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/.

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Antibiotic resistance is one of the biggest threats society is facing around the globe and has been on the rise worldwide. While antibiotic resistances play crucial roles in shaping and coordinating microbial communities in natural environments, they can lead to disastrous results when acquired by pathogens in clinical environments. Effective antibiotics not only enable the functioning and interactions necessary for our highly globalized world, but also drive advances in healthcare and are the deciding factor facilitating life-saving medical intervention such as open-heart surgery, organ transplants and chemotherapy. Increasing resistance antibiotics is threatening the medical status quo, as well as social and economic stability (Chapter 1). Water environments, especially anthropogenically impacted environments such as wastewater treatment plants, are suspected to be - not only - reservoirs for antibiotic resistance genes but also hotspots for horizontal gene transfer. Knowledge about the impact of anthropogenically impacted aqueous environments is needed in order to be able to uncover the processes, parameters and mechanisms underlying and facilitating the transfer of antibiotic resistance genes in order to be able to implement practical, useful and efficient measures in order to reduce the spread of antibiotic resistance and to reduce anthropogenic impact of antibiotic and antibiotic resistance gene pollution in the environmen.@en

The aim of this study was to capture and explain changes in antibiotic resistance gene (ARG) presence and concentration internationally across the Rhine river. Intl1 concentrations and national antibiotic usage were investigated as proxies to predict anthropogenic ARG pollution. Newly-developed multiplex qPCR assays were employed to investigate ARG profiles across 8 locations (L1-L8) in three countries (Switzerland, Germany, the Netherlands) and to detect potential regional causes for variation. Two of these locations were further monitored, over the duration of one month. A total of 13 ARGs, Intl1 and 16S rRNA were quantified. ARG presence and concentrations initially increased from L1(Diepoldsau) to L3(Darmstadt). A continuous increase could not be observed at subsequent locations, with the large river volume likely being a major contributing factor for stability. ARG presence and concentrations fluctuated widely across different locations. L2(Basel) and L3 were the two most polluted locations, coinciding with these locations being well-developed pharmaceutical production locations. We draw attention to the characteristic, clearly distinct ARG profiles, with gene presence being consistent and gene concentrations varying significantly less over time than across different locations. Five genes were Rhine-typical (ermB, ermF, Intl1, sul1 and tetM). Intl1 and sul1 were the genes with highest and second-highest concentration, respectively. Aph(III)a and bla_OXA were permanently introduced downstream of L1, indicating no source of these genes prior to L1. We highlight that correlations between Intl1 and ARG concentrations (R² = 0.72) were driven by correlations to sul1 and disappeared when excluding sul1 from the analysis (R² = 0.05). Intl1 therefore seems to be a good proxy for sul1 concentrations but not necessarily for overall (anthropogenic) ARG pollution. Aminoglycoside usage per country correlated with concentrations of aph(III)a and several unrelated antibiotic resistance genes (bla_OXA ermB, ermF and tetM). This correlation can be explained by co-resistance caused by mobile genetic elements (MGEs), such as Tn1545.

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This study quantified antibiotic and antibiotic resistance gene (ARG) concentrations in hospital and communal wastewaters as well as the influents and effluents of the receiving urban wastewater treatment plants (UWWTP) in two Dutch cities. In only one city, hospital wastewater was treated on-site using advanced technologies, including membrane bioreactor treatment (MBR), ozonation, granulated activated carbon (GAC) and UV-treatment. On-site hospital wastewater (HWW) treatment reduced gene presence of hospital-related antibiotic resistance genes and antibiotic concentrations in the receiving urban wastewater treatment plant. These findings support the need for on-site treatment of high-risk point sources of antibiotic resistance genes. 13 antibiotic resistance genes, Integrase Class 1 and 16S rRNA concentrations were quantified using multiplex quantitative real-time PCR (qPCR) assays and the presence and/or concentration of 711 antibiotics were analyzed. Hospital wastewater contained approximately 25% more antibiotics and gene concentrations between 0.4 log to 1.8-fold higher than communal wastewater (CWW). blaKPC and vanA could be identified as hospital-related genes and were reduced to under the limit of detection (LOD) during on-site treatment. Advanced on-site treatment removed between 0.5 and 3.6-fold more genes than conventional biological urban wastewater treatment (activated sludge). Advanced on-site treatment was able to eliminate 12 out of 19 detected antibiotics, while urban waste water treatment eliminated up to 1 (out of 21 detected). Different advanced treatment technologies were able to target different pollutants to varying extents, making sequential alignment more effective. MBR treatment was most efficient in antibiotic resistance gene reduction and ozonation in antibiotic reduction. blaKPC could only be detected in the influent of the urban wastewater treatment plant receiving untreated hospital wastewater. Similarly, vanA was only consistently detected in this treatment plant. These results indicate a positive effect of on-site treatment of hospital wastewater on the communal sewage system.@en

Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, Germany) in the Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a number of served population with the aim to get a representative holistic view of the pollution status. Samples were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESI-QTOF-MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX® bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins), PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause oxidative stress and induce xenobioticmetabolism was determined using the Nrf2 and PXR CALUX® bioassays, respectively. The signals from each of the bioassays were compared with the recently developed effect-based trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.@en