Using a harmonised study design and quantitative tool-box reveals major inconsistencies when investigating the main drivers of water and biofilm antibiotic resistomes in different rivers.

The spread of antibiotic resistance (ABR) via surface waters is of increasing concern. Large-scale studies investigating ABR drivers in different water bodies and habitats with uniform quantitative methods are largely missing. Here, we present a comprehensive investigation on ABR occurrence and drivers in water and biofilms of four Austrian rivers over a one-year-cycle using a harmonised quantitative tool-box and study-design. At the bacterial community level, human faecal pollution was a main factor driving the aquatic riverine resistome. Despite relatively low concentrations, also antibiotics and metals showed significant correlations, however to a different extent in the different rivers. At the organismic level, a decoupling of the Escherichia coli resistome from the bacterial community resistomes was observed. In biofilms, the relationships with anthropogenic pollution factors were heterogeneous and markedly dampened. Our results clearly show that general conclusions about the role of biofilms, the influence of pollution or the prevalence of resistance genes or phenotypic resistances must be drawn with caution. Results are dependent on the river and local situation of the sampling sites due to the large environmental heterogeneity. International harmonisation of the methodology and general awareness of this problem shall contribute to better understand environmental ABR to develop effective mitigation strategies.