Treating domestic wastewater towards freshwater quality: bacterial community and antibiotic resistance profiles highlight critical steps and improvement opportunities

Ideally, wastewater treatment aims to produce water indistinguishable from freshwater, especially for reuse. This study evaluated bacterial community and antibiotic resistance variations throughout treatment and benchmarked these with freshwater sources. Samples collected from six points of a full-scale wastewater treatment plant, pilot-scale advanced treatment options (non-thermal plasma - NTP, ultrafiltration - UF, UF followed by reverse osmosis- UF+RO), two rivers and a borehole were analyzed for quality parameters (BOD5, TSS, turbidity, Escherichia coli), antibiotic resistance genes (quantitative PCR), class 1 integron variable region composition (Oxford Nanopore sequencing), and bacterial community composition (16S rRNA Illumina sequencing). Secondary treatment followed by sand filters and coagulants caused the highest reduction (~2 log-unit/volume) of all analyzed parameters and the sharpest reduction of diversity of antibiotic resistance genes within class 1 integrons’ variable region. Ultraviolet disinfection triggered minimal bacterial or genes reduction, while among advanced treatments, UF+RO caused the highest, and NTP the lowest. Principal component analysis suggested significant associations between antibiotic resistance (n=32) and genetic recombination elements (n=12) and predominant bacterial families in raw wastewater (Aeromonadaceae, Moraxellaceae, Campylobacteraceae, Lachnospiraceae). For predominant freshwater families (Comamonadaceae, Chitinophagaceae, Flavobacteriaceae) no significant associations were observed. Freshwater differed from UF-treated water by a lower antibiotic resistance abundance, higher bacterial richness (~4000 vs.1200 operational taxonomic units) and distinct predominant families - Alcaligenaceae, Sphingomonadaceae, Chitinophagaceae, and Microbacteriaceae in UF water. The findings underscore the critical role of secondary/post-secondary treatments in shaping resistance and community profiles and suggest that advanced treatment should balance water quality with bacterial diversity preservation for sustainable reuse.