Anthropogenic and natural disturbances along a river and its estuary alter the diversity of pathogens and antibiotic resistance mechanisms

Antibiotic resistance (AMR) in pathogens threatens human health worldwide, and antibiotic-resistant bacteria (ARB) are widespread in the environment. In particular, anthropogenically-disturbed rivers became reservoirs of ARBs and hotspots of antibiotic resistance gene (ARG) transmission. However, the diversity and sources of ARB, and the mechanisms of ARG transmission are not fully known. Here, we used deep metagenomic sequencing to study the dynamics of pathogens and their antibiotic resistance mechanisms along the Alexander River (Israel), affected by sewage and animal farm runoffs. Putative pathogens such as Aeromicrobium marinum and Mycobacterium massilipolynesiensis were enriched in western stations, following the inputs of polluted Nablus River. Aeromonas veronii was dominant in eastern stations in Spring. Several AMR mechanisms showed distinct patterns in Summer-Spring (dry season) and Winter (rainy season). We found low abundance beta-lactamases conferring carbapenem resistance: e.g., OXA-912 was linked to A. veronii in Spring; OXA-119 and OXA-205 to Xanthomonadaceae in Winter. We classified 33 % of ARG-containing contigs as putative plasmid sequences, indicating the high potential for resistome transmission. A limited number of ARGs were linked to putative phages. Our results suggest that this model river is a hotspot for AMR activity and transmission, and highlight the merit of deep sequencing for AMR discovery.