Water quality measurements and antimicrobial resistance in the Musi River, Hyderabad, India, 2022-2023

Water and sediment grab samples were collected at 10 locations along the Musi River, 1 upstream of the city (relatively clean), 8 within the city of Hyderabad (heavily polluted) and 2 downstream (pollution partially cleared), during the wet (18-19 August 2022) and dry (11-12 March 2023) seasons. Sampling occurred on days without rainfall for the preceding five days to minimize storm-related variability. At most sites, duplicate water samples were collected mid-stream from bridges using a bucket suspended on a rope and transferred to pre-sterilised bottles. Sub-samples were preserved immediately with H2SO4 for chemical oxygen demand (COD) analysis, whereas water temperature, pH, dissolved oxygen (DO), TDS and conductivity were measured on-site.

Water quality and sediment physicochemical parameters were measured using standard protocols. Dry matter (DM) and organic matter (OM) fractions were quantified using standard methods to establish a common basis for concentrations of bacteria and genes across sediment samples with different mineral content. Viable counts were taken on different media. E. coli was quantified on Tryptone Bile X-β-D-glucuronide agar as blue-green colonies, incubated at 44°C, as an indicator of faecal pollution and sentinel for AMR prevalence. 'Total' Gram-negative (GN) heterotrophic bacteria were enumerated on SDS-supplemented OECD synthetic sewage medium (OSS) at both 30°C for environmental and 44°C for gut-associated bacteria. Extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant (CARB) GN heterotrophic subpopulations were enumerated on OSS supplemented with appropriate selective antibiotics. These clinically relevant resistance phenotypes were selected as GN bacteria account for the majority of global AMR-attributable mortality. All samples were plated in triplicate within 24 hours of collection, and colony counts recorded after 24 hours of incubation.

DNA from both water and sediment samples was extracted within 24 hours of sampling. Using qPCR, the 16S rDNA gene was quantified as a proxy for total prokaryotes and to compare with culturing data. Antimicrobial resistance genes (ARGS) included genes coding for resistance to fluoroquinolones (qnrS), aminoglycosides (aph(3'')-Ib), sulfonamides (sul2), carbapenems and third-generation cephalosporins (blaNDM and blaCTX-M), macrolides (ermF), and tetracyclines (tetW). Two more genes were included: uidA to quantify E. coli numbers and intI1 to quantify class I integrons as a proxy for mobile genetic elements (MGEs).

Synthetic DNA fragments containing suites of target genes were used as positive controls and to create standard curves, for which ten-fold serial dilutions of gene fragments were performed in DNase/RNase-free water. Every sample was analysed in technical duplicates. Standard curves were included in each PCR plate with at least seven serial dilution points and technical duplicates. A joint standard curve based on standard curves from every run was created for every gene set. Concentrations of genes in the samples were then calculated using this joint standard curve.