Occurrence and Spatial Distribution of Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes in Surface and Groundwater of the Kelani River Basin, Sri Lanka.

Antimicrobial resistance (AMR) in aquatic environments represents a critical global public health threat, yet comprehensive environmental surveillance data remain scarce in low- and middle-income countries. The Kelani River Basin, supplying approximately 80% of Colombo's potable water and Sri Lanka's most industrially impacted waterway, lacks a basin-wide characterization of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) across spatial and seasonal gradients. This study provides the first comprehensive basin-wide assessment of ARB distribution, ARG occurrence, minimum inhibitory concentrations (MICs), and multidrug-resistant (MDR) pathogen diversity across surface water and groundwater during wet and dry seasons. A total of 71 water samples (surface water: 53; groundwater: 18) were screened against ten clinically relevant antibiotics using standard pour-plate and agar dilution methods. ARG profiling was conducted using environmental DNA (eDNA)-based PCR, and MDR isolates were characterized by sequencing of the 16S rRNA gene. The transitional zone was identified as the principal contamination hotspot, with ARB loads significantly elevated during the wet season (p < 0.05). Phenotypic resistance was highest against cloxacillin (20.4%), amoxicillin (14.8%), and cefuroxime (14.4%), while ciprofloxacin and tetracycline exhibited the lowest resistance rates. MIC profiling revealed that 41.9% of isolates tolerated concentrations up to 600 µg/mL, with MDR isolates exhibiting MICs ≥ 540 µg/mL. MAR indices ranged from 0.1 to 1.0, with high MAR (≥ 0.5) predominant in dry (59.9%) and wet (43.5%) seasons. ARG screening detected sul1 (93%), qnrB (66.2%), mefA (43.7%), and blaCTX-M (23.9%) basin-wide across both water compartments. 16S rRNA sequencing confirmed clinically significant pathogens, including Klebsiella pneumoniae, Pseudomonas fluorescens, and Proteus mirabilis among high-level MDR isolates. Water temperature, total phosphorus, and nitrogen compounds were significantly associated with ARB abundance spatially and seasonally (p < 0.05). These findings reveal the Kelani River Basin as a high-risk environmental AMR reservoir, while delivering a low-cost, replicable surveillance framework for resource-limited settings to support evidence-based water quality policy and Sri Lanka's National Action Plan for AMR.