Drivers of antibiotic resistance in two monsoon-impacted Indian urban rivers receiving untreated wastewater

Rivers in India receive 78.7% of untreated sewage from cities and towns making it a potential global "hotspot" for AMR. During monsoon, flooding is common in most Indian cities bringing in fecal contamination in storm runoff. Identifying the major driving factors contributing to the elevated levels of AMR in these rivers is essential to combat AMR proliferation. Determining whether the introduction of ARBs and ARGs through fecal pollution or subsequent (co-) selection for ARGs through chemical pollutants is responsible for the rise and spread of AMR is important in mitigation efforts. To achieve this, we targeted two rivers in northern India which received untreated wastewater and assessed the level of two fecal indicators (E. coli and intI1) and wastewater-associated ARGs (ermF, sul1, sul2, and tetW) along with heavy metals (Ag, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn). Water and sediment samples were collected in triplicates at 5-6 locations for each river. Consequently, to evaluate how seasonality affects the ARG levels as well as their drivers in these heavily polluted rivers, sampling campaigns were carried out during three seasons: in summer (pre-monsoon), during monsoon, and post-monsoon in winter. We here demonstrate that the main source for the river resistome in the Bindal and Rispana is the constant inflow of untreated wastewater throughout the year and not the co-selection of ARGs due to the presence of heavy metals. This may be true for many Indian rivers that receive untreated or partially treated wastewater. The rainfall adds to the ARG abundance of the river instead of diluting it. MGE intI1 is more reliable than E. coli as an indicator for fecal pollution and horizontal gene transfer. Heavy metals, despite being present in the river in higher concentrations than the MCSC, no evidence for co-selection was observed.