The co-occurrence of antibiotic-resistance genes (ARGs) and mobile genetic elements (MGEs) in farm environments can potentially foster the development of antibiotic-resistant pathogens. We studied the resistome of Finnish dairy and swine farms where use of antibiotics is limited to treating bacterial infections and manure is only applied from April to September. The resistome of manure, soil, and tile drainage water from the ditch was investigated from the beginning of the growing season until forage harvest. The relative ARG and MGE abundance was measured using a qPCR array with 363 primer pairs. Manure samples had the highest abundance of ARGs and MGEs, which increased during storage. Immediately following land application, the ARGs abundant in manure were detected in soil, but their abundance decreased over time with many becoming undetectable. This suggests that increases in ARG abundances after fertilizing are temporary and occur annually under agricultural practices that restrict antibiotic use. A few of the ARGs were detected in the ditch water, but most of them were undetected in the manure. Our results document the dissipation and dissemination off farm of ARGs under Finnish limited antibiotic use and suggest that such practices could help reduce the load of antibiotic-resistance genes in the environment.

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The dissemination of antibiotic resistance genes to the environment is an important factor causing increased prevalence of resistant pathogens. Manure is an important fertilizer, but it contains diverse resistance genes. Therefore, its application to fields may lead to increased abundance of resistance genes in the environment. Farming environments exposed to animal manure have not been studied extensively in countries with comparably low antibiotic use, such as Finland. The effects of manure storage and application to fields on the abundance of resistance genes were studied on two dairy cattle farms and two swine farms in southern Finland. Samples were taken from farms during the 2013 cropping season. Copy numbers of carbapenem (), sulfonamide (), and tetracycline () resistance genes were measured with quantitative polymerase chain reaction, and the data were analyzed using linear mixed models. The relative abundance of antibiotic resistance genes increased about fourfold in soil after manure application. Carbapenemase encoding was detected on all of the studied farms, which indicated that the gene is dispersed in the farm environment. The relative abundance of antibiotic resistance genes increased in stored manure compared with fresh manure roughly fivefold. This study shows that antibiotic resistance genes are disseminated on Finnish production animal farms. The spreading of resistance genes in farm-associated environments could possibly be limited by experimenting with new manure handling methods that could reduce the abundance of the genes in manure used for land application.

Large quantities of antibiotics are used in agricultural production, resulting in their release to agroecosystems through numerous pathways, including land application of contaminated manure, runoff from manure-fertilized fields, and wastewater irrigation of croplands. Antibiotics and their transformation products (TPs) exhibit a wide range of physico-chemical and biological properties and thus present substantive analytical challenges. Advances in the measurement of these compounds in various environmental compartments (plants, manure, soil, sediment, and water) have uncovered a previously unrealized landscape of antibiotic residues. These advanced multiresidue methods, designed to measure subng g -1 concentrations in complex mixtures, remain limited by the inherent intricacy of the sample matrices and the difficultly in eliminating interferences that affect antibiotic detection. While efficient extraction methods combined with high sensitivity analysis by liquid chromatography/mass spectrometry can provide accurate quantification of antibiotics and their TPs, measured concentrations do not necessarily reflect their bioavailable fractions and effects in the environment. Consequently, there is a need to complement chemical analysis with biological assays that can provide information on bioavailability, biological activity, and effects of mixtures. Enzyme-linked immunosorbent assays (ELISA), often used as screening tools for antibiotic residues, may be useful for detecting the presence of structurally related antibiotic mixtures but not their effects. Other tools, including bioreporter assays, hold promise in measuring bioavailable antibiotics and could provide insights on their biological activity. Improved assessment of the ecological and human health risks associated with antibiotics in agroecosystems requires continued advances in analytical accuracy and sensitivity through improvements in sample preparation, instrumentation, and screening technologies.

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Even though the use of antibiotics for food-producing animals may contribute to the emergence of antimicrobial resistance, antibiotics are still used as growth promoters. Due to consumer and regulatory pressures, the use of alternatives to antibiotics as growth promoters is increasing, thus more information is needed on their capability to disseminate antimicrobial resistance compared to antibiotics. We investigated the impacts of carbadox (antibiotic), copper sulfate and zinc oxide (metals) and mushroom powder (natural product) on the pig fecal resistome and microbiome. Antibiotic resistance gene (ARG) and mobile genetic element (MGE) abundances were measured using a high-throughput qPCR array with 382 primer pairs. Bacterial community composition was determined by 16S rRNA gene sequencing. More ARGs co-occurred with MGEs in the growth promoter group samples than in the control group samples. Community composition could not be linked to resistome in the growth promoter group samples, indicating a potential decoupling of ARGs and phylogeny. Additionally, machine-learning methods aided in defining the community and resistome differences in response to treatments. Since increased ARG mobility potential was the primary response to the dietary additives used in this study, we suggest that ARG mobility should be considered when designing antimicrobial use policies and antimicrobial resistance surveillances.

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Whole-cell bioreporters are living organisms and thus using them for detecting environmental contaminants would reflect biological effects of these pollutants. However, bioreporters are not widely used in field studies. Many of the bioreporter field protocols are suitable for liquid samples or include pipetting steps, which is a demanding task outside the laboratory. We present a bioreporter protocol without pipetting or sample type requirements. The protocol utilizes polyester swabs, commonly used in cleanroom technology. As an example contaminant, we used tetracycline and generated test samples with known concentrations up to the maximum tetracycline residue limit of milk set by the European Union (EU) regulation. The matrices of the test samples were Milli-Q water, milk and soil. The swabs were first dipped in the bioreporter cell cultures and then to test samples and luminescence was measured after incubation. The standard deviation of measurements from ten replicate swabs was in the same range as commonly in pipetting protocols (4-19%). The test samples with lowest tetracycline concentration (5 ng mL −1 ) were distinguished from the control samples (0 ng mL −1 tetracycline). Our results show that swabs can be used together with luminescent whole cell bioreporters, making it possible to conduct the measurements in field conditions.