Making waves: The NORMAN antibiotic resistant bacteria and resistance genes database (NORMAN ARB&ARG)-An invitation for collaboration to tackle antibiotic resistance.

With the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB&ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB&ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/.

Occurrence and human health risk assessment of antibiotics and their metabolites in vegetables grown in field-scale agricultural systems.

The occurrence of antibiotics (ABs) in four types of commercially grown vegetables (lettuce leaves, tomato fruits, cauliflower inflorescences, and broad bean seeds) was analyzed to assess the human exposure and health risks associated with different agronomical practices. Out of 16 targeted AB residues, seven ABs belonging to three groups (i.e., benzyl pyrimidines, fluoroquinolones, and sulfonamides) were above the method detection limit in vegetable samples ranging from 0.09 ng g-1 to 3.61 ng g-1 fresh weight. Data analysis (quantile regression models, principal component and hierarchical cluster analysis) showed manure application, irrigation with river water (indirect wastewater reuse), and vegetable type to be the most significant factors for AB occurrence in the targeted crops. Metabolites were detected in 70 of the 80 vegetable samples analyzed, and their occurrence was both plant- and compound-specific. In 73 % of the total samples, the concentration of AB metabolites was higher than the concentration of their parent compound. Finally, the potential human health risk estimated using the hazard quotient approach, based on the acceptable daily intake and the estimated daily intake, showed a negligible risk for human health from vegetable consumption. However, canonical-correspondence analysis showed that detected ABs explained 54 % of the total variation in AB resistance genes abundance in the vegetable samples. Thus, further studies are needed to assess the risks of antibiotic resistance promotion in vegetables and the significance of the occurrence of their metabolites.

Effects of prescription antibiotics on soil- and root-associated microbiomes and resistomes in an agricultural context.

The use of treated wastewater for crop irrigation is rapidly increasing to respond to the ever-growing demands for water and food resources. However, this practice may contribute to the spread of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in agricultural settings. To evaluate this potential risk, we analyzed microbiomes and resistomes of soil and Lactuca sativa L. (lettuce) root samples from pots irrigated with tap water spiked with 0, 20, or 100 µg L-1 of a mixture of three antibiotics (Trimethoprim, Ofloxacin, Sulfamethoxazole). The presence of antibiotics induced changes in bacterial populations, particularly in soil, as revealed by 16S rDNA sequence analysis. Parallel shotgun sequencing identified a total of 56 different ARGs conferring resistance against 14 antibiotic families. Antibiotic -treated samples showed increased loads of ARGs implicated in mutidrug resistance or in both direct and indirect acquired resistance. These changes correlated with the prevalence of Xantomonadales species in the root microbiomes. We interpret these data as indicating different strategies of soil and root microbiomes to cope with the presence of antibiotics, and as a warning that their presence may increase the loads of ARBs and ARGs in edible plant parts, therefore constituting a potential risk for human consumers.

Antibiotic resistance gene distribution in agricultural fields and crops. A soil-to-food analysis.

Despite the social concern about the generalization of antibiotic resistance hotspots worldwide, very little is known about the contribution of different potential sources to the global risk. Here we present a quantitative analysis of the distribution of Antibiotic Resistance Genes (ARGs) in soil, rhizospheric soil, roots, leaves and beans in tomato, lettuce and broad beans crops (165 samples in total), grown in nine commercial plots distributed in four geographical zones in the vicinity of Barcelona (North East Spain). We also analyzed five soil samples from a nearby forest, with no record of agricultural activities. DNA samples were analyzed for their content in the ARGs sul1, tetM, qnrS1, blaCTX-M-32, blaOXA-58, mecA, and blaTEM, plus the integron intI1, using qPCR methods. In addition, soil microbiomes from the different plots were analyzed by amplicon-targeted 16S rRNA gene sequencing. Our data show a decreasing gradient of ARG loads from soil to fruits and beans, the latter showing only from 0.1 to 0.01% of the abundance values in soil. The type of crop was the main determinant for both ARG distribution and microbiome composition among the different plots, with minor contributions of geographic location and irrigation water source. We propose that soil amendment and/or fertilization, more than irrigation water, are the main drivers of ARG loads on the edible parts of the crop, and that they should therefore be specifically controlled.

Antibiotic resistance genes distribution in microbiomes from the soil-plant-fruit continuum in commercial Lycopersicon esculentum fields under different agricultural practices.

While the presence of antibiotic resistance genes (ARGs) in agricultural soils and products has been firmly established, their distribution among the different plant parts and the contribution of agricultural practices, including irrigation with reclaimed water, have not been adequately addressed yet. To this end, we analyzed the levels of seven ARGs (sul1, blaTEM, blaCTX-M-32, mecA, qnrS1, tetM, blaOXA-58), plus the integrase gene intl1, in soils, roots, leaves, and fruits from two commercial tomato fields irrigated with either unpolluted groundwater or from a channel impacted by treated wastewater, using culture-independent, quantitative real-time PCR methods. ARGs and intl1 sequences were found in leaves and fruits at levels representing from 1 to 10% of those found in roots or soil. The relative abundance of intl1 sequences correlated with tetM, blaTEM, and sul1 levels, suggesting a high horizontal mobility potential for these ARGs. High-throughput 16S rDNA sequencing revealed microbiome differences both between sample types (soil plus roots versus leaves plus fruits) and sampling zones, and a correlation between the prevalence of Pseudomonadaceae and the levels of different ARGs, particularly in fruits and leaves. We concluded that both microbiome composition and ARGs levels in plants parts, including fruits, were likely influenced by agricultural practices.

Distribution of antibiotic resistance genes in soils and crops. A field study in legume plants (Vicia faba L.) grown under different watering regimes.

Social concern has raised during the last years due to the development of antibiotic resistance hotspots in different environmental compartments, including the edible parts of crops. To assess the influence of the water quality used for watering, we collected samples from soil, roots, leaves and beans from the legume plant Vicia faba (broad beans) in three agricultural peri-urban plots (Barcelona, NE Spain), irrigated with either groundwater, river water, or reclaimed water. Antibiotic resistance genes (ARGs) sul1, tetM, qnrS1, blaCTX-M-32,blaOXA-58, mecA, and blaTEM were quantified by real-time PCR, along with 16S rDNA and intl1 sequences, as proxies for bacterial abundance and integron prevalence, respectively. Microbiome composition of all samples were analyzed by high-throughput DNA sequencing. Results show a gradient of bacterial species diversity and of ARG prevalence from highly diverse soil samples to microbially-poor beans and leaves, in which Rhizobiales essentially displaced all other groups, and that presented very small loads of ARGs and integron sequences. The data suggest that the microbiome and the associated resistome were likely influenced by agricultural practices and water quality, and that future irrigation water legal standards should consider the specific Physiology of the different crop plants.

Antibiotic resistance in wastewater treatment plants: Tackling the black box.

Wastewater is among the most important reservoirs of antibiotic resistance in urban environments. The abundance of carbon sources and other nutrients, a variety of possible electron acceptors such as oxygen or nitrate, the presence of particles onto which bacteria can adsorb, or a fairly stable pH and temperature are examples of conditions favouring the remarkable diversity of microorganisms in this peculiar habitat. The wastewater microbiome brings together bacteria of environmental, human and animal origins, many harbouring antibiotic resistance genes (ARGs). Although numerous factors contribute, mostly in a complex interplay, for shaping this microbiome, the effect of specific potential selective pressures such as antimicrobial residues or metals, is supposedly determinant to dictate the fate of antibiotic resistant bacteria (ARB) and ARGs during wastewater treatment. This paper aims to enrich the discussion on the ecology of ARB&ARGs in urban wastewater treatment plants (UWTPs), intending to serve as a guide for wastewater engineers or other professionals, who may be interested in studying or optimizing the wastewater treatment for the removal of ARB&ARGs. Fitting this aim, the paper overviews and discusses: i) aspects of the complexity of the wastewater system and/or treatment that may affect the fate of ARB&ARGs; ii) methods that can be used to explore the resistome, meaning the whole ARB&ARGs, in wastewater habitats; and iii) some frequently asked questions for which are proposed addressing modes. The paper aims at contributing to explore how ARB&ARGs behave in UWTPs having in mind that each plant is a unique system that will probably need a specific procedure to maximize ARB&ARGs removal.