Antibiotic Resistomes and Microbiomes in the Surface Water along the Code River in Indonesia Reflect Drainage Basin Anthropogenic Activities.

Water and sanitation are important factors in the emergence of antimicrobial resistance in low- and middle-income countries. Drug residues, metals, and various wastes foster the spread of antibiotic resistance genes (ARGs) with the help of mobile genetic elements (MGEs), and therefore, rivers receiving contaminants and effluents from multiple sources are of special interest. We followed both the microbiome and resistome of the Code River in Indonesia from its pristine origin at the Merapi volcano through rural and then city areas to the coast of the Indian Ocean. We used a SmartChip quantitative PCR with 382 primer pairs for profiling the resistome and MGEs and 16S rRNA gene amplicon sequencing to analyze the bacterial communities. The community structure explained the resistome composition in rural areas, while the city sampling sites had lower bacterial diversity and more ARGs, which correlated with MGEs, suggesting increased mobility potential in response to pressures from human activities. Importantly, the vast majority of ARGs and MGEs were no longer detectable in marine waters at the ocean entrance. Our work provides information on the impact of different influents on river health as well as sheds light on how land use contributes to the river resistome and microbiome.

Biological units of antimicrobial resistance and strategies for their containment in animal production.

The increasing prevalence of antimicrobial-resistant bacterial infections has ushered in a major global public health crisis. Judicious or restricted antimicrobial use in animal agriculture, aiming to confine the use for the treatment of infections, is the most commonly proposed solution to reduce selection pressure for resistant bacterial strains and resistance genes. However, a multifaceted solution will likely be required to make acceptable progress in reducing antimicrobial resistance, due to other common environmental conditions maintaining antimicrobial resistance and limited executionary potential as human healthcare and agriculture will continue to rely heavily on antimicrobials in the foreseeable future. Drawing parallels from systematic approaches to the management of infectious disease agents and biodiversity loss, we provide examples that a more comprehensive approach is required, targeting antimicrobial resistance in agroecosystems on multiple fronts simultaneously. We present one such framework, based on nested biological units of antimicrobial resistance, and describe established or innovative strategies targeting units. Some of the proposed strategies are already in use or ready to be implemented, while some require further research and discussion among scientists and policymakers. We envision that antimicrobial resistance mitigation strategies for animal agriculture combining multiple tools would constitute powerful ecosystem-level interventions necessary to mitigate antimicrobial resistance.

Antimicrobial use, biosecurity, herd characteristics, and antimicrobial resistance in indicator Escherichia coli in ten Finnish pig farms.

We investigated connections between antimicrobial use (AMU), biosecurity, and the numbers of pigs and staff in ten Finnish farrow-to-finish herds. Data on AMU in each herd were collected for 12 months. AMU was quantified as treatment incidences per 1000 days at risk (TI) using the consensus defined daily dose calculation. Biosecurity was scored using the Biocheck.UGent™ system. We also examined antimicrobial resistance patterns of indicator E. coli isolated from faeces of selected pigs. In each herd, two groups of five pigs were formed: 1) antimicrobial treatment group (ANT: at least one pig in the litter was identified as sick and treated with antimicrobials) and 2) non-antimicrobial treatment group (NON: the litter was not medicated). Faecal samples were taken from these pigs at 5 and 22 weeks of age, cultured, and indicator E. coli isolates were tested for antimicrobial susceptibilities. The AMU varied considerably between the herds. Altogether, most of the antimicrobial treatment courses were assigned to weaned piglets. When AMU was quantified as TIs, suckling piglets had the highest TI (mean 46.6), which was significantly higher (P < 0.05) than TIs in fatteners and breeders (9.3 and 7.3, respectively). The difference between TI in suckling and TI in weaned piglets (19.1) was not statistically significant. There was a tendency for a negative correlation between the TI in breeders and the number of sows (r = -0.56, P = 0.09). Larger herds had higher external biosecurity scores than smaller herds (LS-means; 72 vs. 66, P < 0.05). The proportions of E. coli isolates resistant to at least one antimicrobial were higher in pigs at 5 weeks than in pigs at 22 weeks of age (Binomial proportion means; 40.5 % vs. 15.5 %, P < 0.05); as well as proportions of isolates resistant to at least three antimicrobial classes (23.0 % vs. 3.7 %, P < 0.01). These proportions did not differ between the ANT and NON groups at either 5 or 22 weeks of age (P> 0.05). We found few connections: enhanced external biosecurity levels found in the large herds co-occurred with lower use of antimicrobials and herds with low biosecurity scores - especially in the internal subcategories - appeared to have higher proportions of resistant isolates. Conclusively, we suggest that enhancing internal biosecurity might contribute to a reduction in the spreading of antimicrobial resistance in pig herds.

Swine growth promotion with antibiotics or alternatives can increase antibiotic resistance gene mobility potential.

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.

Field-compatible protocol for detecting tetracyclines with bioluminescent bioreporters without pipetting steps.

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.

Low black carbon concentration in agricultural soils of central and northern Ethiopia.

Soil carbon (C) represents the largest terrestrial carbon stock and is key for soil productivity. Major fractions of soil C consist of organic C, carbonates and black C. The turnover rate of black C is lower than that of organic C, and black C abundance decreases the vulnerablility of soil C stock to decomposition under climate change. The aim of this study was to determine the distribution of soil C in different pools and impact of agricultural management on the abundance of different species. Soil C fractions were quantified in the topsoils (0-15cm) of 23 sites in the tropical highlands of Ethiopia. The sites in central Ethiopia represented paired plots of agroforestry and adjacent control plots where cereal crops were traditionally grown in clayey soils. In the sandy loam and loam soils of northern Ethiopia, the pairs represented restrained grazing with adjacent control plots with free grazing, and terracing with cereal-based cropping with adjacent control plots without terracing. Soil C contained in carbonates, organic matter and black C along with total C was determined. The total C median was 1.5% (range 0.3-3.6%). The median proportion of organic C was 85% (range 53-94%), 6% (0-41%) for carbonate C and 6% (4-21%) for black C. An increase was observed in the organic C and black C fractions attributable to agroforestry and restrained grazing. The very low concentration of the relatively stable black C fraction and the dominance of organic C in these Ethiopian soils suggest vulnerability to degradation and the necessity for cultivation practices maintaining the C stock.

Influence of Manure Application on the Environmental Resistome under Finnish Agricultural Practice with Restricted Antibiotic Use.

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.

Challenges in the Measurement of Antibiotics and in Evaluating Their Impacts in Agroecosystems: A Critical Review.

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 sub-ng g 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.

Fertilizing with Animal Manure Disseminates Antibiotic Resistance Genes to the Farm Environment.

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.