The influence of urbanization and water reclamation plants on fecal indicator bacteria and antibiotic resistance in the Los Angeles River watershed: A case study with complementary monitoring methods

Urban land use and water reclamation plants (WRPs) can impact fecal indicator bacteria (FIB) and antimicrobial resistance (AMR) in coastal watersheds. However, there is a lack of studies exploring these effects on the US West Coast. Additionally, there is limited research using a complementary approach across culture-, qPCR-, and metagenomics-based techniques for characterizing environmental AMR. In this study, sixteen locations were sampled in the Los Angeles River, encompassing both upstream and downstream of three WRPs discharging into the river. Culture-dependent methods quantified Enterococcus , total coliforms, E. coli , and extended spectrum beta-lactamase-producing E. coli as a low-cost screening tool for AMR, while qPCR measured selected antibiotic resistance genes (ARGs): sul 1, erm F, tet W, bla SHV, along with intI 1 and 16S rRNA genes. Bacteroides HF183 and crAssphage markers were quantified via ddPCR. All samples underwent shotgun sequencing to investigate gene abundance and mobility and an overall risk score for AMR. Results reveal downstream sites contain ARGs at least two orders of magnitude greater than upstream locations. Developed areas had the highest ARG sequence abundances and the most ARG classes as indicated by metagenomic analysis. WRP effluent exhibited elevated ARGs and co-location of ARGs, mobile genetic elements, and pathogens. A culture-based assessment of AR in E. coli and Pseudomonas aeruginosa revealed increased resistance ratios for most antibiotics from upstream to downstream a WRP discharge point. This study highlights the impacts of land use and WRPs on ARGs and FIB, offering a multi-pronged analysis of AMR. • ARGs, fecal indicator bacteria, and human fecal markers increase in abundance through an urbanization gradient. • Water reclamation plants increase ARGs, reduce fecal indicator bacteria, and increase resistant E. coli and P. aeruginosa . • A low-cost culture based screening tool identified AR hotspots, confirmed by qPCR- and metagenomics-based techniques.