Genomic evidence of clinically relevant antimicrobial resistance genes and mobile plasmids in wastewater-associated environmental bacteria

Abstract

Wastewater treatment plants (WWTPs) are increasingly recognized as environmental reservoirs for antimicrobial resistance (AMR), yet genome-level data on non-clinical Gram-negative bacteria in wastewater remain scarce in many low- and middle-income settings. This study characterized the resistome, plasmid content, and genetic diversity of environmentally associated Gram-negative bacteria recovered from wastewater systems to elucidate their contribution to environmental AMR persistence. Influent and effluent samples (n = 30) were collected from three wastewater treatment plants. Sixteen Gram-negative isolates were recovered and identified using the VITEK system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed phenotypically, followed by whole-genome sequencing and bioinformatic analyses to identify antimicrobial resistance genes (ARGs), plasmid replicons, virulence-associated genes, and sequence types.The isolates comprised of Enterobacter cloacae (n = 5), Raoultella planticola (n = 3), Raoultella ornithinolytica (n = 3), Citrobacter freundii (n = 3), and Aeromonas spp . (n = 2). Multidrug resistance was observed phenotypically across influent and effluent samples. Genomic analysis identified clinically relevant ARGs, including bla ACT , bla CTX-M-15 , bla CMY , qnr variants, fosA , aadA , sul1 , and tet genes, frequently associated with IncF, IncQ, IncL/M, and Col-type plasmids and virulent determinants including yersiniabactin and enterobactin . Resistance and plasmid profiles persisted in effluent-derived isolates, suggesting environmental persistence and potential transmission.This study provides genome-level baseline evidence that wastewater-associated, non-clinical Gram-negative bacteria in Namibia harbor clinically relevant AMR determinants, reinforcing the value of wastewater-based genomic surveillance for understanding environmental AMR dissemination, and emphasizing the public health implications of wastewater reuse.