Spatiotemporal profiling of class 1 integrons and IncP-1 plasmids in an aquatic ecosystem heavily impacted by urban and industrial anthropic pressures

Although natural aquatic environments have progressively been recognized as playing a central role in the propagation of antibiotic resistance, they remain difficult to apprehend because of the thousands of existing resistance determinants, thus justifying the use of proxies. Nevertheless, most prevalence studies have focused on searching for anthropic inputs using comparative approaches, paying little attention to the multiple sources of variation observed, whether real or analytical. In this work, we examined in detail the molecular prevalence of two proxies for antibiotic resistance determinants, namely class 1 integrons and IncP-1 plasmids, in a 23 km long river continuum impacted by anthropic activities. A combination of nested and nested-factorial experiments demonstrated that the seasonality dominates over the position of sampling sites along the river in explaining variations in the abundance of these two proxies. Conversely, the sampling depth in the water column as well as analytical parameters, such as the reproducibility of DNA extractions and quantitative PCRs, do not significantly contribute to variation in the proxy abundances. Comparing data obtained from different sampling sites demonstrated that long distances are necessary to observe a significant increase in class 1 integron abundances in the river water and that the two proxies behave independently, thus highlighting both their complementarity as proxies and their distinct origins. The progressive input of integrons along the river continuum, which initially appeared as moderate, became more pronounced when analyzing their content in resistance gene cassettes, as the latter better represent the multiplicity and differences of the pollution sources. Our results provide an integrated view of the fluctuations in the abundance of class 1 integrons and IncP-1 plasmids, and bring up some limitations in using them as proxies for antibiotic resistance.