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 cropThis work has been financially supported by the Basque Government (projects: URAGAN and KONTRAE-Elkartek-KK-2020-00007) and the Spanish Ministry of Science and Innovation (project: PRADA PID2019-110055RB-C21). LJ was the recipient of a predoctoral fellowship from the Department of Economic Development and Infrastructure of the Basque Governmen

Agricultural Soils Amended with Thermally-Dried Anaerobically-Digested Sewage Sludge Showed Increased Risk of Antibiotic Resistance Dissemination

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 alpha-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.This work has been financially supported by the Basque Government (projects: URAGAN and KONTRAE-Elkartek-KK2020-00007) and the Spanish Ministry of Science and Innovation (project: PRADA PID2019-110055RB-C21). LJ was the recipient of a predoctoral fellowship from the Department of Economic Development and Infrastructure of the Basque Government

Antibiotikoen erresistentziak agroekosistemetan

Antibiotics have become an indispensable tool to combat bacterial infections in medicine and veterinary medicine. The abuse and misuse of antibiotics has led to the emergence of one of the most important health challenges, as bacteria have developed widespread resistance to antibiotics. This global problem must be analysed from a One Health perspective, as antibiotic resistance is shared, spread and multiplied between humans, animals and the environment. For example, the application of organic fertilizers to soil can improve soil health and crop yields. However, this practice carries risks, as antibiotics not fully metabolised by humans or animals can find their way to soil. In addition to the antibiotics, the use of such organic fertilisers involves the spread of antibiotic-resistant genes, antibiotic-resistant bacteria and mobile genetic elements. Indeed, the continuous introduction of antibiotics into soils creates a potential threat to indigenous bacteria; antibi-otic-resistant bacteria multiply and alter the biodiversity of indigenous bacterial communities. This wide dispersal makes the use of antibiotics increasingly ineffective in the treatment of pathogenic resistant bacterial infections. This review first tries to explain the origin and mechanisms of antibiotic-resistant genes and antibiotics. Next, the use of organic fertilisers of human or animal origin in soils is mentioned, as they are a major source for the spread of antibiotics and antibiotic resistance in the environment. The environmental impact of antibiotic resistance is then explained, following the exposure pathway from crops to humans. Finally, we suggest using some management processes that could be useful to reduce the risk of antibiotic resistance in livestock-derived organic fertilisers.; Antibiotikoak ezinbesteko tresna bilakatu dira medikuntza eta albaitaritzan bakterio-infekzioei aurre egiteko. Antibiotikoen gehiegizko erabilerak eta erabilera okerrak osasun-erronka garrantzitsuenetako bat bizkortu dute, bakte-rioek antibiotikoekiko erresistentzia zabala garatu baitute. Mundu mailako arazo hau Osasun Bakarraren ikuspegitik aztertu behar da, antibiotikoekiko erresistentziak gizakien, animalien eta ingurumenaren artean partekatu, barreiatu eta ugaritzen baitira. Adibidez, lurzoruetan ongarri organikoak aplikatzeak lurren osasuna eta uzten etekina hobetu ditzake. Hala ere, praktika honek arriskuak dakartza, lurzorura iritsi baitaitezke gizaki nahiz abereek guztiz metabolizatu ez dituzten antibiotikoak. Antibiotikoa hedatzeaz gain, antibiotikoekiko erresistenteak diren geneak, bakterioak eta elementu genetiko mugikorrak ba-rreiatzea dakar mota honetako ongarri organikoen erabilerak. Izan ere, lurzoruetan etengabe antibiotikoak sartzeak mehatxu potentziala sortzen du bertako bakterioentzat; bakterio antibiotikoekiko erresistenteak ugaritu egiten dira eta bertako bakterio-komunitateen bioaniztasuna aldatzen dute. Barreiadura zabal honen ondorioz, gero eta gehiago, antibiotikoen erabilera ez da eraginkorra izaten ari erresistente diren patogenoen infekzioen tratamenduetan. Berrikuspen honetan, lehenik, antibiotikoen nahiz antibiotikoekiko erresistente diren geneen jatorria eta mekanismoak azaltzen dira. Jarraian, lurzoruetan egiten den giza edo animalia-jatorriko ongarri organikoen erabilera aipatzen da, antibiotikoak eta erresistentziak ingurumenean barreiatzeko sarbide garrantzitsuak direlako. Ondoren, antibiotikoekiko erresistentziek ingurumenean duten eragina azaltzen da, esposizio-bidea jarraituz kultiboetatik gizakietara iritsi arte. Azkenik, abereetatik eratorritako ongarri organikoetan anti-biotikoekiko erresistentzien arriskua gutxitzeko erabilgarriak izan litezkeen kudeaketa-prozesuak proposatzen ditugu

Agroecosystem Health Cards: A Practical Tool for Sustainable Management of Grasslands

The traditional grazing activity carried out for centuries in mountainous areas of the Basque Country (Northern Atlantic Spain) facilitated the presence of different extensive pasture habitats, such as those included in the Gorbeia Natural Park and surrounding valleys (43° 02’N, 2° 49’W). Currently, these pastures are highly valued due to the ecosystem services they provide. In this context, one of the main objectives of the LIFE-SOILMONTANA project (ref. LIFE 10 NAT/ES/579) is to develop a practical tool that allows grassland managers (farmers, scientists and authorities) to auto-evaluate the suitability of alternative agronomic practices in relation to the conservation of these ecosystem services through the conservation of their biodiversity, especially soil biodiversity

Zero-valent iron nanoparticles and organic amendment assisted rhizoremediation of mixed contaminated soil using Brassica napus

Soil is one of our most important natural resources. Regrettably, the expansion of human activities has resulted in the degradation of the soil resource due to contamination with a myriad of organic and inorganic compounds. The remediation of mixed contaminated soils, i.e. soils contaminated with both organic compounds and metals, is challenging as it requires actions to simultaneously decrease metal-induced risks and organic contaminant concentrations. Here, we evaluated the effect of the addition of zero-valent iron nanoparticles (nanoremediation) and organic amendments (biostimulation) on the rhizoremediation, using Brassica napus plants, of soil simultaneously contaminated with zinc (2500 mg kg(-1)) and lindane (100 mg kg(-1)). We used a factorial design with three factors (amendment, nZVI, plant) to evaluate the impact of the applied remediation actions on lindane and extractable Zn concentrations, as well as on soil health recovery as manifested by the values of different soil microbial indicators. The studied microbial indicators were not negatively affected by nZVI application. The application of nZVI was the most effective factor regarding the targeted reduction in lindane concentration (51% average reduction in nZVI treated soils). The highest reduction in extractable Zn was achieved in the presence of B. napus, nZVI and organic amendments (99 and 95% reduction in horse manure-amended and sewage sludge-amended soils, respectively). The combination of the three factors led to the highest values of soil microbial indicators (although a significant triple interaction was not observed for all parameters), especially when combined with horse manure amendment: in this case, prokaryotic richness increased by 64%, respiration by 376%, eukaryotic abundance by 333%, and prokaryotic abundance by 437%, compared to untreated soils. The combination of remediation approaches (rhizoremediation with B. napus, nanoremediation with nZVI, biostimulation with organic amendments) can help overcome the limitations of each individual strategy.This work was supported by the European Union through Interreg SUDOE Program (Project Phy2SUDOE SOE4/P5/E1021), the Spanish Ministry of Economy, Industry, and Competitiveness through NANORRIZORREM-2 (AGL2016-76592-R) and PRADA projects (PID2019-110055RB-C21 and PID2019-110055RB-789 C22), MCIN/AEI/10.13039/501100011033/FEDER, UE, and Consolidated Research Group of the Basque Government (GV ITO18-16). JH is the recipient of a predoctoral fellowship from the Spanish Ministry of Science and Innovation. We sincerely thank Dr. Fernando Blanco for technical assistance

The Application of Nanoscale Zero-Valent Iron Promotes Soil Remediation While Negatively Affecting Soil Microbial Biomass and Activity

The use of nanoscale zero-valent iron (nZVI) particles for soil remediation is gaining increased attention. However, there are concerns about the potential adverse effects of nZVI on soil microbial communities and, hence, soil quality. The objective of this study was to assess the impact of the application of nZVI on soil microbial parameters (as bioindicators of soil quality) during the nanoremediation of soil artificially contaminated with lindane (10 mg gamma-HCH kg(-1) DW soil) and zinc (1,500 mg Zn kg(-1) DW soil). nZVI particles were also applied to non-contaminated soil. The following nZVI doses were applied twice: 0, 0.25, 0.5, 1, and 2 mg nZVI g(-1) DW soil. Nine weeks after nZVI application, the following parameters were determined in soil samples: lindane concentration, extractable Zn concentration, microbial biomass carbon (C-MB), bacterial and fungal abundance (gene copy numbers by qPCR), enzyme activities (beta-glucosidase, beta-glucosaminidase, xylosidase, acid phosphatase, arylsulphatase, and Leu-aminopeptidase) and bacterial richness by ARISA profiles. The application of nZVI reduced lindane and extractable Zn concentrations following a dose-dependent pattern. The presence of contaminants reduced soil microbial biomass and activity. The application of nZVI negatively affected the microbial quality of the contaminated soil but not of the non-contaminated soil. This observation might reflect a "stress-on-stress" effect, i.e., the already stressed microbial populations present in the contaminated soil were more sensitive to the application of nZVI (a second stress) than those present in the non-contaminated soil.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness through NANORRIZORREM-2 Project (AGL2016-76592-R)

The Application of Nanoscale Zero-Valent Iron Promotes Soil Remediation While Negatively Affecting Soil Microbial Biomass and Activity

The use of nanoscale zero-valent iron (nZVI) particles for soil remediation is gaining increased attention. However, there are concerns about the potential adverse effects of nZVI on soil microbial communities and, hence, soil quality. The objective of this study was to assess the impact of the application of nZVI on soil microbial parameters (as bioindicators of soil quality) during the nanoremediation of soil artificially contaminated with lindane (10 mg γ-HCH kg−1 DW soil) and zinc (1,500 mg Zn kg−1 DW soil). nZVI particles were also applied to non-contaminated soil. The following nZVI doses were applied twice: 0, 0.25, 0.5, 1, and 2 mg nZVI g−1 DW soil. Nine weeks after nZVI application, the following parameters were determined in soil samples: lindane concentration, extractable Zn concentration, microbial biomass carbon (CMB), bacterial and fungal abundance (gene copy numbers by qPCR), enzyme activities (β-glucosidase, β-glucosaminidase, xylosidase, acid phosphatase, arylsulphatase, and Leu-aminopeptidase) and bacterial richness by ARISA profiles. The application of nZVI reduced lindane and extractable Zn concentrations following a dose-dependent pattern. The presence of contaminants reduced soil microbial biomass and activity. The application of nZVI negatively affected the microbial quality of the contaminated soil but not of the non-contaminated soil. This observation might reflect a “stress-on-stress” effect, i.e., the already stressed microbial populations present in the contaminated soil were more sensitive to the application of nZVI (a second stress) than those present in the non-contaminated soil

Bioindicators of soil quality: methodological tool for the assessment of the efficiency of a phytoremediation process

La fitorremediación es una tecnología emergente que utiliza plantas y microorganismos asociados para descontaminar suelos, aire, sedimentos, y aguas. Esta fitotecnología se basa en la capacidad de algunas especies vegetales para tolerar, absorber, acumular y degradar compuestos contaminantes. El objetivo último de un proceso fitorremediador de suelos contaminados no debe ser solamente eliminar el contaminante sino sobre todo recuperar la calidad del suelo, entendida ésta como la capacidad de este recurso para realizar sus funciones de forma sostenible. Los indicadores biológicos de la calidad del suelo, en especial aquellos relacionados con la biomasa, actividad y biodiversidad de las comunidades microbianas, presentan un enorme potencial como herramienta monitorizadora de la eficacia de un proceso fitorremediador. Estos bioindicadores son de gran utilidad para evaluar el efecto que tienen los procesos fitoextractores de suelos contaminados con metales sobre la calidad del suelo.Phytoremediation is an emerging technology that uses plants and their associated microorganisms to depollute soils, air, sediments and waters. This phytotechnology is based on the capacity of some plant species to tolerate, absorb, accumulate and degrade pollutants. The ultimate goal of a phytoremediation process must be not only to remove the contaminant from the polluted soil but to restore soil quality, i.e. its capacity to function sustainably. Biological indicators of soil quality, especially those related to the biomass, activity and biodiversity of soil microbial communities, have great potential as monitoring tools for the assessment of the efficiency of a phytoremediation process. These bioindicators are particularly useful for the assessment of the effect of metal phytoextraction processes on soil quality

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems

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