Metagenomic exploration reveals a differential patterning of antibiotic resistance genes in urban and peri-urban stretches of a riverine system.

Antimicrobial resistance in the riverine ecosystem of urban areas is an alarming concern worldwide, indicating the importance of molecular monitoring to understand their patterning in urban and peri-urban areas. In the present study, we evaluated the influence of urban rivers on the connected peri-urban rivers of a riverine system of India in the context of antibiotic resistance genes. The rivers traversing through urban (Mula, Mutha, Pawana, and Ramnadi) and peri-urban stretches (Bhima and Indrayani) form the riverine system of Pune district in Maharashtra, India. The MinION-based shotgun metagenomic analysis revealed the resistome against 26 classes of antibiotics, including the last line of antibiotics. In total, we observed 278 ARG subtypes conferring resistance against multiple drugs (40%), bacitracin (10%), aminoglycoside (7.5%), tetracycline (7%), and glycopeptide (5%). Further, the alpha diversity analysis suggested relatively higher ARG diversity in the urban stretches than peri-urban stretches of the riverine system. The NMDS (non-metric multidimensional scaling) analysis revealed significant differences with overlapping similarities (stress value = 0.14, p-value = 0.004, ANOSIM statistic R: 0.2328). These similarities were reasoned by assessing the influence of downstream sites (sites at the outskirts of Pune city; however, directly impacted), which revealed significant differences in the ARG contents of urban and peri-urban stretches (stress value = 0.14, p-value = 0.001, ANOSIM statistic R: 0.6137). Overall, we detected the dissemination of antibiotic resistance genes from the polluted urban rivers into the peri-urban rivers located downstream in the connected riverine system potentially driven by anthropogenic activities.