Substantial antibiotic resistance invasion of pristine river biofilms without specific alteration of the dominant community structure

The dissemination of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in freshwater ecosystems is increasingly recognized as a major environmental and public health concern. However, processes associated with the establishment of anthropogenic ARGs/MGEs in the environment remain poorly understood. In this study, we investigated the spatial dynamics of ARG/MGE entries along two low-impacted headwater rivers from pristine areas to the first signs of human activity. Epilithic biofilm and water samples collected along the river continuums during six sampling campaigns were analyzed using (i) high-throughput qPCR targeting a selection of 45 ARG and MGEs markers, (ii) microbial community dynamics by 16S rRNA gene amplicon sequencing, (iii) microbial source identification, and (iv) fecal coliforms enumeration. Both rivers exhibited a sharp increase in fecal contamination at the first inhabited areas, confirmed by human-specific microbial markers. This transition coincided with the abrupt introduction of anthropogenic ARGs/MGEs into river biofilms, whereas the relative abundance of these markers kept increasing further downstream. Notably, the widely used anthropogenic indicator intI1 exhibited an increasing relative abundance spanning five orders of magnitude starting from the pristine areas of the river, whereas ISEcp1, an insertion sequence involved in the mobilization of several ARGs, appeared immediately at the onset of anthropogenic influence. Although microbial community composition shifted markedly in one river, anthropogenic ARG/MGE invasion occurred with limited changes in community structure, indicating that resistance dissemination is not necessarily accompanied by detectable microbial replacement of the dominant community structure. Overall, these findings suggest that anthropogenic resistance determinants can establish within river biofilms even when the dominant microbial community remains largely unchanged.