International tempo-spatial study of antibiotic resistance genes across the Rhine river using newly developed multiplex qPCR assays

The aim of this study was to capture and explain changes in antibiotic resistance gene (ARG) presence and concentration internationally across the Rhine river. frail 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, frail 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. ARC 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 phanhaceutical 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 Mal and ARG concentrations (R-2 = 072) were driven by correlations to sul1 and disappeared when excluding sull from the analysis (R-2 = 0.05). Mat therefore seems to be a good proxy for sun concentrations but not necessarily for overall (anthropogenic) ARG pollution. Aminoglycosicle usage per country correlated with concentrations of aph(III)a and several unrelated antibiotic resistance genes (bla(OXA), ermB, coal and tetM). This correlation can be explained by co-resistance caused by mobile genetic elements (MGEs), such as Tn1545. (C) 2019 Elsevier B.V. All rights reserved.